Development and use of therapeutic drugs for TSLP-related diseases

Abstract

To provide an antibody that recognizes TSLP protein, and a preparation method and application therefor.SOLUTION: The present invention relates to an antibody binding to a TSLP protein or an antigen binding part thereof, a manufacturing method therefor and an application thereof. The antibody can bind to human TSLP and / or cynomolgus TSLP with high affinity, block the binding of TSLP and TSLPR, and inhibit the transduction of TSLP stimulus signals by means of an STAT5 pathway.SELECTED DRAWING: Figure 10

Description

[Technical Field]

[0001] The present disclosure relates to antibodies that recognize the TSLP protein, methods for their preparation, and uses. [Background technology]

[0002] Thymic stromal lymphopoietin (TSLP), a cytokine of the interleukin-7 family, is produced primarily by epithelial cells and keratinocytes in the skin, intestine, or lungs and is involved in maintaining the stability of the mucosal immune system. After epithelial tissue is stimulated or destroyed by allergens or pathogens, dendritic cells present antigens to naive CD4+ T cells and release the epithelial cytokines, TSLP and IL-33. These important costimulatory cytokines induce the development of allergen-specific Th2 cells, which subsequently produce the cytokines interleukins IL-4, IL-5, and IL-13. These Th2-derived cytokines play an important role in the pathogenesis of allergic diseases.

[0003] TSLP is a multifunctional cytokine that exerts its biological functions through the TSLPR / IL-7Rα receptor on the surface of multiple immune cells, including DCs, CD4+ and CD8+ T cells, B cells, mast cells, basophils, eosinophils, and NKT cells (Ziegler SF, 2013). TSLP stimulates the activation of immature dendritic cells (iDCs), increasing antigen presentation and the production of IL-8 (interleukin 8, or chemokine (C-C motif) ligand 8 (CXCL8)), eosinophil chemokine-2 (eotaxin 2, or chemokine (C-C motif) ligand 24 (CCL24)), TARC (thymus and activation-regulated chemokine, or chemokine (C-C motif) ligand 17 (CCL17)), and MDC (macrophage-derived chemokine, or chemokine (C-C motif) 22 (CCL22)), which attract and aggregate eosinophils, neutrophils, and Th2 cells. TSLP also promotes the differentiation of naive T cells into Th2 cells, a process that relies on the induction of TSLP on DCs expressing OX40L. TSLP induces mast cell proliferation, prolongs the eosinophil life cycle, and specifically releases proinflammatory cytokines and chemokines. TSLP stimulates group 2 innate lymphoid cells (ILC2s), another important group of mucosal immune cells, to secrete the Th2 cytokines IL-4, IL-5, and IL-13, promoting skin inflammation in vivo. Smooth muscle cells also secrete IL-8 and eotaxin upon stimulation with TSLP. TSLP has also been shown to increase cytokine production in multiple innate immune cells, including ILC2s, mast cells, natural killer cells, and eosinophils, and to promote the development and function of basophil subpopulations.Therefore, TSLP may be one of the initiators of the inflammatory cascade, and inhibition of TSLP can intervene at the early stage of inflammation and prevent immune cells from releasing proinflammatory cytokines, which is more effective than inhibiting IL-4, IL-5, or IL-13 alone.

[0004] TSLP transmits signals via the JAK / STAT (JAK kinase-signal transducer and activator of transcription) pathway. TSLP binds to TSLPR on the cell membrane and then binds to IL-7Rα to form a stabilized TSLP-TSLPR-IL7Rα receptor complex. The intracellular segment of the TSLPR receptor in this complex recruits and activates JAK2, which works together with JAK1 recruited to IL7Rα to activate downstream signaling molecules. In human peripheral blood-derived CD11c+ DC cells, TSLP can activate multiple STAT signaling molecules, including STAT1, STAT3, STAT4, STAT5, and STAT6. STAT5 activation signaling is crucial for promoting the differentiation of TH2 cells and the secretion of TH2 factors. TSLP can also stimulate the activation of the JAK / STAT5 signaling pathway and abolish the inhibitory effect of glucocorticoids on ILC2 cells, suggesting that TSLP inhibition may contribute to the restoration of glucocorticoid sensitivity in patients.

[0005] TSLP is closely related to the development of type II inflammatory diseases. Studies have shown that TSLP is highly expressed in damaged skin of patients with atopic dermatitis, but not in undamaged skin. Patients with asthma and chronic obstructive pulmonary disease (COPD) have abundant TSLP mRNA-positive cells in the pulmonary epithelium and submucosa, and alveolar lavage samples from these patients have higher TSLP concentrations than healthy controls. TSLP expression levels in asthma patients are directly correlated with the expression of TH2 cytokines and chemokines and inversely correlated with the patient's residual pulmonary function. Biopsies from patients with allergic rhinitis show increased expression of TSLP in the nasal epithelium, which is associated with TH2 cytokine production and eosinophil infiltration in epithelial-associated tissues.

[0006] Genetic polymorphisms in TSLP and TSLPR are thought to be associated with the pathogenesis of eosinophilic esophagitis (EoE). High expression of TSLP and an increased proportion of basophils (lin-, CD49b+, FcεRI+, c-kit-, 2D7+) have been detected in esophageal samples from EoE patients. Notably, mouse models of EoE depend on TSLP and basophils, but not IgE. Antibody neutralization or basophil depletion using TSLP can effectively alleviate EoE symptoms, suggesting that inhibition of TSLP-basophils, but not IgE, may be an effective method for treating EoE.

[0007] Studies have also shown that TSLP may promote TH1 / TH17-related autoimmune diseases, such as rheumatoid arthritis and multiple sclerosis. TSLP- and TSLPR-expressing cells increase in the joints of patients with rheumatoid arthritis. In a proteoglycan-induced mouse model of rheumatoid arthritis, TSLPR-deficient mice exhibit reduced levels of IL-17, IL-1β, and IL-6, and increased levels of INFγ and IL-10, resulting in alleviated disease symptoms. These findings suggest that TSLP and its receptors may be novel therapeutic targets for rheumatoid arthritis. Summary of the Invention

[0008] The present inventors have obtained high-affinity antibodies that recognize human and cynomolgus monkey TSLP recombinant proteins by immunizing mice with recombinant TSLP protein, a gene gun, or a combination thereof. The antibodies disclosed herein have high affinity and can effectively block the binding of TSLP to its receptor TSLPR. They can also block TSLP-induced STAT5 signaling in reporter gene-expressing cells and simultaneously block TSLP-induced cell proliferation, with higher efficiency than other antibodies of the same type. Therefore, they can be used in the diagnosis and treatment of allergic inflammatory diseases.

[0009] In one aspect, the disclosure provides an antibody, or antigen-binding portion thereof, that binds to a TSLP protein.

[0010] In one aspect, the present disclosure provides a nucleic acid molecule encoding an antibody or antigen-binding portion thereof according to the previous aspect.

[0011] In one aspect, the present disclosure provides a vector comprising the nucleic acid molecule described in the previous aspect.

[0012] In one aspect, the present disclosure provides a cell comprising the vector described in the previous aspect.

[0013] According to the antibody or antigen-binding portion thereof of any one of the preceding aspects, the antibody or antigen-binding portion thereof is humanized.

[0014] In one aspect, the disclosure provides a pharmaceutical composition or kit comprising the antibody or antigen-binding portion thereof of any one of the preceding aspects, or a nucleic acid encoding same, and a pharmaceutically acceptable carrier.

[0015] In one aspect, the disclosure provides a method of treating a TSLP-associated disorder, comprising administering to the mammal a therapeutically effective amount of the antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, cell, and / or pharmaceutical composition of any one of the preceding aspects.

[0016] In one aspect, the disclosure provides the use of an antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, cell, and / or pharmaceutical composition according to any one of the preceding aspects in the manufacture of a medicament or kit for treating a TSLP-related disorder in a mammal.

[0017] The antibody can bind to human TSLP and / or cynomolgus monkey TSLP with high affinity, block the binding of TSLP to TSLPR, and inhibit the transmission of TSLP-stimulated signals via the STAT5 pathway. [Brief explanation of the drawings]

[0018] [Figure 1] FIG. 1 shows TSLP protein recombinantly expressed in prokaryotes. [Figure 2] FIG. 1 shows TSLPR protein recombinantly expressed in HEK293 cells. [Figure 3] FIG. 1 shows detection of TSLP recombinant protein-binding receptor TSLPR by ELISA. [Figure 4] FIG. 1 shows FACS detection of Ba / F3-hTSLPR cells. [Figure 5] FIG. 1 shows detection of Ba / F3-hTSLPR-IL7Rα cell proliferation. [Figure 6] FIG. 1 shows detection of Ba / F3-hTSLPR-IL7Rα-STAT5, a reporter gene in luc cells. [Figure 7] FIG. 1 shows ELISA binding detection of chimeric antibodies. [Figure 8] FIG. 1 shows ELISA blocking detection of chimeric antibodies. [Figure 9] Figure 1 shows cellular blockade of chimeric antibodies. [Figure 10] FIG. 1 shows the inhibition of cell proliferation by chimeric antibodies. [Figure 11] FIG. 1 shows the inhibition of reporter gene expression by chimeric antibodies. [Figure 12]FIG. 1 shows ELISA detection of binding of humanized antibodies to human TSLP and cynomolgus TSLP. [Figure 13] FIG. 1 shows ELISA blocking detection of humanized antibodies. [Figure 14] Figure 1 shows cellular blocking of humanized antibodies. [Figure 15] FIG. 1 shows the inhibition of cell proliferation by humanized antibodies. [Figure 16] FIG. 1 shows the inhibition of reporter gene expression by humanized antibodies. DETAILED DESCRIPTION OF THE INVENTION

[0019] I. Definition In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology used herein are terms and routine procedures commonly used in the corresponding fields. For a better understanding, definitions and explanations of relevant terms are provided below.

[0020] In one aspect, the present disclosure provides antibodies (e.g., monoclonal antibodies) and antigen-binding fragments thereof that specifically bind to TSLP. In a specific aspect, the present disclosure provides monoclonal anti-TSLP antibodies that specifically bind to TSLP, wherein the anti-TSLP antibodies comprise variants of a parent antibody. In a specific aspect, the present disclosure provides antibodies that specifically bind to TSLP (e.g., human TSLP). In a specific aspect, the present disclosure provides anti-TSLP antibodies that contain one or more amino acid residue modifications (e.g., 5-13 amino acid substitutions in the framework regions of the heavy chain variable region), and that maintain affinity for the antigen compared to a parent antibody without such modifications.

[0021] Unless otherwise specified, the terms "about" or "approximately" as used herein refer to ±10% of a particular numerical value or range. When required to be an integer, the term is rounded to the nearest integer within ±10% of the particular numerical value or range.

[0022] With respect to antibody chain polypeptide sequences, the term "substantially identical" is understood to refer to antibody chains that exhibit at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to a control polypeptide sequence. With respect to nucleic acid sequences, the term is understood to refer to a nucleotide sequence that exhibits at least 60% or more, at least 65% or more, at least 70% or more, at least 75% or more, at least 80% or more, at least 85% or more, at least 90% or more, at least 95% or more, at least 96% or more, at least 97% or more, at least 98% or more, at least 99% or more sequence identity to a control nucleic acid sequence.

[0023] Sequence "homology" or "identity" has the meaning well known in the art, and conventional techniques can be used to calculate the percentage of sequence identity between two nucleic acid or polypeptide molecules or domains. Sequence identity can be measured along the entire length of a polynucleotide or polypeptide or along a region of the molecule (see, e.g., Computational Molecular Biology, Lesk, A.M., ed., Oxford University Pres, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, (See, e.g., "Sequence Analysis in Molecular Biology," von Heinje, G., Academic Press, 1987; and "Sequence Analysis Primer," Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there are many methods for determining the identity of two polynucleotides or polypeptides, the term "identity" is well known to those skilled in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988)).

[0024] "Substitutional" variants are those in which at least one amino acid residue in a native sequence has been removed and a different amino acid inserted in its place. The substitutions may be single, where only one amino acid has been substituted in the molecule, or multiple, where two or more amino acids have been substituted in the same molecule. Multiple substitutions may occur at consecutive sites. Similarly, an amino acid may be substituted by multiple residues, and such variants include both substitutions and insertions. Adjacent to an amino acid means attached to the α-carboxyl or α-amino functional group of the amino acid. "Deletion" variants are variants in which one or more amino acids in a native amino acid sequence have been removed. Typically, deletion variants have one or two amino acids deleted in a specific region of the molecule.

[0025] With respect to antibody variable regions, the term "variable" refers to the specific portions of the associated molecule that vary significantly in sequence among antibodies and are used for specific recognition and binding of a particular antibody to a particular target. However, variability is not uniformly distributed throughout the variable regions of antibodies. The variability is concentrated in three segments called complementarity-determining regions (CDRs, i.e., CDR1, CDR2, and CDR3) or hypervariable regions, all found in the light and heavy chain variable regions. The more conserved portions of the variable regions are called framework (FR) regions or framework sequences. Each naturally occurring heavy and light chain variable region contains four FR regions, both primarily of β-sheet structure, connected via three CDRs that form loops that connect, and in some cases, form part of, the β-sheet structure. The CDRs of each chain are usually contiguous and linked by FR regions and, together with the CDRs from the other chain, contribute to the formation of the target binding site (epitope or determinant) of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, MD (1987)). As used herein, immunoglobulin amino acid residues are numbered according to the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise specified. A CDR may have the ability to specifically bind to an associated epitope.

