Development and application of a therapeutic agent for tslp-related disorders

Abstract

The present disclosure relates to an antibody or antigen-binding portion thereof that binds to TSLP protein, and methods of making and using the same. The antibody can bind to human TSLP and / or cynomolgus monkey TSLP with high affinity, can block the binding of TSLP to TSLPR, and can inhibit the transduction of TSLP-stimulated signals through the STAT5 pathway.

Description

Technical Field

[0001] This disclosure relates to an antibody that recognizes TSLP protein, a method for manufacturing the antibody, and its application. Background Technology

[0002] Thymic stromal lymphopoietin (TSLP) is a cytokine belonging to the interleukin-7 family, primarily produced by epithelial and keratinocyte cells located in the skin, intestines, or lungs, and participates in maintaining the stability of the mucosal immune system. When epithelial tissue is stimulated or damaged by allergens or pathogens, dendritic cells present antigens to primitive CD4+ T cells, simultaneously releasing epithelial cytokines, TSLP, and IL-33. These important co-stimulatory cytokines lead to the development of allergen-specific TH2 cells, which then produce the cytokines interleukins IL-4, IL-5, and IL-13. This group of TH2-derived cytokines plays a crucial role in the pathogenesis of allergic diseases.

[0003] TSLP is a multifunctional cytokine that exerts its biological functions through the TSLPR / IL-7Rα receptors on the surface of various immune cells, including dendritic cells (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), increases antigen presentation, and produces IL-8 (Interleukin 8 or Chemokine (CXC motif) ligand 8 (CXCL8)), eosinophil chemokine-2 (eotaxin 2 or Chemokine (CC motif) ligand 24 (CCL24)), TARC (thymus and activation-regulated chemokine or Chemokine (CC motif) ligand 17 (CCL17)), and MDC (macrophage-derived chemokine or Chemokine (CC)). TSLP (CCL22 motif) attracts the aggregation of eosinophils, neutrophils, and Th2 cells. TSLP can promote the differentiation of naive T cells into Th2 cells, a process dependent on TSLP-induced expression of OX40L in dendritic cells (DCs). TSLP can induce mast cell proliferation, prolong the lifespan of eosinophils, and specifically release inflammatory cytokines and chemokines. TSLP can also stimulate another important group of mucosal immune cells—group 2 innate lymphoid cells (ILC2)—to secrete Th2 cytokines IL-4, IL-5, and IL-13, promoting skin inflammation in vivo. Smooth muscle cells stimulated by TSLP also secrete IL-8 and eotaxin. TSLP has also been shown to increase the cytokine production of various innate immune cells (including ILC2, mast cells, natural killer cells, and eosinophils) and promote the development and function of basophil subsets. Therefore, TSLP may be one of the initiators of the inflammatory cascade. Inhibiting TSLP can intervene in the early stage of inflammation and prevent immune cells from releasing pro-inflammatory cytokines, which is more effective than inhibiting IL-4, IL-5 and IL-13 alone.

[0004] TSLP transduces signals via the JAK / STAT (JAK kinase-signal transducer and activator of transcription) pathway. After TSLP binds to TSLPR on the cell membrane, it then binds to IL-7Rα, forming a stable TSLP-TSLPR-IL7Rα receptor complex. The intracellular segment of the TSLPR receptor in this complex recruits and activates JAK2, which, together with JAK1 recruited by IL-7Rα, activates downstream signaling molecules. Studies have shown that in human peripheral blood-derived CD11c+ DC cells, TSLP can activate multiple STAT signaling molecules, including STAT1, STAT3, STAT4, STAT5, and STAT6. STAT5 activation is crucial for promoting TH2 cell differentiation and TH2 factor secretion. Furthermore, TSLP stimulation of the JAK / STAT5 signaling pathway can render ILC2 cells unresponsive to glucocorticoid inhibition, suggesting that inhibiting TSLP may help restore patients' sensitivity to glucocorticoids.

[0005] TSLP is closely associated with the development of type II inflammatory diseases. Studies have shown that TSLP is highly expressed in damaged skin of patients with atopic dermatitis, but undetectable in undamaged skin. High levels of TSLP mRNA-positive cells have also been detected in the lung epithelium and submucosa of patients with asthma and COPD, with higher TSLP concentrations in bronchoalveolar lavage samples compared to healthy controls. TSLP expression levels in asthma patients are directly correlated with TH2 cytokine and chemokine expression, but negatively correlated with retained lung function. In biopsies of patients with allergic rhinitis, increased TSLP expression in the nasal epithelium was found and associated with TH2 cytokine production and eosinophil infiltration in epithelial-associated tissues.

[0006] Genetic polymorphisms of TSLP and TSLPR are thought to be associated with the pathogenesis of eosinophilic esophagitis (EoE). High expression of TSLP and an elevated proportion of basophils (lin-, CD49b+, FcεRI+, c-kit-, 2D7+) have been detected in esophageal samples from EoE patients. Interestingly, the mouse EoE model is dependent on TSLP and basophils, but not on IgE. Using TSLP-neutralizing antibodies or eliminating basophils effectively alleviates EoE symptoms, suggesting that inhibiting TSLP-basophils, rather than IgE, may be an effective approach to treating EoE.

[0007] Furthermore, studies have shown that TSLP may promote TH1 / TH17-related autoimmune diseases, such as rheumatoid arthritis and multiple sclerosis. In the joints of rheumatoid arthritis patients, TSLP and TSLPR expression cells are increased. In a proteoglycan-induced rheumatoid arthritis mouse model, TSLPR-deficient mice showed decreased levels of IL-17, IL-1β, and IL-6, and increased levels of INFγ and IL-10, with reduced disease symptoms. This suggests that TSLP and its receptor may be novel therapeutic targets for rheumatoid arthritis. Summary of the Invention

[0008] The inventors immunized mice with recombinant TSLP protein, a gene gun, or a combination thereof, obtaining multiple high-affinity antibodies that recognize recombinant TSLP protein from humans and cynomolgus monkeys. The antibodies disclosed herein possess high affinity, effectively blocking the binding of TSLP to its receptor TSLPR, and inhibiting TSLP-induced STAT5 signaling in reporter cells. They also block TSLP-induced cell proliferation, exhibiting a stronger blocking efficiency than other similar antibodies, thus enabling their use in the diagnosis and treatment of allergic inflammatory diseases.

[0009] In one aspect, this disclosure provides antibodies or antigen-binding portions thereof that bind to TSLP proteins.

[0010] In one respect, this disclosure provides nucleic acid molecules encoding antibodies or their antigen-binding portions as described above.

[0011] In one respect, this disclosure provides a vector containing nucleic acid molecules as described above.

[0012] In one respect, this disclosure provides cells containing the aforementioned carriers.

[0013] The antibody or its antigen-binding portion according to any of the foregoing aspects, wherein the antibody or its antigen-binding portion is humanized.

[0014] In one aspect, this disclosure provides pharmaceutical compositions or kits comprising an antibody or its antigen-binding portion or its encoded nucleic acid as described above and a pharmaceutically acceptable carrier.

[0015] In one aspect, this disclosure provides a method for treating TSLP-related conditions, comprising the steps of administering a therapeutically effective amount of an antibody or antigen-binding fragment or nucleic acid molecule or carrier or cell and / or pharmaceutical composition thereof to the mammal.

[0016] The use of any of the foregoing antibodies or their antigen-binding fragments or nucleic acid molecules or carriers and / or cells or pharmaceutical compositions in the preparation of a medicament or kit for treating TSLP-related diseases in mammals.

[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 transduction of TSLP stimulation signals through the STAT5 pathway. Attached Figure Description

[0018] Figure 1 TSLP protein expressed by prokaryotic recombinant

[0019] Figure 2 TSLPR protein recombinantly expressed in HEK293 cells

[0020] Figure 3 ELISA detection of TSLP recombinant protein binding receptor TSLPR

[0021] Figure 4 Ba / F3-hTSLPR cell FACS detection

[0022] Figure 5 Ba / F3-hTSLPR-IL7Rα cell proliferation detection

[0023] Figure 6 Ba / F3-hTSLPR-IL7Rα-STAT5, luc cell reporter gene detection

[0024] Figure 7 ELISA binding detection of chimeric antibodies

[0025] Figure 8 ELISA blocking detection of chimeric antibodies

[0026] Figure 9 Chimeric antibody-mediated cell-level blocking

[0027] Figure 10 Inhibition of cell proliferation by chimeric antibodies

[0028] Figure 11 Inhibition of reporter gene expression by chimeric antibodies

[0029] Figure 12 ELISA detection of humanized antibodies binding to human and cynomolgus monkey TSLP

[0030] Figure 13 ELISA blocking detection of humanized antibodies

[0031] Figure 14 Blocking at the level of humanized antibody cells

[0032] Figure 15 Inhibition of cell proliferation by humanized antibodies

[0033] Figure 16Inhibition of reporter gene expression by humanized antibodies Detailed Implementation

[0034] I. Definition

[0035] In this disclosure, unless otherwise stated, 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, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0036] In one aspect, this document provides antibodies (e.g., monoclonal antibodies) that specifically bind TSLP and their antigen-binding fragments. In a more specific aspect, this document provides monoclonal anti-TSLP antibodies that specifically bind TSLP, wherein said anti-TSLP antibodies comprise variants of parental antibodies. In a more specific aspect, this document provides antibodies that specifically bind TSLP (e.g., human TSLP). In a more specific aspect, this document provides anti-TSLP antibodies containing modifications in one or more amino acid residues (e.g., 5-13 amino acid substitutions in the framework region of the heavy chain variable region) that maintain affinity for the antigen compared to parental antibodies without said modifications.

[0037] As used herein and unless otherwise stated, the terms “about” or “approximately” mean within 10% of a given value or range. Where an integer is required, the term means within 10% of a given value or range, rounded up or down to the nearest integer.

[0038] Regarding antibody chain polypeptide sequences, the phrase "substantially identical" can be understood as an antibody chain exhibiting at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a reference polypeptide sequence. Regarding nucleic acid sequences, the term can be understood as a nucleotide sequence exhibiting at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with a reference nucleic acid sequence.

[0039] Sequence “identity” or “identity” has a generally accepted meaning in the art, and the percentage of sequence similarity between two nucleic acid or polypeptide molecules or regions can be calculated using publicly available techniques. Sequence similarity can be measured along the full length of a polynucleotide or polypeptide or along a region of that molecule (see, for example: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, 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, 1994; 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). Although there are many methods for measuring the similarity between two polynucleotides or polypeptides, the term "similarity" is well known to those skilled in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988)).

[0040] A "substitution" variant is a variant in which at least one amino acid residue is removed from the natural sequence and replaced by a different amino acid at the same position. The substitution can be single, where only one amino acid is substituted in the molecule; or it can be multiple, where two or more amino acids are substituted in the same molecule. Multiple substitutions can be located at consecutive sites. Similarly, an amino acid can be substituted by multiple residues, and such variants include both substitution and insertion. An "insertion" variant is a variant in which one or more amino acids are inserted into an amino acid immediately adjacent to a specific position in the natural sequence. An adjacent amino acid is defined as one attached to the α-carboxyl or α-amino functional group of that amino acid. A "deletion" variant is a variant in which one or more amino acids are removed from the natural amino acid sequence. Typically, deletion variants have one or two amino acids missing from a specific region of their molecule.