[0026] As used herein, an "antibody fragment" or "antigen-binding fragment" of an antibody refers to any portion of a full-length antibody that is less than full-length but contains at least a partial variable region of the antibody that binds to an antigen (e.g., one or more CDRs and / or one or more antibody-binding sites), thereby retaining the binding specificity and at least a portion of the specific binding ability of the full-length antibody. Thus, an antigen-binding fragment refers to an antibody fragment that contains an antigen-binding portion that binds to the same antigen as the antibody from which the antibody fragment was generated. Antibody fragments include antibody derivatives produced by enzyme-catalyzed treatment of a full-length antibody, as well as synthetically produced derivatives, e.g., recombinantly produced derivatives. Antibodies include antibody fragments. Specific examples of antibody fragments include Fab, Fab', F(ab')2, single-chain Fv (scFv), Fv, dsFv, diabody, Fd and Fd' fragments, and modified fragments (e.g., see Methods in Molecular Biology, Vol. 207: Recombinant Antibodies Examples of suitable fragments include, but are not limited to, other fragments comprising a single chain (e.g., fragments of a single antibody fragment, see "Chapter 1 for Cancer Therapy Methods and Protocols (2003); p3-25, Kipriyanov). The fragments may comprise, for example, multiple chains linked by disulfide bonds and / or peptide linkers. Antibody fragments usually contain at least or about 50 amino acids, and typically contain at least or about 200 amino acids. Antigen-binding fragments are fragments that, when inserted into an antibody framework (e.g., by substituting the corresponding domain), are immunospecific (i.e., at least 10 7 ~10 8 M -1 Ka of at least about 10 7 ~10 8 M -1A "functional fragment" or "anti-TSLP antibody analog" is a fragment or analog that prevents or substantially reduces the ability of the receptor to bind to a ligand or initiate signaling. As used herein, a functional fragment generally has the same meaning as an "antibody fragment," and, in the case of an antibody, a functional fragment is a fragment or analog that prevents or substantially reduces the ability of the receptor to bind to a ligand or initiate signaling. "Fv" refers to a fragment, such as Fv, Fab, or F(ab')2, that prevents or substantially reduces the ability of the receptor to bind to a ligand or initiate signal transduction. An "Fv" fragment is a dimer (V) formed by the non-covalent association of one heavy chain variable region and one light chain variable region. H -V L In this configuration, the three CDRs of each variable region interact to form a V H -V L The six CDRs define a target-binding site on the surface of the dimer. Together, the six CDRs confer target-binding specificity to the full-length antibody. However, a single variable region (or half of an Fv containing only three target-specific CDRs) can also function to recognize and bind to a target.

[0027] As used herein, the term "bispecific antibody (BsAb)" refers to an antibody and / or antigen-binding molecule that can specifically bind to two different antigenic determinants. Typically, a bispecific antibody and / or antigen-binding molecule contains two antigen-binding sites, each capable of specifically binding to a different antigenic determinant. In some embodiments, the bispecific antibody and / or antigen-binding molecule can simultaneously bind to two antigenic determinants, particularly two antigenic determinants expressed on two different types of cells.

[0028] As used herein, the term "monoclonal antibody" refers to a population of identical antibodies, meaning that each individual antibody molecule in the monoclonal antibody population is identical to all other antibody molecules. This characteristic is the opposite of a polyclonal population of antibodies, which contains antibodies with a variety of different sequences. Monoclonal antibodies can be prepared by a number of known methods (Smith et al. (2004) J. Clin. Pathol. 57, 912-917 and Nelson et al., J. Clin. Pathol (2000), 53, 111-117). For example, monoclonal antibodies can be prepared by immortalizing B cells, e.g., by fusing them with myeloma cells to produce hybridoma cell lines, or by infecting B cells with a virus such as EBV. Recombinant technology can also be used to prepare antibodies in vitro from a clonal population of host cells by transforming the host cells with a plasmid carrying an artificial sequence of nucleotides encoding the antibody.

[0029] As used herein, the term "hybridoma" or "hybridoma cell" refers to a cell or cell line (usually a myeloma or lymphoma cell) produced by the fusion of an antibody-producing lymphocyte with a non-antibody-producing cancer cell. As known to those skilled in the art, a hybridoma can grow and produce a continuous supply of a specific monoclonal antibody. Methods for producing hybridomas are known in the art (see, e.g., Harlow & Lane, 1988). When the term "hybridoma" or "hybridoma cell" is used, it also includes subclones and progeny of hybridomas.

[0030] As used herein, a full-length antibody is an antibody having two full-length heavy chains (e.g., VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4), two full-length light chains (VL-CL), and a hinge region, including, for example, antibodies produced naturally by antibody-secreting B cells and synthetically produced antibodies having the same regions.

[0031] The term "chimeric antibody" refers to an antibody whose variable region sequences are derived from one species and whose constant region sequences are derived from another species, such as, for example, an antibody whose variable region sequences are derived from a murine antibody and whose constant region sequences are derived from a human antibody.

[0032] "Humanized" antibodies are chimeric immunoglobulins, immunoglobulin chains, and fragments thereof (e.g., Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin (e.g., Humanized antibodies preferably refer to antibodies of a human immunoglobulin (recipient antibody) in which complementarity-determining region (CDR) residues of the recipient antibody are substituted by CDR residues from a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity.

[0033] Humanization can also involve mutating amino acid residues in the CDR1, CDR2, and / or CDR3 regions of VH and / or VL to improve one or more binding characteristics (e.g., affinity) of the antibody. For example, mutations can be introduced by PCR-mediated mutagenesis, and their effect on antibody binding or other functional properties can be assessed using in vitro or in vivo assays described herein. Conservative mutations are typically introduced. Such mutations may be amino acid substitutions, additions, or deletions.

[0034] The term "CDR" as used herein refers to a complementarity-determining region, and each heavy and light chain of an antibody molecule is known to have three CDRs. CDRs, also known as hypervariable regions, are present in the variable regions of each heavy and light chain of an antibody, and contain highly variable regions in the primary structure of the CDRs. Herein, the heavy chain CDRs are represented by CDR1, CDR2, and CDR3 at the amino terminus of the amino-terminal sequence derived from the heavy chain, and the light chain CDRs are represented by CDR1, CDR2, and CDR3 at the amino terminus of the amino-terminal sequence derived from the light chain. These regions are adjacent to each other in the tertiary structure and determine the specificity of the antigen binding to the antibody.

[0035] The term "epitope" as used herein refers to any antigenic determinant on an antigen that binds to the paratope of an antibody. Epitopic determinants usually comprise chemically active surface subtypes of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.

[0036] As used herein, the terms "specifically binds" or "immunospecifically binds" with respect to an antibody or antigen-binding fragment thereof are used interchangeably herein and refer to the ability of the antibody or antigen-binding fragment to form one or more non-covalent bonds with a cognate antigen through non-covalent interactions between the antibody and the antibody-binding site of the antigen. The antigen may be isolated or may be present on tumor cells. Typically, antibodies that immunospecifically bind (or specifically bind) to an antigen are present in an amount of about 1×10 7 M -1 or approximately 1 x 10 8 M -1 or a larger affinity constant Ka (or 1 × 10 -7 or 1 x 10 -8The antibody binds to the antigen with a dissociation constant (Kd) of no greater than M. The affinity constant can be measured by standard kinetic methods of antibody reactions, such as immunoassays, surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin. Biotechnol 11:54; Englebienne (1998) Analyst. 123:1599), isothermal titration calorimetry (ITC), or other kinetic interactions known in the art (see, e.g., Paul, ed.; Fundamental Immunology, 2nd ed.; Raven Press, New York, pages 332-336 (1989)). See also U.S. Pat. No. 7,229,619, which describes exemplary SPR and ITC methods for calculating antibody binding affinity). Devices and methods for detecting and monitoring binding kinetics in real time are known and commercially available (BiaCore 2000, Biacore AB, Upsala, Sweden and GE Healthcare Life Sciences, see Malmqvist (2000) Biochem. Soc. Trans. 27:335).

[0037] As used herein, the terms "polynucleotide" and "nucleic acid molecule" refer to an oligomer or polymer containing at least two linked nucleotides or nucleotide derivatives, typically deoxyribonucleic acid (DNA) and ribonucleic acid (RIB) linked by phosphodiester bonds. This includes nucleic acids (RNA). As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. Nucleic acid molecules may be single-stranded or double-stranded, and may be cDNA.

[0038] As used herein, an isolated nucleic acid molecule is a nucleic acid molecule that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid molecule. For example, an "isolated" nucleic acid molecule of a cDNA molecule is substantially free of other cellular materials or culture medium when prepared by recombinant techniques, or substantially free of chemical precursors or other chemical components when chemically synthesized. Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules that encode the provided antibodies or antigen-binding fragments.

[0039] As used herein, "operably linked" with respect to nucleic acid sequences, regions, elements, or domains means that the nucleic acid regions are functionally related to each other. For example, a promoter can be operably linked to a nucleic acid encoding a polypeptide such that it can regulate or mediate transcription of the nucleic acid.

[0040] "Conservative sequence modifications" of the sequences set forth in the sequence listing herein are modifications that do not delete the nucleotide and amino acid sequence encoded by the nucleotide sequence or containing the amino acid sequence that binds to the antigen of the antibody. These conservative sequence modifications include conservative nucleotide and amino acid substitutions, as well as nucleotide and amino acid additions and deletions. For example, modifications can be introduced into the sequence listings set forth herein by standard techniques known in the art (e.g., site-directed mutagenesis and PCR-mediated mutagenesis). Conservative sequence modifications include conservative amino acid substitutions, in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in an anti-TSLP antibody is preferably replaced with another amino acid residue from the same side chain family. Methods for identifying conservative nucleotide and amino acid substitutions that do not eliminate antigen binding are well known in the art (e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al., Biochem. 32: 1180-1187 (1993)). al., Protein Eng. 12(10): 879-884 (1999); see Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997)).

[0041] Alternatively, in another embodiment, for example, saturation mutagenesis can be used to randomly introduce mutations along all or part of the sequence encoding an anti-TSLP antibody, and the resulting modified anti-TSLP antibodies can be screened for improved binding activity.

[0042] As used herein, "expression" refers to the process of producing a polypeptide by transcription and translation of a polynucleotide. The expression level of a polypeptide can be assessed using any method known in the art, including, for example, methods for determining the amount of polypeptide produced from a host cell. Such methods include, but are not limited to, quantitating polypeptide in cell lysates by ELISA, gel electrophoresis followed by Coomassie blue staining, Lowry protein assay, and Bradford protein assay. do not have.

[0043] As used herein, a "host cell" is a cell used to receive, maintain, replicate, and amplify a vector. A host cell can also express a polypeptide encoded by the vector. When the host cell divides, the nucleic acid contained in the vector is replicated, thereby amplifying the nucleic acid. A host cell can be a eukaryotic or prokaryotic cell. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, and HEK cells such as HEK293 cells.

[0044] As used herein, a "vector" refers to a replicable nucleic acid that can express one or more heterologous proteins when transformed into a suitable host cell. Vectors include those into which a nucleic acid encoding a polypeptide or a fragment thereof can be introduced, typically via restriction enzyme digestion and ligation. Vectors also include vectors containing a nucleic acid encoding a polypeptide. A vector introduces a nucleic acid encoding a polypeptide into a host cell, amplifies the nucleic acid, or expresses / displays the polypeptide encoded by the nucleic acid. While vectors are typically free-standing, they may also be engineered into a chromosome, where a gene or a portion thereof is integrated into the genome. Artificial chromosome vectors, such as yeast artificial vectors and mammalian artificial chromosomes, are also contemplated. The selection and use of these vehicles are well known to those skilled in the art.

[0045] As used herein, vector further includes "viral vectors" or "viral vectors." Viral vectors are genetically engineered viruses that are operably linked to exogenous genes so as to transfer the exogenous genes (as vehicles or shuttles) into cells.

[0046] As used herein, "expression vector" includes vectors capable of expressing DNA operably linked to regulatory sequences capable of affecting expression of DNA fragments, such as, for example, promoter regions. Such additional fragments include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, and the like. Expression vectors are typically derived from plasmid or viral DNA, or may contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus, or other vector, that, upon introduction into a suitable host cell, results in expression of cloned DNA. Suitable expression vectors include those capable of replicating in eukaryotic and / or prokaryotic cells, as well as expression vectors in a free state or that integrate into a host cell genome, as is well known to those skilled in the art.

[0047] As used herein, "treating" an individual with a disease or symptoms of a disease means that the individual's symptoms are partially or completely alleviated, or remain unchanged after treatment. Thus, treatment includes prophylaxis, treatment, and / or cure. Prevention refers to preventing underlying disease and / or preventing worsening of symptoms or onset of disease. Treatment includes any pharmaceutical use of the provided antibodies or antigen-binding fragments thereof and compositions provided herein.

[0048] As used herein, "therapeutic effect" refers to the effect of treating an individual to alter, generally improve or ameliorate, or cure a disease or disease symptoms.

[0049] As used herein, a "therapeutically effective amount" or "therapeutically effective dose" refers to a therapeutically effective amount that, after administration to a subject, It refers to the amount of a substance, compound, material, or composition containing a compound that is sufficient to produce at least a therapeutic effect, i.e., the amount needed to prevent, cure, ameliorate, delay, or partially delay a disease or a symptom of a disease.

[0050] As used herein, a "prophylactically effective amount" or "prophylactically effective dose" refers to an amount of a substance, compound, material, or composition containing a compound that, when administered to a subject, has the desired prophylactic effect, e.g., preventing or delaying the onset or recurrence of a disease or condition, or reducing the likelihood of the onset or recurrence of a disease or condition. A fully prophylactically effective dose need not occur by administering a single dose, but may occur after administering a series of doses. Thus, a prophylactically effective amount can be administered in one or more administrations.