[0041] Regarding the variable domains of antibodies, the term "variable" refers to certain portions of related molecules with extensive sequence differences between antibodies, used for the specific recognition and binding of a particular antibody to its specific target. However, variability is not uniformly distributed throughout the entire variable domain of an antibody. Variability is concentrated in three segments known as complementarity-determining regions (CDRs; namely CDR1, CDR2, and CDR3) or hypervariable regions, all located within the variable domains of the light and heavy chains. More conserved portions within the variable domain are called framework (FR) regions or framework sequences. Each variable domain of the natural heavy and light chains includes four FR regions, primarily employing a β-sheet configuration, linked by three CDRs forming loops that connect the β-sheet structure and, in some cases, partially form a β-sheet. CDRs on each chain are typically linked together in proximity by FR regions, and the presence of CDRs from other chains contributes to the formation of antibody target binding sites (epitopes or determinants) (see Kabat et al., Sequences of Proteins of Immunological Interest, National Institute 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 stated. A CDR may have the ability to specifically bind associated epitopes.

[0042] 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 portion of the variable region of the antibody that binds to the antigen (e.g., one or more CDRs and / or one or more antibody binding sites) and thus retains binding specificity as well as at least a portion of the specific binding capacity of the full-length antibody. Therefore, an antigen-binding fragment refers to an antibody fragment containing an antigen-binding portion that binds to the same antigen as the antibody fragment derived from the antibody. Antibody fragments include antibody derivatives produced by enzymatic treatment of a full-length antibody, as well as synthetically produced derivatives, such as recombinant derivatives. Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single-chain Fv (scFv), Fv, dsFv, biantibodies, Fd and Fd' fragments, and other fragments, including modified fragments (see, for example, Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov). The fragment may comprise multiple chains linked together, for example by disulfide bonds and / or by peptide linkers. Antibody fragments generally contain at least or about 50 amino acids, and typically at least or about 200 amino acids. Antigen-binding fragments include any antibody fragment that, upon insertion into an antibody framework (e.g., by replacing the corresponding region), acquires immune-specific binding (i.e., exhibits at least or at least about 10 amino acids). 7 -10 8 Antibodies against the Ka antigen of M-1. A “functional fragment” or “anti-TSLP antibody analog” is a fragment or analog that prevents or substantially reduces the ability of the receptor to bind ligands or initiate signal transduction. As used herein, a functional fragment generally has the same meaning as an “antibody fragment,” and in the context of antibodies, it can refer to a fragment that prevents or substantially reduces the ability of the receptor to bind ligands or initiate signal transduction, such as Fv, Fab, F(ab')2, etc. An “Fv” fragment is a dimer (V) formed by non-covalent binding of a variable domain of a heavy chain and a variable domain of a light chain. H -V L (Dimer) composition. In this configuration, the three CDRs of each variable domain interact to determine V. H -V L The target binding sites on the surface of the dimer are the same as in the case of the intact antibody. The six CDRs collectively confer target binding specificity to the intact antibody. However, even a single variable domain (or half the Fv of only including three target-specific CDRs) can still have the ability to recognize and bind to the target.

[0043] As used herein, the term "bispecific antibody" (BsAb) refers to an antibody and / or antigen-binding molecule that specifically binds to two different antigenic determinants. Typically, a bispecific antibody and / or antigen-binding molecule contains two antigen-binding sites, each specific to a different antigenic determinant. In some embodiments, the bispecific antibody and / or antigen-binding molecule is capable of binding to two antigenic determinants simultaneously, particularly two antigenic determinants expressed on two different cells.

[0044] As used herein, “monoclonal antibody” refers to a population of identical antibodies, meaning that each individual antibody molecule in a population of monoclonal antibodies is identical to the others. This characteristic contrasts with that of a polyclonal population of antibodies, which contains antibodies with a variety of different sequences. Monoclonal antibodies can be prepared by a number of well-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 from immortalized B cells, for instance, by fusing with myeloma cells to generate hybridoma cell lines or by infecting B cells with a virus such as EBV. Recombinant techniques can also be used to prepare antibodies in vitro from a clonal population of host cells by transforming host cells with plasmids carrying artificial sequences of nucleotides encoding the antibody.

[0045] As used herein, the term "hybridoma" or "hybridoma cell" refers to a cell or cell line (typically myeloma or lymphoma cells) resulting from the fusion of antibody-producing lymphocytes and non-antibody-producing cancer cells. As is known to those skilled in the art, a hybridoma can proliferate and continuously supply cells that produce a specific monoclonal antibody. Methods for generating hybridomas are known in the art (see, for example, Harlow & Lane, 1988). When referring to the term "hybridoma" or "hybridoma cell," it also includes subclones and progeny cells of the hybridoma.

[0046] 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) and two full-length light chains (VL-CL) and a hinge region, such as antibodies naturally produced by antibody-secreting B cells and synthetically produced antibodies with the same domains.

[0047] The term "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.

[0048] "Humanized" antibodies refer to non-human (e.g., mouse) antibody forms that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) containing minimal sequences derived from non-human immunoglobulins. Preferably, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues of the complementarity-determining region (CDR) of the recipient antibody are replaced by CDR residues from a non-human species (donor antibody) with the desired specificity, affinity, and capability, such as mouse, rat, or rabbit.

[0049] Furthermore, in humanization, amino acid residues in the CDR1, CDR2, and / or CDR3 regions of VH and / or VL may be mutated to improve one or more binding properties (e.g., affinity) of the antibody. Mutations can be introduced, for example, through PCR-mediated mutations, and their effects on antibody binding or other functional properties can be assessed using the in vitro or in vivo assays described herein. Typically, conserved mutations are introduced. Such mutations can be amino acid substitutions, additions, or deletions. Additionally, mutations within the CDRs typically do not exceed one or two. Therefore, the humanized antibodies described in this disclosure also cover antibodies containing one or two amino acid mutations within the CDRs.

[0050] As used herein, the term "CDR" refers to the 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 located in the variable regions of each heavy and light chain of the antibody and are highly variable sites in the primary structure of the CDR. In this specification, the CDRs of the heavy chain are represented by CDR1, CDR2, and CDR3 from the N-terminal sequence of the heavy chain, and the CDRs of the light chain are represented by CDR1, CDR2, and CDR3 from the N-terminal sequence of the light chain. These sites are adjacent to each other in the tertiary structure and determine the specificity of the antigen to which the antibody binds.

[0051] As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which an antibody binds at its complementary site. Epitope determinants typically comprise chemically active surface subtypes of a molecule, such as amino acid or sugar side chains, and often possess specific three-dimensional structural features as well as specific charge characteristics.

[0052] As used herein, the terms “specific binding” and “immune-specific binding” for antibodies or their antigen-binding fragments are used interchangeably and refer to the ability of an antibody or antigen-binding fragment to form one or more non-covalent bonds with the same antigen through a non-covalent interaction between the antibody and the antigen’s antibody-binding site. The antigen may be an isolated antigen or present in tumor cells. Typically, antibodies that immune-specifically bind (or specifically bind) antigens are present in quantities of approximately 1 × 10⁻⁶. 7 M -1 Or 1x10 8 M -1 Or a larger affinity constant Ka (or 1×10) -7 M or 1×10 -8 The antigen is bound by a dissociation constant (M or lower) of M or lower. The affinity constant can be determined by standard kinetic methods of antibody reaction, such as immunoassay, 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 interaction assays known in the art (see, for example, Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336 (1989); also see U.S. Patent No. 7,229,619, which describes exemplary SPR and ITC methods for calculating the binding affinity of an antibody). Instruments and methods for real-time detection and monitoring of binding rates are known and commercially available (see BiaCore 2000, Biacore AB, Upsala, Sweden and GE Healthcare Life Sciences; Malmqvist (2000) Biochem. Soc. Trans. 27:335).

[0053] As used herein, the terms “polynucleotide” and “nucleic acid molecule” refer to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) typically linked together by phosphodiester bonds. As used herein, the term “nucleic acid molecule” is intended to include both DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded and can be cDNA.

[0054] As used herein, isolated nucleic acid molecules are nucleic acid molecules isolated from other nucleic acid molecules present in natural sources of nucleic acid molecules. “Isolated” nucleic acid molecules, such as cDNA molecules, may be substantially free of other cellular material or culture medium when prepared by recombinant technology, 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 encoding provided antibody or antigen-binding fragments.

[0055] As used herein, “operably linked” in relation to a nucleic acid sequence, region, element, or domain indicates 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, thereby regulating or mediating the transcription of that nucleic acid.

[0056] Also provided are “conserved sequence modifications” of the sequences listed herein, i.e., nucleotide and amino acid sequence modifications that do not eliminate the binding of antibodies to antigens encoded by nucleotide sequences or containing amino acid sequences. These conserved sequence modifications include conserved 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 herein using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conserved sequence modifications include conserved amino acid substitutions, wherein amino acid residues are replaced with amino acid residues having similar side chains. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with non-polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, the predicted non-essential amino acid residues in anti-TSLP antibodies are preferably replaced by another amino acid residue from the same side chain family. Methods for identifying nucleotides and conserved amino acid substitutions that do not eliminate antigen binding are well known in the art (e.g., see Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

[0057] As an alternative, in another embodiment, mutations can be randomly introduced along all or part of the coding sequence of the anti-TSLP antibody, for example, through saturation mutagenesis, and the resulting modified anti-TSLP antibody can be screened for improved binding activity.

[0058] As used herein, “expression” refers to the process by which a polypeptide is produced through the transcription and translation of polynucleotides. The expression level of a polypeptide can be evaluated using any method known in the art, including, for example, methods for determining the amount of polypeptide produced from host cells. Such methods may include, but are not limited to, quantifying polypeptides in cell lysates by ELISA, Coomassie blue staining following gel electrophoresis, Lowry protein assays, and Bradford protein assays.

[0059] As used herein, a “host cell” is a cell used to receive, maintain, replicate, and amplify a vector. Host cells can also be used to express the polypeptide encoded by the vector. When a host cell divides, the nucleic acids contained in the vector replicate, thereby amplifying the nucleic acids. Host cells can be eukaryotic or prokaryotic cells. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, and HEK cells such as HEK 293 cells.

[0060] As used herein, a "vector" is a reproducible nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into a suitable host cell. Vectors include those into which nucleic acids encoding polypeptides or fragments thereof can typically be introduced via restriction enzyme digestion and ligation. Vectors also include those containing nucleic acids encoding polypeptides. Vectors are used to introduce nucleic acids encoding polypeptides into host cells for amplification of nucleic acids or for expression / display of the polypeptide encoded by the nucleic acid. Vectors are typically kept free but can be designed to integrate genes or portions thereof into the chromosome of the genome. Vectors for artificial chromosomes, such as yeast artificial vectors and mammalian artificial chromosomes, are also considered. The selection and use of such vectors are well known to those skilled in the art.

[0061] As used in this article, vectors also include “viral vectors” or “vectors of viruses.” Viral vectors are engineered viruses that are operatively linked to a foreign gene to transfer (as a medium or shuttle) the foreign gene into cells.

[0062] As used herein, "expression vector" includes a vector capable of expressing DNA operatively linked to regulatory sequences, such as promoter regions, that influence the expression of such DNA fragments. These additional fragments may include promoter and terminator sequences and optionally include one or more origins of replication, one or more selection markers, enhancers, polyadenylation signals, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. Therefore, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, bacteriophage, recombinant virus, or other vector, which, when introduced into a suitable host cell, results in the expression of clonal DNA. Suitable expression vectors are well known to those skilled in the art and include reproducible expression vectors in eukaryotic and / or prokaryotic cells, as well as expression vectors that remain free or are integrated into the host cell genome.

[0063] As used herein, “treatment” for an individual suffering from a disease or disease condition means that the individual’s symptoms are partially or completely relieved, or remain unchanged after treatment. Therefore, treatment includes prevention, treatment, and / or cure. Prevention refers to preventing underlying disease and / or preventing the worsening of symptoms or the development of disease. Treatment also includes any antibodies or antigen-binding fragments thereof provided, and any pharmaceutical use of the compositions provided herein.

[0064] As used in this article, “therapeutic effect” refers to the effect resulting from treatment of an individual, which alters, usually improves or enhances the symptoms of a disease or condition, or cures a disease or condition.

[0065] As used herein, "therapeutic effective amount" or "therapeutic effective dose" refers to the amount of a substance, compound, material, or composition containing a compound that, when applied to a subject, is at least sufficient to produce a therapeutic effect. Therefore, it is the amount necessary to prevent, cure, improve, block, or partially block the symptoms of a disease or condition.

[0066] As used herein, "preventive effective dose" or "preventive effective amount" refers to the amount of a substance, compound, material, or composition containing a compound that, when applied to a subject, would have the intended preventive effect, such as preventing or delaying the onset or recurrence of a disease or symptom, or reducing the likelihood of the onset or recurrence of a disease or symptom. A fully preventive effective dose does not necessarily occur through the administration of a single dose and can occur only after a series of doses have been administered. Therefore, a preventive effective dose can be administered in one or more applications.

[0067] As used in this article, the term "patient" refers to mammals, such as humans.

[0068] II. Detailed Implementation

[0069] In one aspect, this disclosure provides an antibody or antigen-binding moiety thereof that binds to TSLP, comprising a heavy chain CDR selected from amino acid sequences SEQ ID NO: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 selected from amino acid sequences SEQ ID NO:8-10, 18-20, 28-30, 38-40, 48-50, 58-60, 68-70, 78-80, 83-85, 133-135, 138-140, 143-145, 148-150, 153-155, 158-160, 163-165 or any variant thereof, and / or selected from amino acid sequences SEQ ID NO:8-10, 18-20, 28-30, 38-40, 48-50, 58-60, 163-165 or any variant thereof, and / or a heavy chain CDR selected from amino acid sequences SEQ ID NO:8-10, 18-20, 28-30, 38-40, 48-50, 58-60, NO: 13-15, 23-25, 33-35, 43-45, 53-55, 63-65, 73-75, 168-170, 173-175 or any variant of light chain CDR.

[0070] In one aspect, this disclosure relates to an antibody or antigen-binding portion thereof according to the preceding aspect, comprising a heavy chain CDR1 selected from amino acid sequences 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 amino acid sequences 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, and ... Heavy chain CDR3 selected from amino acid sequences 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 light chain CDR1 selected from amino acid sequences SEQ ID NO:13, 23, 33, 43, 53, 63, 73, 168, 173 or any variant thereof; light chain CDR2 selected from amino acid sequences SEQ ID NO:14, 24, 34, 44, 54, 64, 74, 169, 173 or any variant thereof; and light chain CDR3 selected from amino acid sequences SEQ ID NO:15, 25, 35, 45, 55, 65, 75, 170, 175 or any variant thereof.

[0071] In one aspect, this disclosure relates to an antibody or antigen-binding portion thereof according to the preceding aspect, comprising a combination of CDRs selected from the heavy and light chains:

[0072] (1) Each contains the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:8-10, and / or contains the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:13-15, respectively;

[0073] (2) Each contains the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:18-20, and / or contains the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:23-25, respectively;

[0074] (3) Containing heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:28-30 respectively, and / or containing light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:33-35 respectively;

[0075] (4) Each contains the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:38-40, and / or contains the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:43-45, respectively;

[0076] (5) Containing heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:48-50 respectively, and / or containing light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:53-55 respectively;

[0077] (6) Containing heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:58-60 respectively, and / or containing light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:63-65 respectively;

[0078] (7) Containing heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:68-70 respectively, and / or containing light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:73-75 respectively;

[0079] (8) Containing heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:78-80 respectively, and / or containing light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:168-170 respectively;

[0080] (9) Containing heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:83-85 respectively, and / or containing light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:168-170 respectively;

[0081] (10) Containing heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:88-90 respectively, and / or containing light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:168-170 respectively;

[0082] (11) Containing heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:93-95 respectively, and / or containing light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:168-170 respectively;

[0083] (12) Each of the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:98-100, and / or each of the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:168-170;

[0084] (13) Containing heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:103-105 respectively, and / or containing light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:168-170 respectively;

[0085] (14) Each of the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:108-110, and / or each of the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:168-170;

[0086] (15) Containing heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:113-115 respectively, and / or containing light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:168-170 respectively;

[0087] (16) Each of the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:118-120, and / or each of the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:168-170;

[0088] (17) Containing heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:123-125 respectively, and / or containing light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:168-170 respectively;

[0089] (18) Each of the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:128-130, and / or each of the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:168-170;

[0090] (19) Containing heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:133-135 respectively, and / or containing light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:168-170 respectively;

[0091] (20) Each of the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:138-140, and / or each of the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:168-170;

[0092] (21) Containing heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:143-145 respectively, and / or containing light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:168-170 respectively;

[0093] (22) Each of the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:148-150, and / or each of the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:168-170;

[0094] (23) Containing heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:153-155 respectively, and / or containing light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:168-170 respectively;

[0095] (24) Each of the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:158-160, and / or each of the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:168-170;

[0096] (25) Each of the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:163-165, and / or each of the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:168-170;

[0097] (26) Containing heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:153-155 respectively, and / or containing light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:173-175 respectively;

[0098] (27) Containing heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:158-160 respectively, and / or containing light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:173-175 respectively;

[0099] (28) Containing heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:163-165 respectively, and / or containing light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:173-175 respectively;

[0100] (29) Containing heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:78-80 respectively, and / or containing light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:173-175 respectively;

[0101] (30) Contains heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:83-85 respectively, and / or contains light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:173-175 respectively;

[0102] (31) Containing heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:98-100 respectively, and / or containing light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:173-175 respectively;

[0103] (32) Each of the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:103-105, and / or each of the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:173-175;

[0104] (33) Containing heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:123-125 respectively, and / or containing light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:173-175 respectively;

[0105] (34) Each contains the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:128-130, and / or contains the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NO:173-175.

[0106] In one aspect, this disclosure relates to an antibody or antigen-binding portion thereof according to the preceding aspect, comprising a heavy chain variable region selected from amino acid sequences SEQ ID NO: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 SEQ ID NO:12, 22, 32, 42, 52, 62, 72, 167, 172 or any variant thereof.

[0107] In one aspect, this 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:7 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO:12 or any variant thereof.

[0108] In one aspect, this 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:17 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO:22 or any variant thereof.

[0109] In one aspect, this 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:27 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO:32 or any variant thereof.

[0110] In one aspect, this 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:37 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO:42 or any variant thereof.

[0111] In one aspect, this 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:47 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO:52 or any variant thereof.

[0112] In one aspect, this 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:57 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO:62 or any variant thereof.

[0113] In one aspect, this 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:67 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO:72 or any variant thereof.

[0114] In one aspect, this 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.

[0115] In one aspect, this 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.

[0116] In one aspect, this 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:87 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO:167 or any variant thereof.

[0117] In one aspect, this 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:92 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO:167 or any variant thereof.

[0118] In one aspect, this 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:167 or any variant thereof.

[0119] In one aspect, this 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:102 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO:167 or any variant thereof.

[0120] In one aspect, this 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:107 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO:167 or any variant thereof.

[0121] In one aspect, this 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:112 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO:167 or any variant thereof.

[0122] In one aspect, this 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:117 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO:167 or any variant thereof.

[0123] In one aspect, this 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:122 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO:167 or any variant thereof.

[0124] In one aspect, this 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.

[0125] In one aspect, this 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:132 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO:167 or any variant thereof.

[0126] In one aspect, this 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:137 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO:167 or any variant thereof.

[0127] In one aspect, this 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:142 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO:167 or any variant thereof.

[0128] In one aspect, this 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:147 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO:167 or any variant thereof.

[0129] In one aspect, this 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:152 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO:167 or any variant thereof.

[0130] In one aspect, this 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:157 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO:167 or any variant thereof.

[0131] In one aspect, this 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.

[0132] In one aspect, this 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:152 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO:172 or any variant thereof.

[0133] In one aspect, this 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:157 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO:172 or any variant thereof.

[0134] In one aspect, this 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:172 or any variant thereof.

[0135] In one aspect, this 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:172 or any variant thereof.

[0136] In one aspect, this 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:172 or any variant thereof.

[0137] In one aspect, this 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.

[0138] In one aspect, this 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:102 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO:172 or any variant thereof.

[0139] In one aspect, this 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:122 or any variant thereof, and a light chain variable region of amino acid sequence SEQ ID NO:172 or any variant thereof.

[0140] In one aspect, this 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:172 or any variant thereof.

[0141] In one aspect, this disclosure provides nucleic acid molecules encoding antibodies or their antigen-binding portions according to any of the foregoing aspects. Preferably, the nucleic acid molecule comprises an antibody heavy chain nucleic acid sequence selected from SEQ ID NO: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 selected from SEQ ID NO:16, 26, 36, 46, 56, 66, 76, 171, 176 or any variant thereof.

[0142] In one aspect, this disclosure relates to an antibody or antigen-binding portion thereof that binds to human TSLP, having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with any of the antibodies or antigen-binding portions thereof in the foregoing.

[0143] In one aspect, this disclosure relates to nucleic acid molecules encoding antibodies or their antigen-binding portions as described above, or nucleic acid molecules having sequence identity with them of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher.

[0144] In one respect, this disclosure provides vectors containing nucleic acids as described above.

[0145] In one respect, this disclosure provides cells containing a carrier as described in any of the foregoing aspects.

[0146] In one aspect, this disclosure provides pharmaceutical compositions comprising an antibody or its antigen-binding portion or its encoded nucleic acid as described above and a pharmaceutically acceptable carrier.

[0147] In one aspect, this disclosure provides a method for treating TSLP-related conditions, comprising the steps of administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof or nucleic acid molecule or carrier or cell or pharmaceutical composition to the mammal.

[0148] In one aspect, this disclosure provides the use of antibodies or antigen-binding fragments or nucleic acid molecules or carriers or cells or pharmaceutical compositions of any of the foregoing aspects in the preparation of medicaments for treating TSLP-related diseases in mammals.

[0149] Optionally, according to any of the foregoing aspects, the TSLP-related condition is a TSLP-related inflammatory condition and an autoimmune disease. Optionally, the antibody is conjugated to other drugs, such as labeled or cytotoxic conjugates.