[0051] As used herein, the term "patient" refers to a mammal, such as a human.

[0052] II. MODE FOR CARRYING OUT THE INVENTION In one aspect, the disclosure provides a heavy chain CDR selected from the amino acid sequences SEQ ID NOs: 8-10, 18-20, 28-30, 38-40, 48-50, 58-60, 68-70, 78-80, 83-85, 88-90, 93-95, 98-100, 103-105, 108-110, 113-115, 118-120, 123-125, 128-130, 133-135, 138-140, 143-145, 148-150, 153-155, 158-160, 163-165, or any variant thereof, and / or The present invention provides an antibody or antigen-binding portion thereof that binds to TSLP, comprising a light chain CDR selected from NOs: 13-15, 23-25, 33-35, 43-45, 53-55, 63-65, 73-75, 168-170, 173-175, or any variant thereof.

[0053] In one embodiment, the antibody or antigen-binding portion thereof according to the previous embodiment comprises a heavy chain CDR1 selected from the amino acid sequence of SEQ ID NO: 8, 18, 28, 38, 48, 58, 68, 78, 83, 88, 93, 98, 103, 108, 113, 118, 123, 128, 133, 138, 143, 148, 153, 158, 163, or any variant thereof; a heavy chain CDR2 selected from the amino acid sequence of SEQ ID NO: 9, 19, 29, 39, 49, 59, 69, 79, 84, 89, 94, 99, 104, 109, 114, 119, 124, 129, 134, 139, 144, 149, 154, 159, 164, or any variant thereof; a heavy chain CDR3 selected from the amino acid sequence of SEQ ID NO: 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, or any variant thereof; and / or a light chain CDR1 selected from the amino acid sequence of SEQ ID NO: 13, 23, 33, 43, 53, 63, 73, 168, 173, or any variant thereof; a light chain CDR2 selected from the amino acid sequence of SEQ ID NO: 14, 24, 34, 44, 54, 64, 74, 169, 173, or any variant thereof; 15, 25, 35, 45, 55, 65, 75, 170, 175 or any variant thereof.

[0054] In one aspect, the present disclosure provides an antibody or antigen-binding portion thereof according to the previous aspect, comprising a combination of heavy and light chain CDRs selected from:

[0055] (1) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 8-10, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 13-15, respectively; (2) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 18-20, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 23-25, respectively; (3) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 28-30, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 33-35, respectively; (4) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 38-40, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 43-45, respectively; (5) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 48-50, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 53-55, respectively; (6) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 58-60, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 63-65, respectively; (7) Heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 68-70, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 73-75, respectively. (8) Heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 78-80, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 168-170, respectively. (9) Heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 83-85, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 168-170, respectively. (10) Heavy chain CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NOs: 88-90, and / or light chain CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NOs: 168-170. (11) Heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 93-95, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 168-170, respectively. (12) Heavy chain CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NO: 98-100, and / or light chain CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NO: 168-170. (13) Heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 103-105, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 168-170, respectively. (14) Heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 108-110, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 168-170, respectively. (15) Heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 113-115, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 168-170, respectively. (16) Heavy chain CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NOs: 118-120, and / or light chain CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NOs: 168-170. (17) Heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 123-125, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 168-170, respectively. (18) Heavy chain CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NOs: 128-130, and / or light chain CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NOs: 168-170. (19) Heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 133-135, respectively, and / or comprising SEQ ID NOs: 168-170, respectively. Light chain CDR1, CDR2 and CDR3 sequences (20) Heavy chain CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NO: 138-140, and / or light chain CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NO: 168-170. (21) Heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 143-145, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 168-170, respectively. (22) Heavy chain CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NOs: 148-150, and / or light chain CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NOs: 168-170. (23) Heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 153-155, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 168-170, respectively. (24) Heavy chain CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NOs: 158-160, and / or light chain CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NOs: 168-170. (25) Heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 163-165, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 168-170, respectively. (26) Heavy chain CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NOs: 153-155, and / or light chain CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NOs: 173-175. (27) Heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 158-160, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 173-175, respectively. (28) Heavy chain CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NOs: 163-165, and / or light chain CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NOs: 173-175. (29) Heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 78-80, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 173-175, respectively. (30) Heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 83-85, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 173-175, respectively. (31) Heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 98-100, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 173-175, respectively. (32) Heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 103-105, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 173-175, respectively. (33) Heavy chain CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NOs: 123-125, and / or light chain CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NOs: 173-175. (34) Heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 128-130, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 173-175, respectively.

[0056] In one embodiment, the antibody or antigen-binding portion thereof according to the previous embodiment is selected from the group consisting of the amino acid sequences SEQ ID NOs: 7, 17, 27, 37, 47, 57, 67, 77, 82, 87, 92, 97, 102, 107, 112, 117, 122, 127, 132, 137, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168 , 147, 152, 157, 162, or any variant thereof, and / or a light chain variable region selected from the amino acid sequences of SEQ ID NOs: 12, 22, 32, 42, 52, 62, 72, 167, 172, or any variant thereof.

[0057] In another aspect, the disclosure relates to an antibody, or antigen-binding portion thereof, that binds human TSLP, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 7 or any variant thereof, and a light chain variable region having the amino acid sequence of SEQ ID NO: 12 or any variant thereof.

[0058] In another aspect, the disclosure relates to an antibody, or antigen-binding portion thereof, that binds to human TSLP, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 17 or any variant thereof, and a light chain variable region having the amino acid sequence of SEQ ID NO: 22 or any variant thereof.

[0059] In another aspect, the disclosure relates to an antibody, or antigen-binding portion thereof, that binds to human TSLP, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 27 or any variant thereof, and a light chain variable region having the amino acid sequence of SEQ ID NO: 32 or any variant thereof.

[0060] In another aspect, the disclosure relates to an antibody, or antigen-binding portion thereof, that binds to human TSLP, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 37 or any variant thereof, and a light chain variable region having the amino acid sequence of SEQ ID NO: 42 or any variant thereof.

[0061] In another aspect, the disclosure relates to an antibody, or antigen-binding portion thereof, that binds to human TSLP, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 47 or any variant thereof, and a light chain variable region having the amino acid sequence of SEQ ID NO: 52 or any variant thereof.

[0062] In another aspect, the disclosure relates to an antibody, or antigen-binding portion thereof, that binds to human TSLP, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 57 or any variant thereof, and a light chain variable region having the amino acid sequence of SEQ ID NO: 62 or any variant thereof.

[0063] In another aspect, the disclosure relates to an antibody, or antigen-binding portion thereof, that binds to human TSLP, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 67 or any variant thereof, and a light chain variable region having the amino acid sequence of SEQ ID NO: 72 or any variant thereof.

[0064] In another aspect, the disclosure relates to an antibody, or antigen-binding portion thereof, that binds to human TSLP, comprising a heavy chain variable region of amino acid sequence SEQ ID NO: 77 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO: 167 or any variant thereof.

[0065] In another aspect, the disclosure relates to an antibody, or antigen-binding portion thereof, that binds to human TSLP, comprising a heavy chain variable region of amino acid sequence SEQ ID NO: 82 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO: 167 or any variant thereof.

[0066] In another aspect, the present disclosure provides a method for the preparation of a human ovarian cancer cell comprising the amino acid sequence SEQ ID NO: 87 or its analogs. and a light chain variable region having the amino acid sequence SEQ ID NO: 167 or any variant thereof.

[0067] In another aspect, the disclosure relates to an antibody, or antigen-binding portion thereof, that binds to human TSLP, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 92 or any variant thereof, and a light chain variable region having the amino acid sequence of SEQ ID NO: 167 or any variant thereof.

[0068] In another aspect, the disclosure relates to an antibody, or antigen-binding portion thereof, that binds to human TSLP, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 97 or any variant thereof, and a light chain variable region having the amino acid sequence of SEQ ID NO: 167 or any variant thereof.

[0069] In another aspect, the disclosure relates to an antibody, or antigen-binding portion thereof, that binds to human TSLP, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 102 or any variant thereof, and a light chain variable region having the amino acid sequence of SEQ ID NO: 167 or any variant thereof.

[0070] In another aspect, the disclosure relates to an antibody, or antigen-binding portion thereof, that binds to human TSLP, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 107 or any variant thereof, and a light chain variable region having the amino acid sequence of SEQ ID NO: 167 or any variant thereof.

[0071] In another aspect, the disclosure relates to an antibody, or antigen-binding portion thereof, that binds to human TSLP, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 112 or any variant thereof, and a light chain variable region having the amino acid sequence of SEQ ID NO: 167 or any variant thereof.

[0072] In another aspect, the disclosure relates to an antibody, or antigen-binding portion thereof, that binds to human TSLP, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 117 or any variant thereof, and a light chain variable region having the amino acid sequence of SEQ ID NO: 167 or any variant thereof.

[0073] In another aspect, the disclosure relates to an antibody, or antigen-binding portion thereof, that binds to human TSLP, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 122 or any variant thereof, and a light chain variable region having the amino acid sequence of SEQ ID NO: 167 or any variant thereof.

[0074] In another aspect, the disclosure relates to an antibody, or antigen-binding portion thereof, that binds to human TSLP, comprising a heavy chain variable region of amino acid sequence SEQ ID NO: 127 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO: 167 or any variant thereof.

[0075] In another aspect, the disclosure relates to an antibody, or antigen-binding portion thereof, that binds to human TSLP, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 132 or any variant thereof, and a light chain variable region having the amino acid sequence of SEQ ID NO: 167 or any variant thereof.

[0076] In another embodiment, the present disclosure provides a method for the treatment of a cancer comprising administering to a patient a therapeutically effective amount of the amino acid sequence SEQ ID NO: 137 or The present invention relates to an antibody or antigen-binding portion thereof that binds to human TSLP, comprising a heavy chain variable region of any variant thereof and a light chain variable region of amino acid sequence SEQ ID NO: 167 or any variant thereof.

[0077] In another aspect, the disclosure relates to an antibody, or antigen-binding portion thereof, that binds to human TSLP, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 142 or any variant thereof, and a light chain variable region having the amino acid sequence of SEQ ID NO: 167 or any variant thereof.

[0078] In another aspect, the disclosure relates to an antibody, or antigen-binding portion thereof, that binds to human TSLP, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 147 or any variant thereof, and a light chain variable region having the amino acid sequence of SEQ ID NO: 167 or any variant thereof.

[0079] In another aspect, the disclosure relates to an antibody, or antigen-binding portion thereof, that binds to human TSLP, comprising a heavy chain variable region having the amino acid sequence SEQ ID NO: 152 or any variant thereof, and a light chain variable region having the amino acid sequence SEQ ID NO: 167 or any variant thereof.

[0080] In another aspect, the disclosure relates to an antibody, or antigen-binding portion thereof, that binds to human TSLP, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 157 or any variant thereof, and a light chain variable region having the amino acid sequence of SEQ ID NO: 167 or any variant thereof.

[0081] In another aspect, the disclosure relates to an antibody, or antigen-binding portion thereof, that binds to human TSLP, comprising a heavy chain variable region of amino acid sequence SEQ ID NO: 162 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO: 167 or any variant thereof.

[0082] In another aspect, the disclosure relates to an antibody, or antigen-binding portion thereof, that binds to human TSLP, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 152 or any variant thereof, and a light chain variable region having the amino acid sequence of SEQ ID NO: 172 or any variant thereof.

[0083] In another aspect, the disclosure relates to an antibody, or antigen-binding portion thereof, that binds to human TSLP, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 157 or any variant thereof, and a light chain variable region having the amino acid sequence of SEQ ID NO: 172 or any variant thereof.

[0084] In another aspect, the disclosure relates to an antibody, or antigen-binding portion thereof, that binds to human TSLP, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 162 or any variant thereof, and a light chain variable region having the amino acid sequence of SEQ ID NO: 172 or any variant thereof.

[0085] In another aspect, the disclosure relates to an antibody, or antigen-binding portion thereof, that binds human TSLP, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 77 or any variant thereof, and a light chain variable region having the amino acid sequence of SEQ ID NO: 172 or any variant thereof.

[0086] In another aspect, the present disclosure provides a method for the production of a polypeptide comprising the amino acid sequence SEQ ID NO: 82 or its derivatives. and a light chain variable region having the amino acid sequence SEQ ID NO: 172 or any variant thereof.

[0087] In another aspect, the disclosure relates to an antibody, or antigen-binding portion thereof, that binds to human TSLP, comprising a heavy chain variable region of amino acid sequence SEQ ID NO: 97 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO: 172 or any variant thereof.

[0088] In another aspect, the disclosure relates to an antibody, or antigen-binding portion thereof, that binds to human TSLP, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 102 or any variant thereof, and a light chain variable region having the amino acid sequence of SEQ ID NO: 172 or any variant thereof.

[0089] In another aspect, the disclosure relates to an antibody, or antigen-binding portion thereof, that binds to human TSLP, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 122 or any variant thereof, and a light chain variable region having the amino acid sequence of SEQ ID NO: 172 or any variant thereof.

[0090] In another aspect, the disclosure relates to an antibody, or antigen-binding portion thereof, that binds to human TSLP, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO: 127 or any variant thereof, and a light chain variable region having the amino acid sequence of SEQ ID NO: 172 or any variant thereof.