[0150] In one aspect, this disclosure also includes kits, such as those comprising antibodies, fragments thereof, homologs thereof, derivatives thereof, etc., of the present disclosure, such as labeled or cytotoxic conjugates, as well as instructions for use of antibodies, conjugates that kill specific cell types, etc. These instructions may include guidance on the use of antibodies, conjugates, etc., in vitro, in vivo, or ex vivo. Antibodies may be in liquid or solid form, typically lyophilized. The kit may contain other suitable reagents, such as buffers, reconstitution solutions, and other necessary components for the intended use. Consideration is given to reagent combinations packaged in predetermined quantities with instructions for their use, such as for therapeutic purposes or for diagnostic assays. When the antibody is labeled, for example, enzyme-labeled, the kit may include substrates and cofactors required for the enzyme (e.g., providing substrate precursors for detecting chromophores or fluorophores). Furthermore, other additives, such as stabilizers, buffers (e.g., blocking buffers or lysis buffers), may also be included. The relative amounts of various reagents can be varied to provide concentrated reagent solutions, which provides user flexibility, space savings, reagent savings, etc. These reagents may also be available in dry powder form, typically lyophilized, and include excipients that, when dissolved, provide a reagent solution of appropriate concentration.

[0151] In one aspect, this disclosure provides the use of antibodies or functional fragments of the foregoing or nucleic acid molecules or carriers or cells or pharmaceutical compositions and / or kits in the preparation of reagents for the preparation of inhibitors that inhibit the binding of TSLP to TSLPR.

[0152] Furthermore, the antibodies disclosed herein can also be used in immunoassays, purification methods, and other methods that utilize immunoglobulins or fragments thereof. Such uses are well known in the art.

[0153] Accordingly, this disclosure also provides compositions comprising an antibody or fragment thereof against TSLP disclosed herein, said antibody being conveniently combined with a pharmaceutically acceptable carrier, diluent or excipient, as is common practice in the art.

[0154] As used in this disclosure, the term "pharmaceutical composition" refers to a formulation of a variety of preparations. Formulations containing a therapeutically effective amount of a multivalent antibody are in the form of a sterile liquid solution, liquid suspension, or lyophilized form, optionally containing a stabilizer or excipient.

[0155] The antibodies disclosed herein can be used as a single-use composition or in combination with other active agents.

[0156] It should be understood that the therapeutic agent according to the described embodiment will be administered together with a suitable pharmaceutically acceptable carrier, excipient, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerability, etc. A large number of suitable formulations can be found in all pharmacopoeias known to medicinal chemists: Remington's Pharmaceutical Sciences (15th edition, 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, and lipid-containing (cationic or anionic) carriers (e.g., Lipofectin). TM ( ), DNA conjugates, anhydrous absorbents, oil-in-water and water-in-oil emulsions, emulsion polyethylene glycol (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing polyethylene glycol. Any of the foregoing mixtures may be used for treatments or therapies according to this disclosure, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerable for the route of administration.

[0157] In one embodiment, the antibody may be used as a therapeutic agent. Such agents are typically used to treat, alleviate, and / or prevent diseases or pathologies associated with abnormal TSLP expression, activity, and / or signaling in a subject. Treatment regimens can be administered using standard methods by identifying the subject, for example, a person who has (or is at risk of developing) a disease or disorder associated with abnormal TSLP expression, activity, and / or signaling, such as a TSLP-related condition. An antibody preparation, preferably one with high specificity and high affinity for its target antigen, is administered to the subject and will typically produce an effect due to its binding to the target. The administered antibody may eliminate or inhibit or impede the expression, activity, and / or signaling function of the target (e.g., TSLP). The administered antibody may eliminate or inhibit or impede the binding of the target (e.g., TSLP) to its naturally bound endogenous ligand. For example, the antibody binds to the target and modulates, blocks, inhibits, reduces, antagonizes, neutralizes, and / or otherwise impedes TSLP expression, activity, and / or signaling. In some implementations, antibodies with heavy and light chain CDRs may be administered to subjects to treat diseases or disorders associated with abnormal TSLP expression.

[0158] As non-limiting examples, TSLP-related conditions associated with abnormal TSLP expression, activity, and / or signaling include TSLP-related inflammatory conditions and autoimmune diseases. TSLP-related inflammatory conditions 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.

[0159] In another embodiment, antibodies against TSLP can be used in methods known in the art related to the localization and / or quantification of TSLP (e.g., for determining the level of TSLP and / or TSLP in appropriate physiological samples, for diagnostic methods, for protein imaging, etc.). In a given embodiment, an antibody that is specific to TSLP or its derivatives, fragments, analogs, or homologues and comprises an antigen-binding domain derived from the antibody is used as a pharmaceutically active compound (hereinafter referred to as a "therapeutic agent").

[0160] In another embodiment, TSLP peptides can be isolated using antibodies specific to TSLP via standard techniques such as immunoaffinity, chromatography, or immunoprecipitation. Antibodies (or fragments thereof) targeting TSLP proteins can be used to detect proteins in biological samples. In some embodiments, the detection of TSLP in biological samples is part of a clinical testing process, for example, to determine the efficacy of a given treatment regimen. Conjugating (i.e., physically linking) an antibody to a detectable substance can facilitate detection. Examples of detectable substances include various enzymes, cofactors, 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 cofactor complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, luciferin, luciferin isothiocyanate, rhodamine, dichlorotriazine fluorescein, dansyl chloride, or phycoerythrin; one example of a luminescent material is luminol; examples of bioluminescent materials include luciferase, luciferin, and jellyfish protein; and examples of suitable radioactive materials include... 125 I, 131 I, 35 S or 3 H.

[0161] In another embodiment, the antibody according to this disclosure can be used as a reagent to detect the presence of TSLP or its protein fragments in a sample. In some embodiments, the antibody contains a detectable label. The antibody is a polyclonal antibody, or more preferably a monoclonal antibody. A complete antibody or fragment thereof (e.g., Fab, scFv, or F(ab')2) is used. The term "label" with respect to the antibody is intended to include both direct labeling of the antibody by conjugation (i.e., physical linking) to the antibody and indirect labeling of the antibody by reaction with another directly labeled reagent. Examples of indirect labeling include detecting a first antibody using a fluorescently labeled second antibody, and end-labeling an antibody with biotin to enable detection with fluorescently labeled streptavidin. 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 the subject. Thus, the term "biological sample" as used includes blood and fractions or components of blood, including serum, plasma, or lymph. In other words, the detection methods of the embodiments described can be used to detect analytes mRNA, protein, or genomic DNA in biological samples in vitro and in vivo. For example, in vitro detection techniques for analyte mRNA include Norhtern hybridization and in situ hybridization. In vitro detection techniques for analyte proteins 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, JRCrowther (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. Furthermore, in vivo detection techniques for analyte proteins involve introducing labeled anti-analyte protein antibodies into the subject. For example, antibodies can be labeled with radioactive markers, and then the presence and location of the radiolabel in the subject's body can be detected using standard imaging techniques.

[0162] The antibodies described herein and their derivatives, fragments, analogs, and homologues may be incorporated into pharmaceutical compositions suitable for administration. The principles and considerations involved in preparing such compositions, as well as guidelines for selecting components, are well known in the art, for example, see Remington's Pharmaceutical Sciences: The Science and Practice of Pharmacy, 19th edition (edited by Alfonso R. Gennaro et al.), Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, and Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances in Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

[0163] Such compositions typically contain an antibody and a pharmaceutically acceptable carrier. When using antibody fragments, the minimally inhibitory fragment that specifically binds to the target protein binding domain 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 chemically synthesized and / or produced by recombinant DNA technology (see, for example, Marasco et al., Proc. Natl. Acad. Sci. USA, 90:7889-7893 (1993)).

[0164] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delayers compatible with drug administration. Suitable pharmaceutically acceptable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, the standard bibliography in the art, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, glucose solution, and 5% human serum albumin. Liposomes and non-aqueous carriers, such as immobilized oils, may also be used. The use of such media and reagents for pharmaceutically active substances is well known in the art. The use of any conventional media or reagent in the composition is contemplated, except that it may be incompatible with the antibody.

[0165] The pharmaceutical composition of the embodiments described herein is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., local), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous administration may include the following components: sterile diluents for injection such as water, saline solutions, fixative oils, polyethylene glycols, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, or phosphates; and osmotic pressure adjusting agents such as sodium chloride or dextran. pH may be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral formulations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0166] Suitable pharmaceutical compositions for injection include sterile aqueous solutions (in this case, water-soluble) or dispersions, as well as sterile powders for immediate preparation of sterile injections or dispersions. For intravenous administration, suitable pharmaceutically acceptable carriers 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 should be fluid enough for easy injection. It must be stable under manufacturing and storage conditions and must be resistant to contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by using a coating such as lecithin to maintain the desired particle size in the dispersion case, and by using surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferred to include isotonic agents in the composition, such as sugars, polyols (such as mannitol, sorbitol), and sodium chloride. Prolonged absorption of the injectable composition can be achieved by including absorbance-retarding agents such as aluminum monostearate and gelatin in the composition.

[0167] As needed, a sterile injectable solution can be prepared by incorporating the antibody in the desired amount into a suitable solvent having one or a combination of the components listed above (as required), followed by filtration sterilization. Generally, a dispersion is prepared by incorporating the antibody into a sterile carrier containing an alkaline dispersion medium and any other desired components listed above. For sterile powders used to prepare sterile injectable solutions, the preparation method involves obtaining a powder containing the active ingredient and any other desired components derived from a sterile filtrate solution of the aforementioned components through vacuum drying and freeze-drying.

[0168] For inhalation administration, the compound is delivered in the form of an aerosol spray from a pressurized container or dispenser or nebulizer containing a suitable propellant such as carbon dioxide.

[0169] Systemic administration can also be achieved via mucosal or transdermal routes. For mucosal or transdermal administration, a permeabilizing agent suitable for the permeability barrier is used in the formulation. Such permeabilizing agents are generally known in the art and include detergents, bile salts, and fusidic acid derivatives, such as those used for mucosal administration. Mucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, one or more of the antibodies can be formulated into ointments, ointments, gels, or creams as commonly known in the art.

[0170] The compound can also be prepared in the form of suppositories (e.g., having a conventional suppository base, such as cocoa butter or other glycerides) or retention enemas for rectal delivery.

[0171] In one embodiment, the antibody can be prepared using a carrier that prevents it from being rapidly eliminated by the body, such as a sustained-release / controlled-release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene-vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art.

[0172] Particularly advantageous is the formulation of parenteral compositions in unit dosage form for ease of administration and dosage consistency. As used herein, unit dosage form refers to physically separable units suitable as unit doses for use in the subject to be treated; each unit contains a predetermined amount of one or more of the antibodies calculated to bind with the desired drug carrier to produce the desired therapeutic effect. The specifications of the unit dosage form of the embodiments are indicated by and directly depend on the unique characteristics of the antibody and the specific therapeutic effect to be achieved, and the inherent limitations in the field of formulation of such antibodies for the individual being treated.

[0173] The pharmaceutical composition may be placed in a container, package, or dispenser together with the instructions for use.

[0174] The formulations described herein may also contain more than one of the antibodies described, depending on the specific condition to be treated, preferably those with complementary activities that do not negatively affect each other. Alternatively or in addition, the composition may, for example, contain agents that enhance its function, such as cytotoxic agents, cytokines, chemotherapeutic agents, or growth inhibitors. Such molecules are appropriately combined in amounts effective for the intended purpose. For example, they may be combined in a kit or in use.