[0091] In another aspect, the disclosure provides a nucleic acid molecule encoding the antibody or antigen-binding portion thereof of any one of the previous aspects, wherein the nucleic acid molecule preferably comprises an antibody heavy chain nucleic acid sequence chosen from SEQ ID NOs: 11, 21, 31, 41, 51, 61, 71, 81, 86, 91, 96, 101, 106, 111, 116, 121, 126, 131, 136, 141, 146, 151, 156, 161, 166, or any variant thereof, and / or an antibody light chain nucleic acid sequence chosen from SEQ ID NOs: 16, 26, 36, 46, 56, 66, 76, 171, 176, or any variant thereof.

[0092] In another aspect, the disclosure provides an antibody or antigen-binding portion thereof that binds to human TSLP and has at least 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or more sequence identity to an antibody or antigen-binding portion thereof of any one of the previous aspects.

[0093] In another aspect, the disclosure relates to a nucleic acid molecule encoding the antibody or antigen-binding portion thereof of any one of the previous aspects, or a nucleic acid molecule having at least 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or more, sequence identity thereto.

[0094] In another aspect, the present disclosure provides a vector comprising the nucleic acid of any one of the previous aspects.

[0095] A cell is provided comprising the vector of any one of the preceding aspects.

[0096] The antibody or antigen-binding portion thereof of any one of the preceding aspects, or a nucleic acid encoding same. The present invention provides a pharmaceutical composition comprising a nucleic acid and a pharmaceutically acceptable carrier.

[0097] In another aspect, the disclosure provides a method of treating a TSLP-related disorder, comprising administering to the mammal a therapeutically effective amount of the antibody or antigen-binding fragment, nucleic acid molecule, vector, cell, or pharmaceutical composition of any one of the preceding aspects.

[0098] In another aspect, the disclosure provides the use of an antibody or antigen-binding fragment thereof, nucleic acid molecule, vector, cell, or pharmaceutical composition described in any one of the preceding aspects in the preparation of a medicament for treating a TSLP-related disorder in a mammal.

[0099] According to any one of the above aspects, the TSLP-associated disorder may be a TSLP-associated inflammatory disorder or an autoimmune disorder. The antibody may be coupled to another agent, e.g., a labeled or cytotoxic conjugate.

[0100] In one embodiment, the present disclosure further includes a kit. For example, the kit includes an antibody, a fragment, a homolog, a derivative, or the like of the present disclosure, such as a labeled or cytotoxic conjugate, and a protocol for using the antibody, a conjugate that kills a specific type of cell, or the like. The protocol may include a guide for using the antibody, conjugate, or the like in vitro, in vivo, or ex vivo. The antibody may be liquid or solid and is typically lyophilized. The kit may further include other appropriate reagents, such as buffers, reconstitution solutions, and other necessary components depending on the intended use. A combination of packaged reagents in predetermined amounts and a protocol for using them for therapeutic or diagnostic assay applications is also contemplated. If the antibody is labeled, for example, with an enzyme, the kit may include a substrate and a cofactor required for the enzyme (e.g., a substrate precursor that provides a detectable chromophore or fluorophore). Other additives, such as stabilizers and buffers (e.g., a blocking buffer or lysis buffer), may also be included. The relative amounts of the various reagents can be varied to provide a concentrated reagent solution, thereby providing user flexibility, space savings, reagent savings, etc. These reagents can also be provided as dry powders, typically provided lyophilized, and contain excipients that, when dissolved, provide a solution of the appropriate concentration of the reagent.

[0101] In one aspect, the disclosure provides the use of an antibody or functional fragment thereof, nucleic acid molecule, vector, cell, pharmaceutical composition, and / or kit described in any one of the preceding aspects in the preparation of a reagent for inhibiting the binding of TSLP to TSLPR.

[0102] The antibodies of the present invention may also be used in immunoassays, purification methods and other methods that use immunoglobulins or fragments thereof, such uses being well known to those skilled in the art.

[0103] The present disclosure also provides compositions comprising an anti-TSLP antibody or fragment thereof of the present disclosure, which can be suitably combined with a pharmaceutically acceptable carrier, diluent, or excipient, as is well known in the art.

[0104] The term "pharmaceutical composition" as used in this disclosure refers to a formulation of various preparations. The formulations containing a therapeutically effective amount of a multivalent antibody may be in sterile liquid solution, liquid suspension, or lyophilized form, and may contain stabilizers or excipients.

[0105] The antibodies of the present disclosure may be used as the sole administered composition or may be used in combination with other active agents.

[0106] It should be understood that the therapeutic agents of the embodiments are administered with suitable pharmaceutically acceptable carriers, excipients, and other agents that are incorporated into the formulation to provide improved transport, delivery, tolerability, etc. Many suitable formulations are described in pharmacopoeias known to medicinal chemists, such as Remington's Pharmaceutical Sciences (15th ed., Mack Publishing Company, Easton, Pa. (1975)), particularly Chapter 87 of Blaug and Seymour. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid (cationic or anionic)-containing carriers (e.g., Lipofectin TM), DNA conjugates, anhydrous slurries, oil-in-water and water-in-oil emulsions, emulsion polyethylene glycol (polyethylene glycol of various molecular weights), semi-solid gels, and polyethylene glycol-containing semi-solid mixtures. Any of the above mixtures may be used in the treatment or method of treatment of the present invention, provided that the active ingredient in the formulation is not inactivated by the formulation, the formulation is physiologically compatible, and the route of administration is acceptable.

[0107] In one embodiment, the antibodies can be used as therapeutic agents. Such agents are typically used to treat, alleviate, and / or prevent diseases or conditions associated with aberrant TSLP expression, activity, and / or signaling in a subject. Treatment regimens can be implemented by identifying a subject, e.g., a human patient, having (or at risk for, or experiencing) a disease or disorder associated with aberrant TSLP expression, activity, and / or signaling, e.g., a TSLP-associated disorder, using standard methods. An antibody preparation, preferably one with high specificity and high affinity for a target antigen, is administered to the subject and typically exerts its effect by binding to the target. The administered antibody may eliminate, inhibit, or disrupt the expression, activity, and / or signaling function of the target (e.g., TSLP). The administered antibody may eliminate, inhibit, or disrupt the binding of the target (e.g., TSLP) to its natural endogenous ligand. For example, the antibody binds to the target and modulates, blocks, inhibits, reduces, antagonizes, neutralizes, and / or otherwise disrupts TSLP expression, activity, and / or signaling. In some embodiments, an antibody having heavy and light chain CDRs can be administered to a subject to treat a disease or disorder associated with aberrant TSLP expression.

[0108] Non-limiting examples of TSLP-associated diseases associated with abnormal TSLP expression, activity, and / or signaling include TSLP-associated inflammatory diseases and autoimmune diseases. TSLP-associated inflammatory diseases include allergic inflammation, asthma, chronic obstructive pulmonary disease, atopic dermatitis, and eosinophilic esophagitis. Allergic inflammation includes allergic rhinitis, allergic sinusitis, and allergic conjunctivitis. Autoimmune diseases include rheumatoid arthritis and multiple sclerosis.

[0109] In another embodiment, antibodies to TSLP are used in methods known in the art related to the localization and / or quantification of TSLP (e.g., measuring TSLP and / or levels of TSLP in appropriate physiological samples, diagnostic methods, protein imaging). In certain embodiments, antibodies containing an antigen-binding domain derived from an antibody specific for TSLP or a derivative, fragment, analog, or homolog thereof are used as pharmaceutically active compounds (hereinafter referred to as "therapeutic agents").

[0110] In another embodiment, antibodies specific for TSLP can be used to isolate TSLP polypeptides by standard techniques, e.g., immunoaffinity, chromatography, or immunoprecipitation. Antibodies (or fragments thereof) against the TSLP protein are used to detect the protein in biological samples. In some embodiments, TSLP is detected in biological samples as part of a clinical trial procedure, e.g., to determine the effectiveness of a particular therapeutic regimen. The antibody is coupled to a detectable substance (i.e., Physically binding (i.e., physically binding) is advantageous for detection. Detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazine aminofluorescein, dansylamide, or phycoerythrin. An example of a luminescent material includes luminol. Examples of bioluminescent materials include luciferase, luciferin, and aequorin. Examples of suitable radioactive materials include 125 I, 131 I, 35 S, or 3 Contains H.

[0111] In another embodiment, antibodies of the present disclosure can be used as reagents for detecting the presence of TSLP or its protein fragments in a sample. In some embodiments, the antibody comprises a detectable label. The antibody is a polyclonal antibody, or more preferably, a monoclonal antibody. Full-length antibodies or fragments thereof (e.g., Fab, scFv, or F(ab')2) can be used. The term "labeling" with respect to antibodies includes direct labeling of antibodies by coupling (i.e., physically linking) a detectable substance to the antibody, and indirect labeling of antibodies by reaction with another directly labeled reagent. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody, such as one that allows detection with fluorescently labeled streptavidin, and antibodies end-labeled with biotin. The term "biological sample" is intended to include tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and fluids present within a subject. Accordingly, the term "biological sample" includes blood and blood fractions or components, including serum, plasma, or lymph. In other words, the detection methods of the embodiments are used for analyte mRNA, protein, or genomic DNA in biological samples in vitro and in vivo. For example, in vitro detection techniques for analyte mRNA include Northern hybridization and in situ hybridization. In vitro detection techniques for analyte protein include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, and immunofluorescence. In vitro detection techniques for analyte genomic DNA include Southern hybridization.Procedures for performing immunoassays are described, for example, in "ELISA: Theory and Practice: Methods in Molecular Biology," Vol. 42, J.R.Crowther (ed.), Human Press, Totowa, NJ, 1995; "Immunoassay," E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, Calif., 1996; and "Practice and Theory of Enzyme Immunoassays," P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. In vivo detection techniques for analyte proteins also include introducing a labeled anti-analyte protein antibody into a subject. For example, the antibody can be labeled with a radioactive label, and the presence and location of the radioactive label in the subject can then be detected by standard imaging techniques.

[0112] The antibodies and derivatives, fragments, analogs, and homologs thereof described herein can be incorporated into pharmaceutical compositions suitable for administration. The principles, precautions, and guidelines for the preparation of such compositions, as well as the selection of their components, are well known in the art and can be found, for example, in Remington's Pharmaceutical Sciences: Science and Practice of Pharmacy, 19th Edition (Alfonso R. Gennaro et al., eds.), Mack Pub. Co., Easton Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, and Tr See Ends, Harwood Academic Publishers, Langhorne, Pa., 1994, and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

[0113] Such compositions typically comprise an antibody and a pharmaceutically acceptable carrier. When an antibody fragment is used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, peptide molecules that retain the ability to bind to the target protein sequence can be designed based on the variable region sequence of the antibody. Such peptides can be produced by chemical synthesis and / or recombinant DNA technology (see, for example, Marasco et al., Proc. Natl. Acad. Sci. USA, 90:7889-7893 (1993)).

[0114] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are acceptable for pharmaceutical administration. Suitable pharmaceutically acceptable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous carriers (e.g., fixed oils) can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Other than conventional media or agents that are not acceptable for antibodies, their use in the compositions is contemplated.

[0115] Pharmaceutical compositions according to the embodiments are formulated to suit their intended route of administration. Exemplary routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous administration may contain an injectable sterile diluent such as water, saline, fixed oils, polyethylene glycols, glycerol, propylene glycol, or other synthetic solvents; an antibacterial agent such as benzyl alcohol or methyl 4-hydroxybenzoate; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid (EDTA); a buffer such as acetate, citrate, or phosphate; and an agent for adjusting osmolality such as sodium chloride or dextrose. pH can be adjusted with acids or bases, e.g., hydrochloric acid or sodium hydroxide. Parenteral formulations may be packaged in ampoules, disposable syringes, or glass or plastic multiple-dose vials.

[0116] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water-soluble solutions) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Pharmaceutically acceptable carriers suitable for parenteral administration include physiological saline, antibacterial water, Cremophor EL TM(BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and fluid for easy syringability. It must be stable under the conditions of manufacture and storage and must be capable of preventing the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. A coating, for example, lecithin, can be used to maintain a desired particle size in the case of dispersion, and surfactants can be used to maintain the proper fluidity. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parahydroxybenzoates, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. Isotonic agents, such as sugars, polyols (for example, mannitol, sorbitol), sodium chloride, and the like, are often used. Prolonged absorption of the injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.

[0117] Sterile injectable solutions can be prepared by incorporating the antibody, if necessary, in the required amount in an appropriate solvent with one or a combination (if necessary) of the ingredients listed above, followed by sterilization by filtration. Typically, dispersions are prepared by incorporating the antibody in a sterile carrier containing a basic dispersion medium and the required other ingredients listed above. Regarding sterile powders for preparing sterile injectable solutions, methods for preparing them include vacuum drying and freeze-drying, which yield powders containing the active ingredient and any additional desired ingredients, and the active ingredient and any additional desired ingredients are derived from these sterile-filtered solutions.

[0118] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container, dispenser, or nebulizer which contains a suitable propellant, eg, a gas such as carbon dioxide.

[0119] Systemic administration may also be achieved by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant appropriate to the barrier to be permeated is used in the formulation. Such penetrants are well known in the art and include, for example, detergents for transmucosal administration, cholate salts, and fusidic acid derivatives. Transmucosal administration can be achieved using nasal sprays or suppositories. For transdermal administration, one or more antibodies can be formulated into pastes, ointments, gels, or creams, as are well known in the art.

[0120] The compounds can also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter or other glycerides) or retention enemas for rectal delivery.

[0121] In one embodiment, the antibody can be prepared with a carrier that will protect it from rapid excretion from the body, for example, a sustained / controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, for example, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparing such formulations will be apparent to those skilled in the art.