[0175] In one embodiment, one or more of the antibodies may be administered in combination therapy, i.e., in combination with other agents, such as therapeutic agents (which can be used to treat pathological conditions or disorders, such as various forms of cancer, autoimmune disorders, and inflammatory diseases). The term "combination" herein refers to the administration of the agents substantially synchronously, simultaneously, or sequentially. If administered sequentially, the first of the two compounds is still preferably detected at an effective concentration at the therapeutic site when the second compound is initiated. In one case, "combination" may also mean that the reagent kit simultaneously contains the antibodies of this disclosure and other therapeutic agents.

[0176] For example, combination therapy may comprise one or more antibodies described herein 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 growth inhibitors, as detailed below). Such combination therapies can advantageously utilize lower doses of the administered therapeutic agent, thus avoiding the potential toxicities or complications associated with various monotherapy approaches.

[0177] For the purpose of clarity and concise description, the features are described herein as part of some identical or separate embodiments; however, it will be understood that the scope of this disclosure may include some embodiments having a combination of all or some of the features described.

[0178] Example

[0179] Example 1: Preparation of TSLP and TSLPR recombinant proteins

[0180] The cDNA of human TSLP (Uniprot: Q969D9, SEQ ID NO: 1) and cynomolgus monkey TSLP (Uniprot: A0A2K5TXV0, SEQ ID NO: 2) was synthesized and cloned into the eukaryotic expression vector pCMV3 (purchased from Beijing Sinobiological, catalog number CG90911-UT). The signal peptide MDMRVPAQLLGLLLLWLRGARS was inserted at the N-terminus. At the same time, a tag containing 6 histidine residues was fused to the C-terminus of TSLP to obtain the plasmids pSect-hTSLP-cHis and pSect-cyTSLP-cHis. HEK293.6E cells (ATCC CRL-1573) were transfected with these plasmids, and the cell supernatant was collected. However, the recombinant protein of the corresponding length was not obtained by nickel column affinity chromatography. The full-length TSLP fragment was then cloned into the prokaryotic expression plasmid pET-30a. This plasmid was then transformed into BL21 E. coli, 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. Results are as follows... Figure 1 As shown, recombinant human TSLP and cynomolgus monkey TSLP proteins were purified.

[0181] Human TSLPR cDNA (Uniprot: Q9HC73, SEQ ID NO: 3, purchased from Beijing Sinobiological, 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 a human IgG1 Fc(knob) fragment was fused to the C-terminus to obtain the pHC1-TSLPR-hFc-knob plasmid. Simultaneously, a plasmid containing only the human IgG1 Fc(hole) fragment, pHC1-hFc-hole, was constructed. This eukaryotic expression plasmid was then mixed with the pHC1-TSLPR-hFc-knob plasmid and co-transfected into HEK293.6E eukaryotic cells to obtain the human TSLPR-Fc fusion protein. Similarly, the cDNA (Uniprot: G8F663, SEQ ID NO: 4) of the synthesized cynomolgus monkey TSLPR was amplified by PCR to obtain the gene fragment encoding the extracellular domain (Gln23-Lys231) of the cynomolgus monkey TSLPR. This gene fragment was cloned into a eukaryotic expression plasmid containing a human IgG1 Fc(knob) fragment, fusing the cynomolgus monkey TSLPR extracellular domain with the N-terminus of the human IgG1 Fc(knob). Then, this eukaryotic expression plasmid was mixed with a plasmid containing only the human IgG1 Fc(hole) fragment and co-transfected into HEK293.6E eukaryotic cells to obtain the cynomolgus monkey TSLPR-Fc fusion protein. After transfection of HEK293.6E cells, the cells were cultured for 5-7 days, and the cell supernatant was collected. The recombinant proteins of the human TSLPR (SEQ ID NO: 5) and cynomolgus monkey TSLPR (SEQ ID NO: 6) extracellular domains were purified by Protein A affinity chromatography. The results are as follows: Figure 2 As shown.

[0182] The binding of recombinant human TSLP protein to recombinant human TSLPR extracellular domain protein was detected by ELISA. The results showed that its EC50... 50 =0.11 nM, indicating that recombinant human TSLP binds to human TSLPR with high affinity. Figure 3 ).

[0183] Example 2: Construction of TSLPR-stabilized cells

[0184] 1) Construction of Ba / F3-hTSLPR stable cell line

[0185] 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 packaged into the virus using a Lenti-Pac HIV lentiviral packaging kit (Guangzhou Funeng Gene Co., Ltd.), and the virus was transfected into Ba / F3 cells. Puromycin was added for resistance selection to obtain the cell line Ba / F3-hTSLPR, which stably expresses human TSLPR. Figure 4 The results showed that by detecting the binding of biotinylated human TSLP protein to TSLPR on the cell surface, the constructed cells expressed human TSLPR, and their EC50 was confirmed. 50 =0.22nM.

[0186] 2) Construction of a stable Ba / F3-hTSLPR-IL7Rα cell line

[0187] IL-7Rα participates in the signal transduction of the TSLP receptor complex. When TSLP binds to TSLPR, it forms a heterodimeric receptor complex with IL-7Rα. JAK1 in the intracellular domain of IL-7Rα and JAK2 in the intracellular domain of TSLPR are phosphorylated, activating downstream signaling molecules and thus transducing activation signals into the cell. The IRES gene (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 plasmid was transfected into Ba / F3 cells to construct stable cell lines with a complete human TSLP signal transduction pathway.

[0188] 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. Using a Lenti-Pac HIV lentiviral packaging kit, human TSLPR and IL-7Rα co-expression plasmids were packaged into lentiviruses. Ba / F3 cells were transduced with culture supernatant containing the recombinant virus. Puromycin was added for resistance selection to obtain the cell line Ba / F3-hTSLPR-IL7Rα, which stably expresses human TSLPR and IL-7Rα. Cell proliferation activity assays showed that the constructed Ba / F3-hTSLPR-IL7Rα cell line had a dose-dependent proliferation response to human TSLP, and its EC50 = 0.22 nM. Figure 5 ).

[0189] 3) Construction of Ba / F3-hTSLPR-IL7Rα-STAT5 luc reporter gene cell line

[0190] In the Ba / F3-hTSLPR-IL7Rα stable cell line, the STAT5-luc reporter gene was introduced 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. Simultaneously, the hygro resistance gene was cloned into the vector and replaced the Puro resistance gene, yielding pGL4.22-luc2p STAT5 RE-hygro. Next, 10 μg of pGL4.22-luc2p STAT5 RE-hygro plasmid was mixed with 5 × 10⁻⁶ cells... 6 Ba / F3-hTSLPR-IL7Rα stable transfected cells were mixed and then electrotransfected at 300V and 950μF. 48-72 hours after transfection, 200μg / mL hygromycin B solution was added, and the selection medium was changed every 3-5 days. After a period of pressure selection, stable transfected cells formed clones. Single clones obtained from limiting dilutions were stimulated with different concentrations of TSLP for 6 hours, after which fluorescence signals could be detected. Figure 6 The results showed that the fluorescence signal gradually increased with increasing TSLP concentration, indicating that TSLP can activate the STAT5 signaling pathway in stable cells Ba / F3-hTSLPR-IL7Rα-STAT5 luc, and the EC50 of stable cells Ba / F3-hTSLPR-IL7Rα-STAT5 luc was 0.22 nM.

[0191] Example 3: Animal Immunization

[0192] Six-week-old female Balb / C mice were immunized, with each mouse receiving 50 μg of antigen per immunization. The antigen was administered alternately with human and cynomolgus monkey TSLP. An equal volume of antigen and Freund's adjuvant was mixed and administered subcutaneously every two weeks. After four immunizations, blood was collected from the tail, and the serum titer and the inhibitory effect of the serum on the binding of TSLP and TSLPR were detected by ELISA. Alternatively, after the initial immunization, booster immunizations were performed every week using a gene gun. Human TSLP expression plasmid mixed with gold powder was bombarded on the bare skin of the mouse abdomen at 400 psi, for a total of nine immunizations.

[0193] Example 4: Antibody Screening

[0194] 1) Fab phage library

[0195] Animals immunized in Example 3 were given a pulse immunization three weeks after the last immunization, via tail vein injection of 10 μg / 100 μL / animal human TSLP recombinant protein. Four days later, mouse lymph nodes and spleen cells were collected, and RNA was extracted from the cells using the TRNzol lysis method. Single-stranded cDNA was then synthesized via reverse transcription, and this cDNA was used as a template to amplify the variable region sequence of the antibody. A TSLP immune library with a capacity >5 × 10⁻⁶ was constructed on the Fab antibody fragment phage display platform. 9 Ninety-six monoclonal antibodies were randomly selected and induced to express the antibody. Western blot analysis showed that the expression rates of both the light and heavy chains were higher than 95%, and sequencing revealed good sequence diversity. Human TSLP recombinant protein or cynomolgus monkey TSLP recombinant protein was coated onto Maxisorp immunotubes for screening of the TSLP antibody library. After two rounds of screening, several positive clones were obtained that specifically recognized TSLP and efficiently blocked TSLP-TSLPR binding. The monoclonal antibody Fab was expressed in TG1 competent cells to further verify the binding of TSLP to human and cynomolgus monkey TSLP and the blocking of TSLP / TSLPR. Six clones with high affinity were obtained, capable of simultaneously binding to both human and cynomolgus monkey TSLP and efficiently blocking TSLP-TSLPR binding (results are shown in Table 1).

[0196] Table 1 ELISA screening of Fab phage libraries

[0197]

[0198] 2) Mouse hybridoma

[0199] Mice were given a pulse immunization via tail vein injection of 10 μg / 100 μL / mouse human TSLP recombinant protein. Four days later, lymph nodes and spleens were collected from mice and ground in DMEM to obtain a B cell suspension. An appropriate amount of the B cell suspension was mixed with SP2 / 0 and cell fusion was performed using an electrofusion apparatus. The fused cells were then cultured in DMEM complete medium containing HAT at 5% CO2 and 37°C.

[0200] 3) Screening of hybridomas

[0201] Enzyme-linked immunosorbent assay (ELISA) screening for binding with human TSLP

[0202] 1 μg / mL of recombinant human TSLP protein was coated onto a 96-well microplate using carbonate buffer and incubated overnight at 4°C. The plate was washed three times with PBS, and each well was blocked with 200 μL of PBS containing 2% skim milk for 1 hour. After one wash with PBS, 100 μL of hybridoma cell supernatant or phage supernatant was added to each well, and the plate was incubated at room temperature for 60 minutes. The plate was then washed three times each with PBST (PBS + 0.05% Tween-20) and PBS, and each well was incubated with 100 μL of HRP-labeled anti-human IgG Fc secondary antibody for 60 minutes at room temperature. After three washes with PBST and PBS, 100 μL of TMB substrate was added to each well, and the plate was developed at 37°C for 10 minutes. The reaction was terminated with 50 μL of 2M sulfuric acid solution per well, and the absorbance was read at 450 nm.

[0203] Combining hybridoma cell ELISA screening with cynomolgus monkey TSLP

[0204] 1 μg / mL of cynomolgus monkey TSLP recombinant protein was coated onto a 96-well microplate using carbonate buffer and incubated overnight at 4°C. The plate was washed three times with PBS, and each well was blocked with 200 μL of PBS containing 2% skim milk for 1 hour. After one wash with PBS, 100 μL of hybridoma cell supernatant or phage supernatant was added to each well, and the plate was incubated at room temperature for 60 minutes. After washing three times each with PBST and PBS, 100 μL of HRP-labeled anti-human IgG Fc secondary antibody was added to each well, and the plate was 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 the plate was developed at 37°C for 10 minutes. The reaction was terminated with 50 μL of 2M sulfuric acid solution per well, and the absorbance was read at 450 nm.