[0122] It is particularly advantageous to prepare parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to a suitable unit physically discretely provided as a unitary dosage for a subject to be treated, each unit containing a predetermined amount of one or more of the antibodies calculated to achieve the desired therapeutic effect in combination with the necessary pharmaceutical carrier. The specifications for the dosage unit form according to the embodiment are determined and directly depend on the inherent characteristics of the antibody and the particular therapeutic effect to be achieved, as well as the limitations inherent in the technology for preparing such antibodies for the treatment of an individual.

[0123] The pharmaceutical compositions may be included in a container, pack, or dispenser together with an administration protocol.

[0124] The formulations described herein preferably contain one or more of the above antibodies, depending on the specific therapeutic situation, and preferably contain antibodies with complementary activities that do not adversely affect each other. Additionally, the compositions may contain agents that enhance the function of the composition, such as cytotoxins, cytokines, chemotherapeutic agents, or growth inhibitors. Such molecules are appropriately combined in amounts effective for the intended purpose. For example, they can be combined in a kit or for use.

[0125] In one embodiment, one or more of the antibodies are used in combination therapy, i.e., with other agents, e.g., therapeutic agents (e.g., agents for treating a disease state or disorder, e.g., various forms of cancer, autoimmune disorders, and the like). The antibody of the present invention and another therapeutic agent (used in the treatment of inflammatory diseases) can be administered in combination with other therapeutic agents. As used herein, the term "combination" refers to the administration of agents substantially simultaneously, simultaneously, or sequentially. When administered sequentially, preferably, the first of the two compounds can still be detected at effective concentrations at the treatment site when administration of the second compound begins. In one embodiment, a "combination" may also refer to the simultaneous inclusion of an antibody of the present invention and another therapeutic agent in a kit.

[0126] For example, a combination therapy can be used in which one or more antibodies described herein are co-formulated and / or co-administered with one or more additional therapeutic agents (e.g., one or more cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors, and / or cytotoxins or cell proliferation inhibitors, as described in more detail below). Such combination therapies can be preferred because they may utilize lower dosages of the administered therapeutic agents, thereby avoiding possible toxicities or complications associated with various monotherapies. For clarity and conciseness of description, technical features are described herein as part of the same or separate embodiments, but it is understood that the scope of the present invention includes several embodiments having all the described features or combining several features. [Example]

[0127] Example 1. Preparation of TSLP and TSLPR Recombinant Proteins cDNAs for human TSLP (Uniprot: Q969D9, SEQ ID NO: 1) and cynomolgus monkey TSLP (Uniprot: A0A2K5TXV0, SEQ ID NO: 2) were synthesized and cloned into the eukaryotic expression vector pCMV3 (purchased from Beijing Sino Biological Inc., catalog number CG90911-UT). The signal peptide MDMRVPAQLLGLLLLWLRGARS was inserted at the N-terminus, and a six-histidine tag was fused to the carboxyl terminus of TSLP to obtain the pSect-hTSLP-cHis and pSect-cyTSLP-cHis plasmids. HEK293.6E cells (ATCC CRL-1573) were transfected with these plasmids, and the cell supernatants were collected. Recombinant proteins of the corresponding lengths were isolated by nickel column affinity chromatography. The full-length fragment of TSLP was then cloned into the prokaryotic expression plasmid pET-30a. BL21 E. coli was transformed with the plasmid, and after induction of expression, the recombinant protein was found to be expressed in inclusion bodies. The inclusion body protein was refolded and purified to obtain recombinant human TSLP and cynomolgus monkey TSLP proteins. As a result, recombinant human TSLP and cynomolgus monkey TSLP proteins were obtained by purification, as shown in Figure 1.

[0128] Human TSLPR cDNA (Uniprot: Q9HC73, SEQ ID NO: 3, purchased from Beijing Sino Biological Inc., catalog number HG18720-UT) was amplified by PCR to obtain a gene fragment encoding the extracellular domain (Gln23-Lys231) of human TSLPR. This gene fragment was cloned downstream of the promoter of the eukaryotic expression vector pCMV3, and the human IgG1 Fc (knob) fragment was fused to the C-terminus to produce the pHC1-TSLPR-hFc-knob plasmid. A plasmid containing only the human IgG1 Fc (hole) fragment, pHC1-hFc-hole, was then constructed. This eukaryotic expression plasmid was then mixed with the pHC1-TSLPR-hFc-knob plasmid and co-transfected into HEK293.6E eukaryotic cells to produce the human TSLPR-Fc fusion protein. Similarly, the synthesized cDNA of cynomolgus monkey TSLPR (Uniprot: G8F663, SEQ ID NO: 4) was amplified by PCR to obtain a gene fragment encoding the cynomolgus monkey TSLPR extracellular domain (Gln23-Lys231). The gene fragment was then cloned into a eukaryotic expression plasmid containing a human IgG1 Fc (knob) fragment, and the cynomolgus monkey TSLPR extracellular domain was expressed as a human IgG1 Fc (knob) fragment. The human TSLPR (SEQ ID NO: 5) and cynomolgus TSLPR (SEQ ID NO: 6) extracellular domain recombinant proteins were obtained by fusion to the N-terminus of IgG1 Fc (knob). The eukaryotic expression plasmid was then mixed with a plasmid containing only the human IgG1 Fc (hole) fragment and co-transfected into eukaryotic HEK293.6E cells. After transfection of the plasmid into HEK293.6E cells and culturing for 5-7 days, the cell supernatant was collected and purified by Protein A affinity chromatography to obtain human TSLPR (SEQ ID NO: 5) and cynomolgus TSLPR (SEQ ID NO: 6) extracellular domain recombinant proteins. The results are shown in Figure 2.

[0129] The binding of recombinant human TSLP protein and recombinant human TSLPR extracellular domain protein was detected by ELISA, and the EC50 = 0.11 nM, indicating that recombinant human TSLP binds to human TSLPR with high affinity (Fig. 3).

[0130] Example 2: Construction of TSLPR-constantly expressing cells 1) Construction of a cell line stably expressing Ba / F3-hTSLPR Ba / F3 cells were maintained in RPMI-1640 medium containing 10% fetal bovine serum, 50 μM 2-mercaptoethanol, 2 mM L-glutamine, 50 μg / mL penicillin-streptomycin, and 10 ng / mL mouse IL-3. A lentiviral expression vector expressing human TSLPR (Guangzhou Funeng Gene Co., Ltd., EX-W0156-Lv105-B) was transfected into the cells using a Lenti-Pac HIV lentiviral packaging kit (Guangzhou Funeng Gene Co., Ltd.). The virus was packaged using a ELISA kit (Promega Inc., 2000). The virus was transfected into Ba / F3 cells, and the cells were screened for resistance to puromycin to obtain the cell line Ba / F3-hTSLPR, which stably expresses human TSLPR. As shown in Figure 4, the binding of biotinylated human TSLP protein to cell surface TSLPR was detected, confirming that the constructed cells express human TSLPR, with an EC50 of 0.22 nM.

[0131] 2) Construction of a cell line stably expressing Ba / F3-hTSLPR-IL7Rα IL-7Rα is involved in signal transduction via the TSLP receptor complex. Upon binding to TSLPR, TSLP forms a heterodimeric receptor complex with IL-7Rα, phosphorylating JAK1 in the IL-7Rα intracellular segment and JAK2 in the TSLPR intracellular segment, activating downstream signaling molecules and transmitting an activation signal inside the cell. An IRES (GenBank KM077140.1, 99-745) and the human IL-7Rα gene were cloned into a lentiviral expression vector expressing human TSLPR, resulting in the pLenti-CMV-TSLPR-IRES-IL7Rα-Puro dual gene co-expression plasmid. This vector was then transfected into Ba / F3 cells to construct a stable cell line expressing the intact human TSLP signaling pathway.

[0132] Ba / F3 cells were maintained in RPMI-1640 medium containing 10% fetal bovine serum, 2 mM L-glutamine, 50 μg / mL penicillin-streptomycin, and 2 ng / mL mouse IL-3. A human TSLPR and IL-7Rα dual gene co-expression plasmid was packaged into lentivirus using the Lenti-Pac HIV Lentivirus Packaging Kit. Ba / F3 cells were transduced with the culture supernatant containing the recombinant virus and screened for resistance by adding puromycin to obtain the cell line Ba / F3-hTSLPR-IL7Rα, which stably expresses human TSLPR and IL-7Rα. Cell proliferation assays showed that the constructed Ba / F3-hTSLPR-IL7Rα cell line exhibited a dose-dependent growth response to human TSLP, with an EC50 of 0.22 nM (Figure 5).

[0133] 3) Ba / F3-hTSLPR-IL7Rα-STAT5 luc reporter gene cells Strain construction The STAT5-luc reporter gene was introduced into the Ba / F3-hTSLPR-IL7Rα constant-expressing cell line to screen for STAT5 luciferase reporter gene cell lines. First, a nucleotide sequence containing five STAT5 response elements (AGTTCTGAGAAAAGT) was synthesized and cloned into pGL4.22-luc2P (Promega, E6761) using restriction endonucleases KpnI and HindIII. At the same time, the hygro resistance gene was cloned into the vector, replacing the Puro resistance gene to obtain pGL4.22-luc2p STAT5 RE-hygro. Next, 10 μg of pGL4.22-luc2p STAT5 RE-hygro plasmid and 5 × 10 6 The cells were electrotransfected with Ba / F3-hTSLPR-IL7Rα stably expressing cells. The voltage was set at 300 V and the capacitance at 950 μF. After 48–72 hours of transfection, 200 μg / mL hygromycin B solution was added, and the screening medium was changed every 3–5 days. After a period of pressure screening, the stably expressing cells formed clones. Monoclonal lines obtained by limiting dilution were stimulated with different concentrations of TSLP for 6 hours, and then fluorescent signals were detected. As shown in Figure 6, the fluorescent signal gradually increased with increasing TSLP concentration. This indicates that TSLP can activate the STAT5 signaling pathway in the stably expressing cells Ba / F3-hTSLPR-IL7Rα-STAT5 luc, with an EC50 of 0.22 nM for the stably expressing cells Ba / F3-hTSLPR-IL7Rα-STAT5 luc.

[0134] Example 3, Animal Immunization Six-week-old female Balb / C mice were selected and immunized with 50 μg of antigen per immunization. Human TSLP and cynomolgus TSLP were used as antigens for exchange immunization. Equal volumes of the antigen and Freund's adjuvant were mixed and immunized subcutaneously every two weeks. After four immunizations, tail blood was collected and ELISA was used to detect the titer of the mouse serum and the inhibitory effect of the serum on the binding of TSLP to TSLPR. Alternatively, booster immunizations were performed weekly after the first immunization using a gene gun. A human TSLP expression plasmid was mixed with gold powder and impacted on the bare skin of the mouse's abdomen at 400 psi, for a total of nine immunizations.

[0135] Example 4: Antibody screening 1) Fab phage library The immunized animals in Example 3 were harvested, and three weeks after the final immunization, rush immunotherapy was performed by injecting 10 μg / 100 μL / animal of human TSLP recombinant protein into the tail vein. Four days later, lymph node and spleen cells were harvested from the mice, and cellular RNA was extracted using the TRNzol lysis method. It was then reverse-transcribed to synthesize single-stranded cDNA, which was then used as a template to amplify the antibody variable region sequences. A library of 5×10 Fab antibody fragments was displayed on the phage display platform. 9We constructed a TSLP immune library of over 1000, and randomly selected 96 monoclonal clones for induction and expression. Western blot analysis revealed that both the light and heavy chains exhibited expression rates exceeding 95%, and sequencing demonstrated good sequence diversity. The TSLP antibody library was screened using immunotubes (Maxisorp immunotubes) coated with human TSLP recombinant protein or cynomolgus TSLP recombinant protein. After two rounds of screening, we obtained several positive clones that specifically recognized TSLP and efficiently blocked TSLP-TSLPR binding. We then expressed monoclonal antibody Fabs in TG1 competent cells and confirmed their binding to human and cynomolgus TSLP and their blocking of TSLP / TSLPR. We obtained six high-affinity antibodies that simultaneously bound to human and cynomolgus TSLP and efficiently blocked the binding of TSLP to the TSLPR clones (results are shown in Table 1).

[0136] [Table 1]

[0137] 2) Mouse hybridoma Human TSLP recombinant protein (10 μg / 100 μL / mouse) was injected into the tail vein via the Rush technique. After 4 days, lymph node and spleen cells were harvested from the mice and ground into DMEM to obtain a B cell suspension. An appropriate amount of the B cell suspension was mixed with SP2 / 0 and then fused using an electrofusion device. The fused cells were cultured in complete DMEM medium containing HAT at 37°C with 5% CO2.

[0138] 3) Hybridoma screening Screening for binding to human TSLP by enzyme-linked immunosorbent assay (ELISA) 96-well plates were coated overnight at 4°C with 1 μg / mL human TSLP recombinant protein using carbonate buffer. After washing three times with PBS, 200 μL of PBS containing 2% nonfat dry milk was added to each well and blocked for 1 hour. After washing once with PBS, 100 μL of hybridoma or phage supernatant was added to each well and incubated for 60 minutes at room temperature. After washing three times with PBST (PBS + 0.05% Tween-20) and PBS, 100 μL of HRP-conjugated anti-human IgG Fc secondary antibody was added to each well and incubated for 60 minutes at room temperature. After washing three times with PBST and PBS, 100 μL of TMB substrate was added to each well and allowed to develop for 10 minutes at 37°C. The reaction was stopped with 50 μL / well of 2M sulfuric acid solution, and the absorbance was read at 450 nm.