[0205] ELISA screening for the blocking of human TSLP and human TSLPR

[0206] 1 μg / mL human TSLPR recombinant protein was coated onto a 96-well microplate using carbonate buffer and incubated overnight at 4°C. The plate was washed three times with PBS, and each well was blocked with 200 μL of PBS containing 2% skim milk for 1 hour. Hybridoma cell supernatant or phage supernatant was then incubated with 60 ng / mL biotinylated human TSLP protein in a blocked U-shaped 96-well plate at room temperature for 30 minutes. The plate was washed three times with PBS, and each well was incubated with 100 μL of the incubated mixture for 1 hour 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 the plate was incubated at room temperature in the dark for 30 minutes. After washing three times with PBST and PBS, 100 μL of TMB substrate was added to each well, and the plate was developed at 37°C for 10 minutes. The reaction was terminated with 50 μL of 2M sulfuric acid solution per well, and the absorbance was read at 450 nm.

[0207] ELISA screening for the blocking of cynomolgus monkey TSLP and cynomolgus monkey TSLPR

[0208] 1 μg / mL of cynomolgus monkey TSLPR recombinant protein was coated onto a 96-well microplate using carbonate buffer and incubated overnight at 4°C. The plates were washed three times with PBS, and each well was blocked with 200 μL of PBS containing 2% skim milk for 1 hour. Hybridoma cell supernatant or phage supernatant was then incubated with 60 ng / mL biotinylated cynomolgus monkey TSLP protein in a blocked U-shaped 96-well plate at room temperature for 30 minutes. The plates were washed three times with PBS, and each well was incubated with 100 μL of the pre-incubated mixture for 1 hour at room temperature. After washing three times with PBST and PBS, each well was incubated with 100 μL of SA-HRP at room temperature in the dark for 30 minutes. After washing three times with PBST and PBS, each well was incubated with 100 μL of TMB substrate at 37°C for 10 minutes. The reaction was terminated with 50 μL of 2M sulfuric acid solution per well, and the absorbance was read at 450 nm.

[0209] Cellular level blockade

[0210] Ba / F3-hTSLPR cells were added to 96-well U-shaped plates at a density of 5 × 10⁶ m². 4 / well. The antibody was incubated with biotinylated human TSLP protein at 4°C for 30 minutes and then added to cells. After incubation at 4°C for 1 hour, streptavidin-conjugated phycoerythrin fluorescein (SA-PE) was added and the cells were incubated at 4°C for 45 minutes. The blocking effect of the antibody at the cellular level was detected by flow cytometry.

[0211] A total of 712 hybridoma clones that bound human TSLP were screened by ELISA. Among them, 52 hybridoma clones could block the binding of human TSLP and human TSLPR in both ELISA and cell-level assays. Six positive clones bound to cynomolgus monkey TSLP and blocked the binding of cynomolgus monkey TSLP and cynomolgus monkey TSLPR (results are shown in Table 2).

[0212] Table 2 ELISA screening of hybridoma supernatant

[0213]

[0214] In Table 2, ND: Not detected.

[0215] Example 5: Obtaining the antibody sequence

[0216] Based on the results of hybridoma screening, positive monoclonal cells were centrifuged at 1000 rpm, collected, and total RNA was extracted with Trizol. Using this RNA as a template, first-strand cDNA was synthesized, and the variable region DNA sequence corresponding to the hybridoma cells was amplified using the first-strand cDNA as a subsequent template. In a 50 μL reaction system, 1 μL of cDNA, 5 μL of 10×PCR buffer, 1 μL each of forward and reverse primers, 1 μL of dNTP, 1 μL of 25 mmol MgCl2, 39 μL of H2O, and 1 μL of Taq enzyme were added. The mixture was pre-denatured at 95℃ for 10 minutes, followed by temperature cycling for PCR amplification. The reaction conditions were: denaturation at 94℃ for 1 minute, annealing at 58℃ for 1 minute, extension at 72℃ for 15 seconds, for a total of 30 cycles, followed by incubation at 72℃ for 10 minutes. Based on the phage 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 were as follows:

[0217] Cloner: 71G4 SEQ ID Nos: 7-16

[0218] Heavy chain VH

[0219]

[0220] Nucleic acid sequence

[0221]

[0222] Light Chain VK

[0223]

[0224] Nucleic acid sequence

[0225]

[0226] Clone: ​​79D6 SEQ ID Nos: 17-26

[0227] Heavy chain VH

[0228]

[0229] Nucleic acid sequence

[0230]

[0231] Light Chain VK

[0232]

[0233] Nucleic acid sequence

[0234]

[0235] Clone: ​​76A8 SEQ ID Nos: 27-36

[0236] Heavy chain VH

[0237]

[0238] Nucleic acid sequence

[0239]

[0240] Light Chain VK

[0241]

[0242] Nucleic acid sequence

[0243]

[0244] Clone: ​​80D12 SEQ ID Nos: 37-46

[0245] Heavy chain VH

[0246]

[0247] Nucleic acid sequence

[0248]

[0249] Light Chain VK

[0250]

[0251] Nucleic acid sequence

[0252]

[0253] Clone: ​​80E11 SEQ ID Nos: 47-56

[0254] Heavy chain VH

[0255]

[0256] Nucleic acid sequence

[0257]

[0258] Light Chain VK

[0259]

[0260] Nucleic acid sequence

[0261]

[0262] Clone: ​​80B12 SEQ ID Nos: 57-66

[0263] Heavy chain VH

[0264]

[0265] Nucleic acid sequence

[0266]

[0267] Light Chain VK

[0268]

[0269] Nucleic acid sequence

[0270]

[0271] Clone: ​​39G5-H8 SEQ ID Nos: 67-76

[0272] Heavy chain VH

[0273]

[0274] Nucleic acid sequence

[0275]

[0276] Light Chain VK

[0277]

[0278] Nucleic acid sequence

[0279]

[0280] Example 6: Expression of anti-TSLP chimeric antibody

[0281] The variable region sequence fragments of the heavy and light chains obtained in Example 5 were amplified by PCR. The variable region of the heavy chain was cloned into a vector containing the constant region of the human heavy chain to express the complete IgG1 heavy chain in mammalian cells. Similarly, the variable region of the light chain was cloned into a vector containing the constant region of the human light chain to express the complete kappa light chain in mammalian cells. After correct sequencing, the cells were transfected into HEK293.6E mammalian cells. IgG1 was expressed and secreted into the culture medium. The supernatants were collected, filtered, and purified. IgG was purified by Protein A chromatography. The eluted protein was concentrated by ultrafiltration, and the concentration of IgG was determined by spectrophotometry. The purity of IgG was analyzed by SDS-PAGE. Chimeric antibodies of 39G5-H8, 71G4, 79D6, 80B12, 80D12, and 80E11 were obtained.

[0282] Example 7: Affinity determination of chimeric antibodies

[0283] 1) Binding of antibody to human TSLP

[0284] 1 μg / mL of recombinant human TSLP protein was coated onto a 96-well microplate using carbonate buffer and incubated overnight at 4°C. The plate was washed three times with PBS, and each well was blocked with 200 μL of PBS containing 2% skim milk for 1 hour. After one wash with PBS, 100 μL of anti-TSLP antibody was added to each well, and the plate was incubated at room temperature for 60 minutes. After washing three times each with PBST and PBS, 100 μL of HRP-labeled anti-human IgG Fc secondary antibody was added to each well, and the plate was 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 the plate was developed at 37°C for 10 minutes. The reaction was terminated with 50 μL of 2M sulfuric acid solution per well, and the absorbance was read at 450 nm. Figure 7 As shown in -a and Table 3, all chimeric antibodies obtained in Example 6 can bind to human TSLP.

[0285] 2) Binding of antibodies to TSLP in cynomolgus monkeys

[0286] 1 μg / mL of cynomolgus monkey TSLP recombinant protein was coated onto a 96-well microplate using carbonate buffer and incubated overnight at 4°C. The plate was washed three times with PBS, and each well was blocked with 200 μL of PBS containing 2% skim milk for 1 hour. After one wash with PBS, 100 μL of anti-TSLP antibody was added to each well, and the plate was incubated at room temperature for 60 minutes. After washing three times each with PBST and PBS, 100 μL of HRP-labeled anti-human IgG Fc secondary antibody was added to each well, and the plate was 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 the plate was developed at 37°C for 10 minutes. The reaction was terminated with 50 μL of 2M sulfuric acid solution per well, and the absorbance was read at 450 nm. Figure 7 -b and Table 3 show that all the chimeric antibodies obtained in Example 6 can bind to cynomolgus monkey TSLP.

[0287] Table 3. ELISA detection of chimeric antibodies

[0288]

[0289] 3) BIAcore Affinity Measurement

[0290] The kinetic binding activity of anti-TSLP antibody to human TSLP was measured using a BIAcore T200 system. 2 μg / ml of anti-TSLP antibody was immobilized onto the Protein A sensor chip, and a run was performed at a flow rate of 10 μL / min for 40 seconds using 1×HBS-EP buffer. Different concentrations of human TSLP, ranging from 0 to 21.7 nM, were 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 with 10 mM glycine (pH 2.0) regeneration buffer at a flow rate of 30 μL / min.

[0291] The obtained data were fitted to the Langmuir 1:1 Kinetic theoretical model, and the binding rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant KD were analyzed (BIAevaluation software). Table 4 shows that each chimeric antibody binds to human TSLP with high affinity.

[0292] Table 4. Affinity of chimeric antibodies

[0293]

[0294] Example 8: Blocking at the ELISA Level

[0295] 1) Blocking of antibody binding to human TSLP and human TSLPR

[0296] 1 μg / mL human TSLPR recombinant protein was coated onto a 96-well microplate using carbonate buffer and incubated overnight at 4°C. The plate was washed three times with PBS, and each well was blocked with 200 μL of PBS containing 2% skim milk for 1 hour. Anti-TSLP antibody and 60 ng / mL biotinylated human TSLP protein were incubated in a blocked U-shaped 96-well plate at room temperature for 30 minutes. The plate was washed three times with PBS, and each well was incubated with 100 μL of the pre-incubated mixture for 1 hour at room temperature. After washing three times with PBST and PBS, each well was incubated with 100 μL of SA-HRP at room temperature in the dark for 30 minutes. After washing three times with PBST and PBS, each well was incubated with 100 μL of TMB substrate at 37°C for 10 minutes. The reaction was terminated with 50 μL of 2M sulfuric acid solution per well, and the absorbance was read at 450 nm. Figure 8 -a and Table 3 show that all chimeric antibodies can block the binding of human TSLP and human TSLPR.

[0297] 2) Blocking of antibody binding to cynomolgus monkey TSLP and cynomolgus monkey TSLPR

[0298] 1 μg / mL of cynomolgus monkey TSLPR recombinant protein was coated onto a 96-well microplate using carbonate buffer and incubated overnight at 4°C. The plate was washed three times with PBS, and each well was blocked with 200 μL of PBS containing 2% skim milk for 1 hour. Anti-TSLP antibody and 60 ng / mL biotinylated cynomolgus monkey TSLP protein were incubated in a blocked U-shaped 96-well plate at room temperature for 30 minutes. The plate was washed three times with PBS, and each well was incubated with 100 μL of the pre-incubated mixture for 1 hour at room temperature. After washing three times with PBST and PBS, each well was incubated with 100 μL of SA-HRP at room temperature in the dark for 30 minutes. After washing three times with PBST and PBS, each well was incubated with 100 μL of TMB substrate at 37°C for 10 minutes. The reaction was terminated with 50 μL of 2M sulfuric acid solution per well, and the absorbance was read at 450 nm. Figure 8 -b and Table 3 show that each chimeric antibody can block the binding of cynomolgus monkey TSLP and cynomolgus monkey TSLPR.