[0139] Screening of hybridoma cells for binding to cynomolgus monkey TSLP by ELISA 96-well plates were coated overnight at 4°C with 1 μg / mL recombinant cynomolgus monkey TSLP protein using carbonate buffer. After washing three times with PBS, 200 μL of PBS containing 2% nonfat dry milk was added to each well and blocked for 1 hour. After washing once with PBS, 100 μL of hybridoma or phage supernatant was added to each well and incubated for 60 minutes at room temperature. After washing three times with PBST and PBS, 100 μL of HRP-conjugated anti-human IgG Fc secondary antibody was added to each well and incubated for 60 minutes at room temperature. After washing three times with PBST and PBS, 100 μL of TMB substrate was added to each well and allowed to develop for 10 minutes at 37°C. The reaction was stopped with 50 μL / well of 2M sulfuric acid solution, and the absorbance was read at 450 nm.

[0140] Screening for blockade of human TSLP and human TSLPR by ELISA Using carbonate buffer as the coating solution, 1 μg / mL of human TSLPR recombinant protein was coated onto a 96-well plate overnight at 4°C. After washing three times with PBS, 200 μL of PBS containing 2% nonfat dry milk was added to each well and blocked for 1 hour. In the blocked U-shaped 96-well plate, hybridoma supernatant or phage supernatant was incubated with 60 ng / mL of biotinylated human TSLP protein at room temperature for 30 minutes. After washing the 96-well plate three times with PBS, 100 μL of the incubation mixture was added to each well and incubated at room temperature. After washing three times with PBST and PBS, 100 μL of streptavidin-conjugated horseradish peroxidase (SA-HRP) was added to each well and incubated at room temperature for 30 minutes in the dark. After washing three times with PBST and PBS, 100 μL of TMB substrate was added to each well and allowed to develop at 37°C for 10 minutes. The reaction was stopped with 50 μL / well of 2M sulfuric acid solution, and the absorbance was read at 450 nm.

[0141] Screening for blockade of cynomolgus TSLP and cynomolgus TSLPR by ELISA Using carbonate buffer as the coating solution, 1 μg / mL of cynomolgus monkey TSLPR recombinant protein was coated onto a 96-well plate overnight at 4°C. After washing three times with PBS, 200 μL of PBS containing 2% nonfat dry milk was added to each well and blocked for 1 hour. In the blocked U-shaped 96-well plate, hybridoma supernatant or phage supernatant was incubated with 60 ng / mL of biotinylated cynomolgus monkey TSLP protein for 30 minutes at room temperature. After washing the 96-well plate three times with PBS, 100 μL of the incubation mixture was added to each well and incubated for 1 hour at room temperature. After washing three times with PBST and PBS, 100 μL of SA-HRP was added to each well and incubated for 30 minutes at room temperature in the dark. After washing three times with PBST and PBS, 100 μL of TMB substrate is added to each well, and the color is developed at 37° C. for 10 minutes. The reaction is stopped with 50 μL / well of 2 M sulfuric acid solution, and the absorbance is read at a wavelength of 450 nm.

[0142] Blockade at the cellular level 5×10 4 Ba / F3-hTSLPR cells were added to a U-shaped 96-well plate at 1 / well. The antibody and biotinylated human TSLP protein were incubated at 4°C for 30 minutes, then added to the cells and incubated at 4°C for 1 hour. Streptavidin-conjugated phycoerythrin-fluorescein (SA-PE) was added and incubated at 4°C for 45 minutes. The blocking effect of the antibody at the cellular level was detected by flow cytometry.

[0143] ELISA screening yielded a total of 712 hybridoma clones that bound to human TSLP, of which 52 hybridoma clones were able to block the binding of human TSLP to human TSLPR in ELISA and cell-level assays, and 6 positive clones were able to bind to cynomolgus TSLP and block the binding of cynomolgus TSLP to cynomolgus TSLPR (results are shown in Table 2).

[0144] [Table 2]

[0145] Example 5. Obtaining antibody sequences According to the results of hybridoma screening, positive monoclonal cells were centrifuged at 1000 rpm to collect the cells, and total RNA was extracted with Trizol. After synthesizing the first-strand cDNA, the variable region DNA sequence corresponding to the hybridoma cells was amplified using the first-strand cDNA as a template. To a 50μL reaction system, add 1μL of cDNA, 5μL of 10x PCR buffer, 1μL each of forward and reverse primers, 1μL of dNTPs, 1μL of 25mmol MgCl2, 39μL of H2O, and 1μL of Taq enzyme. Pre-denaturation was performed at 95°C for 10 minutes, followed by temperature cycling and PCR amplification. The reaction conditions were denaturation at 94°C for 1 minute, annealing at 58°C for 1 minute, and extension at 72°C for 15 seconds, repeated 30 times, followed by incubation at 72°C for 10 minutes. Based on the hybridoma screening results, the variable region sequences of positive clones were amplified. After sequencing, the heavy and light chain variable region sequences of the candidate positive clones obtained were as follows:

[0146] Clone: 71G4 SEQ ID Nos: 7-16 Heavy chain VH [ka] Nucleic acid sequence [ka] Light chain VK [ka] Nucleic acid sequence [ka]

[0147] Clone: 79D6 SEQ ID Nos: 17-26 Heavy chain VH [ka] Nucleic acid sequence [ka] Light chain VK [ka] Nucleic acid sequence [ka]

[0148] Clone: 76A8 SEQ ID Nos: 27-36 Heavy chain VH [ka] Nucleic acid sequence [ka] Light chain VK [ka] Nucleic acid sequence [ka]

[0149] Clone: 80D12 SEQ ID Nos: 37-46 Heavy chain VH [ka] Nucleic acid sequence [ka] Light chain VK [ka] Nucleic acid sequence [ka]

[0150] Clone: 80E11 SEQ ID Nos: 47-56 Heavy chain VH [ka] Nucleic acid sequence [ka] Light chain VK [ka] Nucleic acid sequence [ka]

[0151] Clone: 80B12 SEQ ID Nos: 57-66 Heavy chain VH [ka] Nucleic acid sequence [ka] Light chain VK [ka] Nucleic acid sequence [ka]

[0152] Clone: 39G5-H8 SEQ ID Nos:67-76 Heavy chain VH [ka] Nucleic acid sequence [ka] Light chain VK [ka] Nucleic acid sequence [ka]

[0153] Example 6. Expression of anti-TSLP chimeric antibodies Fragments of the heavy and light chain variable region sequences obtained in Example 5 were PCR amplified, and the heavy chain variable region was cloned into a vector containing a human heavy chain constant region to express a complete IgG1 heavy chain in mammalian cells. Similarly, the light chain variable region was cloned into a vector containing a human light chain constant region to express a complete kappa light chain in mammalian cells. After sequencing, the fragments were transfected into HEK293.6E mammalian cells, where IgG1 was expressed and secreted into the culture medium. The supernatant was collected, filtered, and purified. The IgG was purified by Protein A chromatography, and the eluted protein was concentrated by ultrafiltration. The IgG concentration was measured spectrophotometrically, and the IgG purity was analyzed by SDS-PAGE. 39G5-H8, 71G4, 79D6, 80B12, 80D12, and 80E11 chimeric antibodies were obtained.

[0154] Example 7: Measurement of the affinity of chimeric antibodies 1) Binding of antibody to human TSLP 96-well plates were coated overnight at 4°C with 1 μg / mL of human TSLP recombinant protein using carbonate buffer. After washing three times with PBS, 200 μL of 2% nonfat dry milk in PBS was added to each well and blocked for 1 hour. After washing once with PBS, 100 μL of anti-TSLP antibody was added to each well and incubated at room temperature for 60 minutes. After washing three times with PBST and PBS, 100 μL of HRP-conjugated anti-human IgG Fc secondary antibody was added to each well and incubated at room temperature for 60 minutes. After washing three times with PBST and PBS, 100 μL of TMB substrate was added to each well and allowed to develop at 37°C for 10 minutes. The reaction was stopped with 50 μL / well of 2M sulfuric acid solution, and the absorbance was measured at 450 nm. As shown in Figure 7-a and Table 3, all of the chimeric antibodies obtained in Example 6 bind to human TSLP.

[0155] 2) Binding of antibodies to cynomolgus monkey TSLP Using carbonate buffer as the coating solution, 1 μg / mL of cynomolgus monkey TSLP recombinant protein was coated onto a 96-well plate overnight at 4°C. After washing three times with PBS, 200 μL of PBS containing 2% nonfat dry milk was added to each well and blocked for 1 hour. After washing once with PBS, 100 μL of anti-TSLP antibody was added to each well and incubated at room temperature for 60 minutes. After washing three times with PBST and PBS, each well was then treated with HRP-conjugated anti-human IgG. 100 μL of Fc secondary antibody was added and incubated at room temperature for 60 minutes. After washing three times with PBST and three times with PBS, 100 μL of TMB substrate was added to each well and allowed to develop at 37°C for 10 minutes. The reaction was stopped with 50 μL / well of 2M sulfuric acid solution, and the absorbance was measured at 450 nm. As shown in Figure 7-b and Table 3, all of the chimeric antibodies obtained in Example 6 bind to cynomolgus monkey TSLP.

[0156] [Table 3]

[0157] 3) Affinity determination by BIAcore The dynamic binding activity of anti-TSLP antibodies to human TSLP was measured using the BIAcore T200 system. 2 μg / ml of anti-TSLP antibody was applied to the sensor chip Protein Protein A was immobilized on the chip, and the chip was run for 40 seconds at a flow rate of 10 μL / min using 1x HBS-EP buffer as the running buffer. Human TSLP at different concentrations (0-21.7 nM) was injected onto the immobilized anti-TSLP antibody surface at a flow rate of 30 μL / min. After each injection cycle, the Protein A chip surface was regenerated using 10 mM glycine (pH 2.0) as the regeneration buffer at a flow rate of 30 μL / min.

[0158] The obtained data was fitted to a Langmuir 1:1 kinetic model to analyze the association rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (KD) (BIAevaluation software). As shown in Table 4, each chimeric antibody binds to human TSLP with high affinity.

[0159] [Table 4]

[0160] Example 8, ELISA-level blocking 1) Blockade of the binding between human TSLP and human TSLPR by antibodies 96-well plates were coated overnight at 4°C with 1 μg / mL of human TSLPR recombinant protein using carbonate buffer. After washing three times with PBS, 200 μL of 2% nonfat dry milk in PBS was added to each well and blocked for 1 hour. Anti-TSLP antibody and 60 ng / mL biotinylated human TSLP protein were incubated in the blocked U-shaped 96-well plate for 30 minutes at room temperature. After washing the 96-well plate three times with PBS, 100 μL of the incubation mixture was added to each well and incubated for 1 hour at room temperature. After washing three times with PBST and PBS, 100 μL of SA-HRP was added to each well and incubated for 30 minutes at room temperature in the dark. After washing three times with PBST and PBS, 100 μL of TMB substrate was added to each well and incubated for 10 minutes at 37°C. The reaction was stopped with 50 μL / well of 2M sulfuric acid solution, and the absorbance was read at 450 nm. As shown in Figure 8-a and Table 3, each of the chimeric antibodies can block the binding of human TSLP to human TSLPR.

[0161] 2) Blockade of binding between cynomolgus monkey TSLP and cynomolgus monkey TSLPR by antibodies Using carbonate buffer as the coating solution, 1 μg / mL of cynomolgus monkey TSLPR recombinant protein was coated onto a 96-well plate overnight at 4°C. After washing three times with PBS, 200 μL of PBS containing 2% nonfat dry milk was added to each well and blocked for 1 hour. In the blocked U-shaped 96-well plate, anti-TSLP antibody and 60 ng / mL biotinylated cynomolgus monkey TSLP protein were incubated at room temperature for 30 minutes. After washing the 96-well plate three times with PBS, 100 μL of the incubation mixture was added to each well and incubated for 1 hour at room temperature. After washing three times with PBST and PBS, 100 μL of AA-HRP was added to each well and incubated for 30 minutes at room temperature in the dark. After washing three times with PBST and three times with PBS, 100 μL of TMB substrate was added to each well and allowed to develop for 10 minutes at 37°C. The reaction was stopped with 50 μL / well of 2 M sulfuric acid solution, and the absorbance was measured at 450 nm. As shown in Figure 8-b and Table 3, each chimeric antibody was able to block the binding of cynomolgus monkey TSLP and cynomolgus monkey TSLPR.

[0162] Example 9: Cellular-level blocking Harvest Ba / F3-hTSLPR-IL7Rα cells, wash them three times with PBS, centrifuge them at 300g for 3 minutes, add 3% BSA / PBS, and block for 30 minutes. After resuspending, 5 x 10 cells were placed in a U-shaped 96-well plate. 4 The antibody was incubated with 60 ng / mL biotinylated human TSLP protein at room temperature for 10 minutes, then added to the cells. The cells were incubated at 4°C for 45 minutes and washed twice with 0.5% BSA / PBS. SA-PE was added, followed by incubation at 4°C for 45 minutes and washing twice with 0.5% BSA / PBS. PI dye was added, incubated at 4°C for 10 minutes, and washed twice with 0.5% BSA / PBS. The cells were resuspended in PBS, and the blocking effect of the antibodies on the cellular level was detected by flow cytometry. As shown in Figure 9, each chimeric antibody was able to block the binding of TSLP to the cell surface TSLPR.

[0163] Example 10. Inhibition of TSLP-stimulated Ba / F3-hTSLPR-IL7Rα cell proliferation by chimeric antibodies Inhibition tests of TSLP antibodies on TSLP-induced Ba / F3-hTSLPR-IL7Rα cell proliferation will be performed in RPMI-1640, 10% fetal bovine serum.

[0164] One day before, 2 × 10 Ba / F3-hTSLPR-IL7Rα cells were cultured in advance. 4 The diluted anti-TSLP antibody and 60 μL of 5 ng / mL human TSLP were added to a 96-well plate at a concentration of 100 μL / well. Sixty μL of diluted anti-TSLP antibody and 60 μL of 5 ng / mL human TSLP were incubated at room temperature for 15 minutes. The pre-incubated antibody-cytokine mixture was added to the cell culture plate at 100 μL / well. The 96-well plate was incubated in a 5% CO2, 37°C incubator for 72 hours. After the incubation period, CellTiter-Glo was added to each well and incubated for 10 minutes. Luminescence readings were taken from the black 96-well plate. As shown in Figure 10 and Table 5, all antibodies were able to inhibit cell proliferation, with 71G4 showing a significant inhibitory effect.

[0165] [Table 5]

[0166] Example 11: Inhibition of TSLP-activated STAT5 signaling by chimeric antibodies Ba / F3-hTSLPR-IL7Rα-STAT5luc cells were starved overnight and the next day, 5 × 10 4 Human TSLP was diluted before inoculating 50 μL / well into a 96-well plate and incubating at 37°C for 30 minutes. 50 μL of the ligand / antibody mixture was pipetted into the cell plate, mixed, and incubated at 37°C for 6 hours. 100 μL of fluorescent detection reagent was added, and luminescence readings from each well were read using a microplate reader. As shown in Figure 11 and Table 6, each antibody was able to inhibit reporter gene expression, but the inhibitory effect of 71G4 was the most pronounced.

[0167] [Table 6]

[0168] Example 12. Humanization of antibodies The TSLP antibodies 39G5, 71G4, 76A8, 79D6, 80B12, 80D12, and 80E11 are derived from the same or similar mouse germline genes, and the mouse antibodies 39G5 and 71G4 with the strongest neutralizing activity were selected and their variable region sequences were humanized.

[0169] First, the mouse antibody 39G5 sequence was compared with the human antibody germline sequence. The human germline light chain genes VK_1_39 and IGKJ1*01 and the human germline heavy chain genes VH-1-69 and IGHJ4*01, which have high homology, identical key amino acid sequences in the antibody structural core (upper hydrophobic core), and are frequently expressed in humans, were selected for mouse antibody CDR grafting. The mouse anti-71G4 light and heavy chains share the same origin as 39G5, with only one or two amino acids in CDR3 being of the same type. Therefore, the CDR1 and CDR2 of the 71G4 heavy chain were replaced with the humanized 39G5 heavy chain, respectively. To improve affinity and molecular surface charge distribution, the 71G4 humanized light chain or 39G5 humanized light chain was paired with the 39G5 humanized heavy chain and expressed, optimizing the region surrounding the core binding site. Subsequently, computer homology modeling was performed to simultaneously analyze the CDR regions and their surrounding framework amino acid sequences, molecular surface charge, and hydrophobic region distribution to confirm the generation of different heavy and light chain derivatives. Twenty-seven humanized variants of the TSLP antibody, Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab20, Ab21, Ab22, Ab23, Ab24, Ab25, Ab26, and Ab27, were obtained. The heavy and light chains of the 27 humanized variants are listed in Table 7.

[0170] [Table 7]

[0171] The complete sequences for the light and heavy chain derivatives were synthesized and then cloned into vectors containing the antibody kappa chain constant region Ckappa or the human IgG1 constant regions CH1-CH3. After pairing the light and heavy chain derivative plasmids, they were transfected into HEK293.6E cells and expressed for 5-6 days. The supernatant was collected and purified using a Protein A column.

[0172] The humanized antibody heavy / light chain variable region sequences are as follows:

[0173] h39G5VH v1:SEQ ID Nos:77-81 [ka] Nucleic acid sequence [ka]

[0174] h39G5VH v2:SEQ ID Nos: 82-86 [ka] Nucleic acid sequence [ka]

[0175] h39G5VH v3:SEQ ID Nos: 87-91 [ka] Nucleic acid sequence [ka]

[0176] h39G5VH v4:SEQ ID Nos: 92-96

change

change

[0177] h39G5VH v5:SEQ ID Nos: 97-101

change

change

[0178] h39G5VH v6:SEQ ID Nos: 102-106

change

change

[0179] h39G5VH v7:SEQ ID Nos: 107-111

change

change

[0180] h39G5VH v8:SEQ ID Nos: 112-116 [ka] Nucleic acid sequence [ka]

[0181] h39G5VH v9:SEQ ID Nos: 117-121 [ka] Nucleic acid sequence [ka]

[0182] h39G5VH v10:SEQ ID Nos: 122-126 [ka] Nucleic acid sequence [ka]

[0183] h39G5VH v11:SEQ ID Nos: 127-131 [ka] Nucleic acid sequence [ka]

[0184] h39G5VH v12:SEQ ID Nos: 132-136 [ka] Nucleic acid sequence [ka]

[0185] h39G5VH v13:SEQ ID Nos: 137-141 [ka] Nucleic acid sequence [ka]

[0186] h39G5VH v14:SEQ ID Nos: 142-146 [ka] Nucleic acid sequence [ka]

[0187] h39G5VH v15:SEQ ID Nos: 147-151 [ka] Nucleic acid sequence [ka]

[0188] h71G4VH v1:SEQ ID Nos: 152-156 [ka] Nucleic acid arrangement

change

[0189] h71G4VH v2:SEQ ID Nos: 157-161

change

change

[0190] h71G4VH v3:SEQ ID Nos: 162-166

change

change

[0191] h39G5VK v1:SEQ ID Nos: 167-171

change

change

[0192] h71G4VK v1:SEQ ID Nos: 172-176

change

change

[0193] Example 13: Measurement of affinity of humanized antibodies 1) Binding of humanized antibodies to human TSLP 96-well plates were coated overnight at 4°C with 1 μg / mL human TSLP recombinant protein in carbonate buffer. After washing three times with PBS, 200 μL of 2% nonfat dry milk in PBS was added to each well and blocked for 1 hour. After washing once with PBS, 100 μL of anti-TSLP antibody was added to each well and incubated at room temperature for 60 minutes. After washing three times with PBST and PBS, 100 μL of HRP-conjugated anti-human IgG Fc secondary antibody was added to each well and incubated at room temperature for 60 minutes. After washing three times with PBST and PBS, 100 μL of TMB substrate was added to each well and developed at 37°C for 10 minutes. The reaction was stopped with 50 μL / well of 2M sulfuric acid solution, and the absorbance was measured at 450 nm. As shown in Figures 12a, c, and e and Table 8, all of the humanized antibodies obtained in Example 12 were able to bind to human TSLP, with Ab20 showing the strongest binding activity.

[0194] 2) Binding of humanized antibodies to cynomolgus monkey TSLP Using carbonate buffer as the coating solution, 1 μg / mL of cynomolgus monkey TSLP recombinant protein was coated onto a 96-well plate overnight at 4°C. After washing three times with PBS, 200 μL of PBS containing 2% nonfat dry milk was added to each well and blocked for 1 hour. After washing once with PBS, 100 μL of anti-TSLP antibody was added to each well and incubated at room temperature for 60 minutes. After washing three times each with PBST and PBS, 100 μL of TMB substrate was added to each well and color development was allowed to occur at 37°C for 10 minutes. The reaction was stopped with 50 μL / well of 2M sulfuric acid solution. The absorbance at a wavelength of 450 nm was measured. As shown in Figure 12-b, 12-d, and 12-f and Table 8, all antibodies except Ab14 and Ab15 bound to cynomolgus monkey TSLP, but Ab20 had the strongest binding effect.

[0195] [Table 8]

[0196] 3) Affinity measurement by Biacore The dynamic binding activity of anti-TSLP antibodies to human TSLP and cynomolgus TSLP was measured by surface plasmon resonance (SPR) using a BIAcore T200 system. Approximately 100 RU of anti-TSLP antibody was immobilized on a sensor chip Protein A, and the running buffer was 1x HBS-EP buffer at a flow rate of 10 μL / min. Human or cynomolgus TSLP at different concentrations (0-21.7 nM) was injected over the immobilized anti-TSLP antibody surface at a flow rate of 30 μL / min. After each injection cycle, the Protein A chip surface was regenerated using 10 mM glycine (pH 2.0) as the regeneration buffer at a flow rate of 30 μL / min. The obtained data were fitted to a Langmuir 1:1 kinetic model to analyze the association rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (KD) (BIAevaluation software).

[0197] As shown in Table 9, each antibody had strong affinity for both human and cynomolgus monkey TSLP, but antibodies Ab22, Ab23, Ab24, and Ab26 exhibited higher affinities, with binding affinities for human TSLP of 0.06 pM, 0.08 pM, 0.02 pM, and 2 pM, respectively, and for cynomolgus monkey TSLP of 0.04 pM, 0.007 pM, 0.18 pM, and 0.31 pM, respectively.

[0198] [Table 9]

[0199] Example 14, ELISA level blocking 1) Blocking the binding of human TSLP and human TSLPR by humanized antibodies 96-well plates were coated overnight at 4°C with 1 μg / mL of human TSLPR recombinant protein using carbonate buffer. After washing three times with PBS, 200 μL of PBS containing 2% nonfat dry milk was added to each well and blocked for 1 hour. Anti-TSLP antibody and 60 ng / mL biotinylated human TSLP protein were incubated in the blocked U-shaped 96-well plate for 30 minutes at room temperature. After washing the 96-well plate three times with PBS, 100 μL of the incubation mixture was added to each well and incubated for 1 hour at room temperature. After washing three times with PBST and PBS, 100 μL of SA-HRP was added to each well and incubated for 30 minutes at room temperature in the dark. After washing three times with PBST and PBS, 100 μL of TMB substrate was added to each well and incubated for 10 minutes at 37°C. The reaction was stopped with 50 μL / well of 2M sulfuric acid solution, and the absorbance was read at 450 nm. As shown in Figure 13-a, c, e and Table 10, humanized antibodies Ab1, Ab2, Ab3, Ab4, Ab5, Ab8, Ab10, Ab13, Ab19, Ab20, Ab21, Ab22, Ab23, Ab24, Ab25, and Ab26 can all block the binding of human TSLP to TSLPR.

[0200] 2) Using carbonate buffer as a blocking coating solution for the binding of the humanized antibody to cynomolgus monkey TSLP and cynomolgus monkey TSLPR, 1 μg / mL of cynomolgus monkey TSLPR recombinant protein was coated onto a 96-well plate overnight at 4°C. After washing three times with PBS, 200 μL of PBS containing 2% nonfat dry milk was added to each well and blocked for 1 hour. In the blocked U-shaped 96-well plate, anti-TSLP antibody and 60 ng / mL biotinylated cynomolgus monkey TSLP protein were incubated at room temperature for 30 minutes. After washing the 96-well plate three times with PBS, 100 μL of the incubation mixture was added to each well and incubated for 1 hour at room temperature. After washing three times with PBST and PBS, 100 μL of SA-HRP was added to each well and incubated in the dark at room temperature for 30 minutes. After washing three times with PBST and three times with PBS, 100 μL of TMB substrate was added to each well and allowed to develop for 10 minutes at 37°C. The reaction was stopped with 50 μL / well of 2 M sulfuric acid solution, and the absorbance was read at 450 nm. As shown in Figure 13-b, 13-d, and 13-f and Table 10, humanized antibodies Ab1, Ab2, Ab3, Ab4, Ab5, Ab8, Ab10, Ab13, Ab19, Ab20, Ab21, Ab22, Ab23, Ab24, Ab25, and Ab26 all blocked the binding of cynomolgus TSLP to TSLPR.

[0201] [Table 10]

[0202] Example 15. FACS detection of blocking of humanized antibodies binding to TSLP and TSLPR Ba / F3-hTSLPR-IL7Rα cells were harvested, washed three times with PBS, centrifuged at 300 g for 3 minutes, and blocked with 3% BSA / PBS for 30 minutes. After resuspending the cells, they were diluted to 5 × 10 4Cells were added to a U-shaped 96-well plate at 1000 x 1000 μL / well. Antibodies were incubated with 60 ng / mL biotinylated human TSLP protein at room temperature for 10 minutes, then added to the cells. The cells were incubated at 4°C for 45 minutes and washed twice with 0.5% BSA / PBS. SA-PE was added, incubated at 4°C for 45 minutes, washed twice with 0.5% BSA / PBS, PI dye was added, incubated at 4°C for 10 minutes, washed twice with 0.5% BSA / PBS, and the cells were resuspended in PBS. The blocking effects of the antibodies at the cellular level were detected by flow cytometry. As shown in Figure 14 and Table 11, the humanized antibodies Ab19, Ab20, Ab21, Ab22, Ab23, Ab24, Ab25, and Ab26 all blocked the binding of TSLP to the cell surface TSLPR, with no significant differences between the individual antibodies.

[0203] [Table 11]

[0204] Example 16: Inhibition of TSLP-stimulated Ba / F3-hTSLPR-IL7Rα cell proliferation by humanized antibodies A short-term proliferation bioassay (utilizing cells expressing recombinant human TSLPR) was applied to evaluate the ability of different anti-TSLP antibodies to biologically neutralize human TSLP. One day before, 2 x 10 cells were cultured in advance. 4 Add 60 μL of diluted anti-TSLP antibody and 60 μL of 5 ng / mL human TSLP to a 96-well cell culture plate at a concentration of 100 μL / well. Preincubate 60 μL of diluted anti-TSLP antibody with 60 μL of 5 ng / mL human TSLP at room temperature for 15 minutes. Add 100 μL of the preincubated antibody-cytokine mixture to the cell culture plate at a concentration of 100 μL / well. Incubate the 96-well plate in a 5% CO2, 37°C incubator for 72 hours. After the incubation period, add CellTiter-Glo to each well and allow to react for 10 minutes. Read the luminescence values in the black 96-well plate.