[0299] Example 9: Blockage at the cellular level

[0300] Ba / F3-hTSLPR-IL7Rα cells were washed three times with PBS, centrifuged at 300g for 3 minutes, and blocked with 3% BSA / PBS for 30 minutes. After resuspending the cells, they were added to 96-well U-plates at 5×10⁻⁶ cells / well. 4 / well. The antibody was incubated with 60 ng / mL biotinylated human TSLP protein at room temperature for 10 minutes, then added to cells and incubated at 4°C for 45 minutes. After washing twice with 0.5% BSA / PBS, SA-PE was added and incubated at 4°C for 45 minutes. After washing twice with 0.5% BSA / PBS, PI dye was added and incubated at 4°C for 10 minutes. After washing twice with 0.5% BSA / PBS, the cells were resuspended in PBS, and the blocking effect of the antibody at the cellular level was detected by flow cytometry. Figure 9 The results showed that all chimeric antibodies could block the binding of TSLP to TSLPR on the cell surface.

[0301] Example 10: Inhibition of TSLP-stimulated Ba / F3-hTSLPR-IL7Rα cell proliferation by chimeric antibody

[0302] The assay of TSLP antibody inhibition of TSLP-induced Ba / F3-hTSLPR-IL7Rα cell proliferation was performed in RPMI-1640, 10% fetal bovine serum.

[0303] Ba / F3-hTSLPR-IL7Rα cells were added to 96-well cell culture plates one day in advance at a concentration of 2×10⁻⁶. 4CellTiter-Glo was added to each well. 60 μL of diluted anti-TSLP antibody was pre-incubated with 60 μL of 5 ng / mL human TSLP for 15 minutes at room temperature. 100 μL of the pre-incubated antibody-cytokine mixture was added to each well of a 96-well cell culture plate. The 96-well plate was incubated at 37°C with 5% CO2 for 72 hours. After the incubation period, CellTiter-Glo was added to each well and reacted for 10 minutes. The luminescence value was read from the black 96-well plate. Results are as follows: Figure 10 As shown in Table 5, all antibodies can inhibit cell proliferation, with 71G4 showing the most significant inhibition.

[0304] Table 5. IC50 of chimeric antibodies against cell proliferation

[0305]

[0306]

[0307] Example 11: Inhibition of TSLP-activated STAT5 signaling by chimeric antibodies

[0308] Ba / F3-hTSLPR-IL7Rα-STAT5 luc cells were starved overnight, and the next day, they were injected with 5×10⁻⁶ cells. 4 Seed 50 μL of human TSLP per well in a 96-well plate; pre-dilute human TSLP and incubate at 37°C for 30 minutes; add 50 μL of ligand / antibody mixture to the plate and mix well, then incubate at 37°C for 6 hours; add 100 μL of fluorescence detection reagent; read the fluorescence value of each well using a microplate reader. Results are as follows: Figure 11 As shown in Table 6, all antibodies can inhibit the expression of reporter genes, with 71G4 showing the most significant inhibition.

[0309] Table 6. IC50 of chimeric antibodies against STAT5 reporter gene inhibition

[0310] IC50(nM) 76A8 9.78 79D6 9.61 71G4 1.67 80B12 5.79 80E11 13.57 80D12 3.84 39G5 3.33

[0311] Example 12, Antibody Humanization

[0312] TSLP antibodies 39G5, 71G4, 76A8, 79D6, 80B12, 80D12 and 80E11 are derived from the same or similar mouse germline genes. The mouse antibodies 39G5 and 71G4 with the strongest neutralizing activity were selected for humanization of the variable region sequence.

[0313] First, the mouse antibody 39G5 sequence was compared with the human antibody germline sequence to identify human germline light chain genes VK_1_39, IGKJ1*01 and human germline heavy chain genes VH-1-69, IGHJ4*01 with good homology, identical key amino acid sequences maintaining the upper hydrophobic core of the antibody structure, and high frequency of occurrence in humans. CDR transplantation of these genes into the mouse antibody was then performed. The mouse antibody 71G4 light and heavy chains are derived from the same source as 39G5, and CDR3 has only one or two different amino acid residues of the same type. Therefore, CDR1 and CDR2 of the 71G4 heavy chain were substituted into the humanized 39G5 heavy chain, respectively. Simultaneously, the 71G4 humanized light chain or the 39G5 humanized light chain was paired with the 39G5 humanized heavy chain for expression, optimizing the region surrounding the core binding site to improve affinity and molecular surface charge distribution. Subsequently, computer-aided homology modeling was performed to analyze the framework amino acid sequence, molecular surface charge, and hydrophobic region distribution in and around the CDR region. This identified different heavy and light chain derivatives, resulting in 27 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. The heavy and light chains of these 27 humanized variants are shown in Table 7.

[0314] Table 7 Humanization design of TSLP antibodies

[0315]

[0316]

[0317] After synthesizing the full sequences of the light and heavy chain derivatives, they were cloned into vectors containing the constant region Ckappa of the antibody kappa chain or the constant region CH1-CH3 of human IgG1. The light and heavy chain derivative plasmids were then combined and transfected into HEK293.6E cells. After expression for 5-6 days, the supernatant was collected and purified using Protein A column purification.

[0318] The variable region sequences of the heavy / light chain of the humanized antibody are as follows:

[0319] h39G5VH v1:SEQ ID Nos:77-81

[0320]

[0321] Nucleic acid sequence

[0322]

[0323] h39G5VH v2:SEQ ID Nos:82-86

[0324]

[0325] Nucleic acid sequence

[0326]

[0327] h39G5VH v3:SEQ ID Nos:87-91

[0328]

[0329] Nucleic acid sequence

[0330]

[0331] h39G5VH v4:SEQ ID Nos:92-96

[0332]

[0333] Nucleic acid sequence

[0334]

[0335] h39G5VH v5:SEQ ID Nos:97-101

[0336]

[0337] Nucleic acid sequence

[0338]

[0339] h39G5VH v6:SEQ ID Nos:102-106

[0340]

[0341] Nucleic acid sequence

[0342]

[0343] h39G5VH v7:SEQ ID Nos:107-111

[0344]

[0345] Nucleic acid sequence

[0346]

[0347] h39G5VH v8:SEQ ID Nos:112-116

[0348]

[0349] Nucleic acid sequence

[0350]

[0351] h39G5VH v9:SEQ ID Nos:117-121

[0352]

[0353] Nucleic acid sequence

[0354]

[0355] h39G5VH v10:SEQ ID Nos:122-126

[0356]

[0357] Nucleic acid sequence

[0358]

[0359] h39G5VH v11:SEQ ID Nos:127-131

[0360]

[0361] Nucleic acid sequence

[0362]

[0363] h39G5VH v12:SEQ ID Nos:132-136

[0364]

[0365] Nucleic acid sequence

[0366]

[0367] h39G5VH v13:SEQ ID Nos:137-141

[0368]

[0369] Nucleic acid sequence

[0370]

[0371] h39G5VH v14:SEQ ID Nos:142-146

[0372]

[0373] Nucleic acid sequence

[0374]

[0375] h39G5VH v15:SEQ ID Nos:147-151

[0376]

[0377] Nucleic acid sequence

[0378]

[0379] h71G4VH v1:SEQ ID Nos:152-156

[0380]

[0381] Nucleic acid sequence

[0382]

[0383] h71G4VH v2:SEQ ID Nos:157-161

[0384]

[0385] Nucleic acid sequence

[0386]

[0387] h71G4VH v3:SEQ ID Nos:162-166

[0388]

[0389] Nucleic acid sequence

[0390]

[0391] h39G5VK v1:SEQ ID Nos:167-171

[0392]

[0393] Nucleic acid sequence

[0394]

[0395] h71G4VK v1:SEQ ID Nos:172-176

[0396]

[0397] Nucleic acid sequence

[0398]

[0399] Example 13: Affinity determination of humanized antibodies

[0400] 1) Binding of humanized antibodies to human TSLP

[0401] 1 μg / mL of recombinant human TSLP protein was coated onto a 96-well microplate using carbonate buffer and incubated overnight at 4°C. The plate was washed three times with PBS, and each well was blocked with 200 μL of PBS containing 2% skim milk for 1 hour. After one wash with PBS, 100 μL of anti-TSLP antibody was added to each well, and the plate was incubated at room temperature for 60 minutes. After washing three times each with PBST and PBS, 100 μL of HRP-labeled anti-human IgG Fc secondary antibody was added to each well, and the plate was 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 the plate was developed at 37°C for 10 minutes. The reaction was terminated with 50 μL of 2M sulfuric acid solution per well, and the absorbance was read at 450 nm. Figure 12 As shown in -a, c, e and Table 8, all humanized antibodies obtained in Example 12 bind to human TSLP, with Ab20 exhibiting the strongest binding.

[0402] 2) Binding of humanized antibodies to cynomolgus monkey TSLP

[0403] 1 μg / mL of cynomolgus monkey TSLP recombinant protein was coated onto a 96-well microplate using carbonate buffer and incubated overnight at 4°C. The plate was washed three times with PBS, and each well was blocked with 200 μL of PBS containing 2% skim milk for 1 hour. After one wash with PBS, 100 μL of anti-TSLP antibody was added to each well, and the plate was incubated at room temperature for 60 minutes. After washing three times each with PBST and PBS, 100 μL of HRP-labeled anti-human IgG Fc secondary antibody was added to each well, and the plate was 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 the plate was developed at 37°C for 10 minutes. The reaction was terminated with 50 μL of 2M sulfuric acid solution per well, and the absorbance was read at 450 nm. Figure 12 -b, d, f and Table 8 show that, except for Ab14 and Ab15, all other antibodies bind to cynomolgus monkey TSLP, with Ab20 showing the strongest binding.

[0404] Table 8. Binding affinity of humanized antibodies to human and cynomolgus monkey TSLP as determined by ELISA.

[0405]

[0406]

[0407] 3) BIAcore Affinity Measurement

[0408] The kinetic binding activity of anti-TSLP antibodies to human and cynomolgus monkey TSLP was measured using a BIAcore T200 system via surface plasmon resonance (SPR). Approximately 100 RU of anti-TSLP antibody was immobilized onto the Protein A sensor chip, with 1×HBS-EP buffer as the run buffer at a flow rate of 10 μL / min. Different concentrations of human or cynomolgus monkey TSLP, ranging from 0 to 21.7 nM, were 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 with 10 mM glycine (pH 2.0) regeneration buffer at a flow rate of 30 μL / min. The obtained data were fitted to the Langmuir 1:1 kinetics theoretical model using BIAevaluation software to analyze the binding rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (KD).

[0409] Table 9 shows that all antibodies have strong affinity for human and cynomolgus monkey TSLP. Among them, antibodies Ab22, Ab23, Ab24 and Ab26 have the highest affinity, with affinity for human TSLP of 0.06 pM, 0.08 pM, 0.02 pM and 2 pM respectively, and affinity for cynomolgus monkey TSLP of 0.04 pM, 0.007 pM, 0.18 pM and 0.31 pM respectively.