[0205] As shown in Figure 15 and Table 12, four humanized antibodies, Ab22, Ab23, Ab24, and Ab26, were able to significantly inhibit TSLP-stimulated proliferation of Ba / F3-hTSLPR-IL7Rα cells (IC50 of 2.20 nM, 1.10 nM, 1.04 nM, and 1.50 nM, respectively). At the same time, compared with the same type of antibody, A5 (see US Patent No. US10287348B2), the inhibitory effects of Ab22, Ab23, Ab24, and Ab26 were all superior to A5 (measured IC50 = 16.55).

[0206] [Table 12]

[0207] Example 17: Reporter Gene Assay Detection of Blockade of TSLP-Induced STAT5 Pathway Signaling by Humanized Antibodies Ba / F3-hTSLPR-IL7Rα-STAT5 luc cells were starved overnight and the next day, 5 × 10 4 Human TSLP was diluted before inoculating 50 μL / well into a 96-well plate and incubating at 37°C for 30 minutes. 50 μL of the ligand / antibody mixture was pipetted into the cell plate, mixed, and incubated at 37°C for 6 hours. 100 μL of fluorescent detection reagent was added, and luminescence readings for each well were read using a microplate reader.

[0208] As shown in Figure 16 and Table 13, four humanized antibodies, Ab22, Ab23, Ab24, and Ab26, significantly inhibited TSLP-stimulated signal transduction through the STAT5 pathway (IC50 = 0.17 nM, 0.17 nM, 0.15 nM, and 0.18 nM), respectively, with inhibitory effects superior to those of Ab5 (measured IC50 = 1.93 nM, see IC50 = 1.4 nM reported in Kenneth V, 2017).

[0209] [Table 13]

[0210] Various modifications and equivalents may be made to the embodiments disclosed herein without departing from the spirit and scope of the present disclosure. Any feature, step or embodiment may be used in combination with any other feature, step or embodiment unless the context dictates otherwise.

[0211] (Addendum) The present disclosure includes the following aspects. Section 1: heavy chain CDRs selected from the amino acid sequences SEQ ID NOs: 8-10, 18-20, 28-30, 38-40, 48-50, 58-60, 68-70, 78-80, 83-85, 88-90, 93-95, 98-100, 103-105, 108-110, 113-115, 118-120, 123-125, 128-130, 133-135, 138-140, 143-145, 148-150, 153-155, 158-160, 163-165, or any variant thereof; and / or amino acid sequences SEQ ID NOs: An antibody or antigen-binding portion thereof that binds to TSLP, comprising a light chain CDR selected from NO: 13-15, 23-25, 33-35, 43-45, 53-55, 63-65, 73-75, 168-170, 173-175, or any variant thereof. Section 2: a heavy chain CDR1 selected from the amino acid sequence SEQ ID NO: 8, 18, 28, 38, 48, 58, 68, 78, 83, 88, 93, 98, 103, 108, 113, 118, 123, 128, 133, 138, 143, 148, 153, 158, 163, or any variant thereof; a heavy chain CDR2 selected from the amino acid sequence SEQ ID NO: 9, 19, 29, 39, 49, 59, 69, 79, 84, 89, 94, 99, 104, 109, 114, 119, 124, 129, 134, 139, 144, 149, 154, 159, 164, or any variant thereof; a heavy chain CDR3 selected from the amino acid sequence of SEQ ID NO: 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, or any variant thereof; and / or a light chain CDR1 selected from the amino acid sequence of SEQ ID NO: 13, 23, 33, 43, 53, 63, 73, 168, 173, or any variant thereof; a light chain CDR2 selected from the amino acid sequence of SEQ ID NO: 14, 24, 34, 44, 54, 64, 74, 169, 174, or any variant thereof; The antibody or antigen-binding portion thereof according to Item 1, comprising a light chain CDR3 selected from 15, 25, 35, 45, 55, 65, 75, 170, 175, or any variant thereof. Section 3: Item 3. The antibody or antigen-binding portion thereof according to Item 1 or 2, comprising a combination of heavy chain and light chain CDRs selected from the group consisting of: (1) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 8-10, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 13-15, respectively; (2) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 18-20, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 23-25, respectively; (3) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 28-30, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 33-35, respectively; (4) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 38-40, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 43-45, respectively; (5) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 48-50, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 53-55, respectively; (6) heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 58-60, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 63-65, respectively; (7) heavy chain CDR1, CDR2 and CDR3 containing SEQ ID NOs: 68-70, respectively; DR3 sequence, and / or light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 73-75, respectively. (8) Heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 78-80, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 168-170, respectively. (9) Heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 83-85, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 168-170, respectively. (10) Heavy chain CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NOs: 88-90, and / or light chain CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NOs: 168-170. (11) Heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 93-95, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 168-170, respectively. (12) Heavy chain CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NO: 98-100, and / or light chain CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NO: 168-170. (13) Heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 103-105, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 168-170, respectively. (14) Heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 108-110, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 168-170, respectively. (15) Heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 113-115, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 168-170, respectively. (16) Heavy chain CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NOs: 118-120, and / or light chain CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NOs: 168-170. (17) Heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 123-125, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 168-170, respectively. (18) Heavy chain CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NOs: 128-130, and / or light chain CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NOs: 168-170. (19) Heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 133-135, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 168-170, respectively. (20) Heavy chain CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NO: 138-140, and / or light chain CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NO: 168-170. (21) Heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 143-145, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 168-170, respectively. (22) Heavy chain CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NOs: 148-150, and / or light chain CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NOs: 168-170. (23) Heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 153-155, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 168-170, respectively. (24) Heavy chain CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NOs: 158-160, and / or light chain CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NOs: 168-170. (25) Heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 163-165, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 168-170, respectively. (26) Heavy chain CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NOs: 153-155, and / or light chain CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NOs: 173-175. (27) Heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 158-160, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 173-175, respectively. (28) Heavy chain CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NOs: 163-165, and / or light chain CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NOs: 173-175. (29) Heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 78-80, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 173-175, respectively. (30) Heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 83-85, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 173-175, respectively. (31) Heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 98-100, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 173-175, respectively. (32) Heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 103-105, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 173-175, respectively. (33) Heavy chain CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NOs: 123-125, and / or light chain CDR1, CDR2, and CDR3 sequences each comprising SEQ ID NOs: 173-175. (34) Heavy chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 128-130, respectively, and / or light chain CDR1, CDR2, and CDR3 sequences comprising SEQ ID NOs: 173-175, respectively. Section 4: an antibody or antigen-binding portion thereof, comprising a heavy chain variable region selected from amino acid sequences of SEQ ID NOs: 7, 17, 27, 37, 47, 57, 67, 77, 82, 87, 92, 97, 102, 107, 112, 117, 122, 127, 132, 137, 142, 147, 152, 157, 162, or any variant thereof; and / or a light chain variable region selected from amino acid sequences of SEQ ID NOs: 12, 22, 32, 42, 52, 62, 72, 167, 172, or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO: 7 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO: 12 or any variant thereof, Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO: 17 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO: 22 or any variant thereof, Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO: 27 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO: 32 or any variant thereof; Preferably, the heavy chain variable fragment of the amino acid sequence SEQ ID NO: 37 or any variant thereof a light chain variable region having the amino acid sequence SEQ ID NO: 42 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO: 47 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO: 52 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO: 57 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO: 62 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO: 67 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO: 72 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO: 77 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO: 167 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO: 82 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO: 167 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO: 87 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO: 167 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO: 92 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO: 167 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO: 97 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO: 167 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO: 102 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO: 167 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO: 107 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO: 167 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO: 112 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO: 167 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO: 117 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO: 167 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO: 122 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO: 167 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO: 127 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO: 167 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO: 132 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO: 167 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO: 137 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO: 167 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO: 142 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO: 167 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO: 147 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO: 167 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO: 152 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO: 167 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO: 157 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO: 167 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO: 162 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO: 167 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO: 152 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO: 172 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO: 157 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO: 172 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO: 162 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO: 172 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO: 77 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO: 172 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO: 82 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO: 172 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO: 97 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO: 172 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO: 102 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO: 172 or any variant thereof; Preferably, the heavy chain variable region has the amino acid sequence SEQ ID NO: 122 or any variant thereof, and the light chain variable region has the amino acid sequence SEQ ID NO: 172 or any variant thereof; Preferably, the antibody or antigen-binding portion thereof according to item 1 or 2 comprises a heavy chain variable region having the amino acid sequence SEQ ID NO: 127 or any variant thereof, and a light chain variable region having the amino acid sequence SEQ ID NO: 172 or any variant thereof. Section 5: A nucleic acid molecule encoding the antibody or antigen-binding portion thereof according to any one of Aspects 1 to 4, or a nucleic acid molecule having at least 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or more, sequence identity thereto, preferably the nucleic acid molecule is an antibody heavy chain selected from SEQ ID NOs: 11, 21, 31, 41, 51, 61, 71, 81, 86, 91, 96, 101, 106, 111, 116, 121, 126, 131, 136, 141, 146, 151, 156, 161, 166, or any variant thereof. a nucleic acid molecule comprising an antibody light chain nucleic acid sequence and / or an antibody light chain nucleic acid sequence selected from SEQ ID NOs: 16, 26, 36, 46, 56, 66, 76, 171, 176 or any variant thereof. Item 6: A vector comprising the nucleic acid molecule of item 5. Section 7: A cell comprising the nucleic acid molecule of Item 6 or the vector of Item 8. Section 8: A composition comprising the antibody or antigen-binding portion thereof according to any one of Aspects 1 to 4, the nucleic acid molecule according to Aspect 5, the vector according to Aspect 6, and / or the cell according to Aspect 7. Section 9: A kit comprising the antibody or antigen-binding portion thereof according to any one of Aspects 1 to 4, the nucleic acid molecule according to Aspect 5, the vector according to Aspect 6, the cell according to Aspect 7, and / or the composition according to Aspect 8. Section 10: A use of the antibody or antigen-binding portion thereof according to any one of Aspects 1 to 4, the nucleic acid molecule according to Aspect 5, the vector according to Aspect 6, the cell according to Aspect 7, and / or the composition according to Aspect 8 in the preparation of a medicament or kit for treating a TSLP-related disease, wherein the TSLP-related disease is preferably selected from TSLP-related inflammatory diseases and autoimmune diseases, preferably the TSLP-related inflammatory disease is selected from allergic inflammation, asthma, chronic obstructive pulmonary disease, atopic dermatitis, and eosinophilic esophagitis, preferably the allergic inflammation is selected from allergic rhinitis, allergic sinusitis, and allergic conjunctivitis, and preferably the autoimmune disease is selected from rheumatoid arthritis and multiple sclerosis.

Claims

1. An antibody or antigen-binding portion thereof that binds to TSLP, wherein the antibody or antigen-binding portion thereof comprises: heavy chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 123-125, respectively; and An antibody or antigen-binding portion thereof that binds TSLP, comprising light chain CDR1, CDR2 and CDR3 sequences comprising SEQ ID NOs: 173-175, respectively.

2. 2. The antibody or antigen-binding portion thereof of claim 1, comprising a heavy chain variable region of amino acid sequence SEQ ID NO: 122 or a variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO: 172 or a variant thereof.

3. A nucleic acid molecule encoding the antibody or antigen-binding portion thereof of claim 1 or 2.

4. The nucleic acid molecule of claim 3 , wherein the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 126 or a variant thereof, and the nucleic acid sequence of SEQ ID NO: 176 or a variant thereof.

5. A vector comprising the nucleic acid molecule of claim 3 or 4.

6. A cell comprising the nucleic acid molecule of claim 3 or 4, or the vector of claim 5.

7. A composition comprising an antibody or antigen-binding portion thereof according to claim 1 or 2, a nucleic acid molecule according to claim 3 or 4, a vector according to claim 5, or a cell according to claim 6.

8. A kit comprising an antibody or antigen-binding portion thereof according to claim 1 or 2, a nucleic acid molecule according to claim 3 or 4, a vector according to claim 5, a cell according to claim 6 or a composition according to claim 7.

9. An antibody or antigen-binding portion thereof according to claim 1 or 2, a nucleic acid molecule according to claim 3 or 4, a vector according to claim 5, a cell according to claim 6 or a composition according to claim 7 for use in the treatment of a TSLP-related disease.

10. The antibody or antigen-binding portion thereof, nucleic acid molecule, vector, cell or composition of claim 9, wherein the TSLP-associated disease is a TSLP-associated inflammatory disease or autoimmune disease.

11. The antibody or antigen-binding portion thereof, nucleic acid molecule, vector, cell or composition of claim 10, wherein the TSLP-associated inflammatory disease is selected from allergic inflammation, asthma, chronic obstructive pulmonary disease, atopic dermatitis and eosinophilic esophagitis.

12. The antibody or antigen-binding portion thereof, nucleic acid molecule, vector, cell or composition of claim 11, wherein the allergic inflammation is selected from allergic rhinitis, allergic sinusitis and allergic conjunctivitis.

13. The antibody or antigen-binding portion thereof, nucleic acid molecule, vector, cell or composition that binds to TSLP according to claim 10, wherein the autoimmune disease is selected from rheumatoid arthritis and multiple sclerosis.

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