[0410] Table 9 Affinity of Humanized Antibodies

[0411]

[0412] Example 14: Blocking at the ELISA Level

[0413] 1) Blocking of binding of human TSLP and human TSLPR by humanized antibodies

[0414] 1 μg / mL human TSLPR recombinant protein was coated onto a 96-well microplate using carbonate buffer and incubated overnight at 4°C. The plate was washed three times with PBS, and each well was blocked with 200 μL of PBS containing 2% skim milk for 1 hour. Anti-TSLP antibody and 60 ng / mL biotinylated human TSLP protein were incubated in a blocked U-shaped 96-well plate at room temperature for 30 minutes. The plate was washed three times with PBS, and each well was incubated with 100 μL of the pre-incubated mixture for 1 hour at room temperature. After washing three times with PBST and PBS, each well was incubated with 100 μL of SA-HRP at room temperature in the dark for 30 minutes. After washing three times with PBST and PBS, each well was incubated with 100 μL of TMB substrate at 37°C for 10 minutes. The reaction was terminated with 50 μL of 2M sulfuric acid solution per well, and the absorbance was read at 450 nm. Figure 13 -a, c, e and Table 10 show that 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.

[0415] 2) Blocking of binding of humanized antibodies to cynomolgus monkey TSLP and cynomolgus monkey TSLPR

[0416] 1 μg / mL of cynomolgus monkey TSLPR recombinant protein was coated onto a 96-well microplate using carbonate buffer and incubated overnight at 4°C. The plate was washed three times with PBS, and each well was blocked with 200 μL of PBS containing 2% skim milk for 1 hour. Anti-TSLP antibody and 60 ng / mL biotinylated cynomolgus monkey TSLP protein were incubated in a blocked U-shaped 96-well plate at room temperature for 30 minutes. The plate was washed three times with PBS, and each well was incubated with 100 μL of the pre-incubated mixture for 1 hour at room temperature. After washing three times with PBST and PBS, each well was incubated with 100 μL of SA-HRP at room temperature in the dark for 30 minutes. After washing three times with PBST and PBS, each well was incubated with 100 μL of TMB substrate at 37°C for 10 minutes. The reaction was terminated with 50 μL of 2M sulfuric acid solution per well, and the absorbance was read at 450 nm. Figure 13 -b, d, f and Table 10 show that 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 TSLP and TSLPR in cynomolgus monkeys.

[0417] Table 10. Blockade of TSLP and TSLPR binding by humanized antibodies

[0418]

[0419]

[0420] Example 15: FACS detection of the blocking effect of humanized antibodies on the binding of TSLP and TSLPR

[0421] Ba / F3-hTSLPR-IL7Rα cells were washed three times with PBS, centrifuged at 300g for 3 minutes, and blocked with 3% BSA / PBS for 30 minutes. After resuspending the cells, they were added to 96-well U-plates and incubated at 5 × 10⁻⁶ cells / well. 4 / well. The antibody was incubated with 60 ng / mL biotinylated human TSLP protein at room temperature for 10 minutes, then added to cells and incubated at 4°C for 45 minutes. After washing twice with 0.5% BSA / PBS, SA-PE was added and incubated at 4°C for 45 minutes. After washing twice with 0.5% BSA / PBS, PI dye was added and incubated at 4°C for 10 minutes. After washing twice with 0.5% BSA / PBS, the cells were resuspended in PBS, and the blocking effect of the antibody at the cellular level was detected by flow cytometry. Figure 14 As shown in Table 11, humanized antibodies Ab19, Ab20, Ab21, Ab22, Ab23, Ab24, Ab25, and Ab26 can all block the binding of TSLP to TSLPR on the cell surface, and there is no significant difference between the antibodies.

[0422] Table 11. Blocking effect of humanized antibodies on the binding of TSLPR and TSLP on cells.

[0423] Humanized antibodies Heavy chain / light chain Cell blocking IC50 (nM) Ab19 h71G4VHv1 / h71G4VKv1 0.45 Ab20 h71G4VHv2 / h71G4VKv1 0.33 Ab21 h71G4VHv3 / h71G4VKv1 0.36 Ab22 h39G5VHv1 / h71G4VKv1 0.39 Ab23 h39G5VHv2 / h71G4VKv1 0.33 Ab24 h39G5VHv5 / h71G4VKv1 0.36 Ab25 h39G5VHv6 / h71G4VKv1 0.35 Ab26 h39G5VHv10 / h71G4VKv1 0.39 Mouse Anti-71G4 mAb71G4 0.27 Mouse Anti-39G5 mAb39G5 0.36 isotype control KLH hIgG1 -

[0424] Example 16: Inhibition of TSLP-stimulated Ba / F3-hTSLPR-IL7Rα cell proliferation by humanized antibodies

[0425] The ability of different anti-TSLP antibodies to biologically neutralize human TSLP was assessed by applying a short-term proliferation bioassay (which utilizes cells expressing recombinant human TSLPR).

[0426] Add cells to 96-well cell culture plates one day in advance at a concentration of 2×10⁻⁶. 4 Cells / well. Pre-incubate 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 pre-incubated antibody-cytokine to each well of a cell culture plate. 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 react for 10 minutes. Read the luminescence value in the black 96-well plate.

[0427] Figure 15The results in Table 12 show that the four humanized antibodies Ab22, Ab23, Ab24, and Ab26 significantly inhibited the proliferation of TSLP-stimulated Ba / F3-hTSLPR-IL7Rα cells (IC50 ≈ 2.20 nM, 1.10 nM, 1.04 nM, and 1.50 nM, respectively). Furthermore, compared with the similar antibody A5 (refer to patent US10287348B2), Ab22, Ab23, Ab24, and Ab26 all showed superior inhibitory effects (IC50 = 16.55).

[0428] Table 12 Inhibition of Cell Proliferation by Humanized Antibodies

[0429]

[0430]

[0431] Example 17: Detection of the blocking effect of humanized antibody on TSLP signal transduction via the STAT5 pathway using reporter gene assay

[0432] Ba / F3-hTSLPR-IL7Rα-STAT5 luc cells were starved overnight, and the next day, they were injected with 5×10⁻⁶ cells. 4 Seed 50 μL of human TSLP into each well of a 96-well plate; pre-dilute human TSLP and incubate at 37°C for 30 minutes; add 50 μL of ligand / antibody mixture to the cell plate and mix well, then incubate at 37°C for 6 hours; add 100 μL of fluorescence detection reagent; read the fluorescence value of each well using a microplate reader.

[0433] Figure 16 The results in Table 13 show that the four humanized antibodies Ab22, Ab23, Ab24 and Ab26 significantly inhibited the transduction of TSLP stimulation signals through the STAT5 pathway (IC50 = 0.17 nM, 0.17 nM, 0.15 nM and 0.18 nM), and the inhibitory effect was better than that of A5 (IC50 = 1.93 nM, referring to Kenneth V, 2017, IC50 = 1.4 nM).

[0434] Table 13. Inhibition IC50 of humanized antibodies on TSLP-induced STAT5 signaling pathway activation

[0435] Humanized antibodies Heavy chain / light chain STAT5 signal suppression IC50 (nM) Ab22 h39G5VHv1 / h71G4VKv1 0.17 Ab23 h39G5VHv2 / h71G4VKv1 0.17 Ab24 h39G5VHv5 / h71G4VKv1 0.15 Ab26 h39G5VHv10 / h71G4VKv1 0.18 Mouse Anti-71G4 mAb71G4 1.14 Mouse Anti-39G5 mAb39G5 4.92 A5 H5 / L5 1.93 isotype control KLH hIgG1 -

[0436] Various changes and equivalent substitutions may be made to the embodiments disclosed in this application without departing from the spirit and scope of this disclosure. Unless the context otherwise requires, any feature, step, or embodiment of the embodiments disclosed herein may be combined with any other feature, step, or embodiment. sequence list <110> Connoya Biomedical Technology (Chengdu) Co., Ltd. Shanghai Lingyue Biomedical Technology Co., Ltd. <120> Development and application of a treatment agent for TSLP-related conditions <130> MTI19043-D1 <160> 176 <170> SIPOSequenceListing 1.0 <210> 1 <211> 393 <212> DNA <213> Homo sapiens <400> 1 tacgacttca ctaactgtga ctttgagaag attaaagcag cctatctcag tactatttct 60 aaagacctga ttacatatat gagtgggacc aaaagtaccg agttcaacaa caccgtctct 120 tgtagcaatc ggccacattg ccttactgaa atccagagcc taaccttcaa tcccaccgcc 180 ggctgcgcgt cgctcgccaa agaaatgttc gccatgaaaa ctaaggctgc cttagctatc 240 tggtgcccag gctattcgga aactcagata aatgctactc aggcaatgaa gaagaggaga 300 aaaaggaaag tcacaaccaa taaatgtctg gaacaagtgt cacaattaca aggattgtgg 360 cgtcgcttca atcgaccttt actgaaacaa cag 393 <210> 2 <211> 393 <212> DNA <213> Macaca fascicularis <400> 2 tacgacttca ccaactgcga cttccagaag atcgaggccg actacctgag aaccatcagc 60 aaggacctga tcacctacat gagcggcacc aagagcaccg acttcaacaa caccgtgtcc 120 tgcagcaaca gaccccactg cctgacagag atccagagcc tgacattcaa ccccacacct 180 agatgtgcca gcctggccaa agagatgttc gccagaaaga ccaaggccac actggccctg 240 tggtgtcctg gctactctga gacacagatc aacgccacac aggccatgaa gaaaaggaga 300 aaaaggaaag tcaccaccaa caagtgcctg gaacaggtgt cccagctgct tggactgtgg 360 cggagattca tcagaaccct gctgaagcag cag 393 <210> 3 <211> 1116 <212> DNA <213> Homo sapiens<00,01139><400> 3 atggggcggc tggttctgct gtggggagct gccgtctttc tgctgggagg ctggatggct 60 ttggggcaag gaggagcagc agaaggagta cagattcaga tcatctactt caatttagaa 120 accgtgcagg tgacatggaa tgccagcaaa tactccagga ccaacctgac tttccactac 180 agattcaacg gtgatgaggc ctatgaccag tgcaccaact accttctcca ggaaggtcac 240 acttcagggt gcctcctaga cgcagagcag cgagacgaca ttctctattt ctccatcagg 300 aatgggacgc accccgtttt caccgcaagt cgctggatgg tttattacct gaaacccagt 360 tccccgaagc acgtgagatt ttcgtggcat caggatgcag tgacggtgac gtgttctgac 420 ctgtcctacg gggatctcct ctatgaggtt cagtaccgga gccccttcga caccgagtgg 480 cagtccaaac aggaaaatac ctgcaacgtc accatagaag gcttggatgc cgagaagtgt 540 tactctttct gggtcagggt gaaggctatg gaggatgtat atgggccaga cacataccca 600 agcgactggt cagaggtgac atgctggcag agaggcgaga ttcgggatgc ctgtgcagag 660 acaccaacgc ctcccaaacc aaagctgtcc aaatttatttt taatttccag cctggccatc 720 cttctgatgg tgtctctcct ccttctgtct ttggaaat tatggagagt gaagaagtttt 780 ccattccca gcgtgccaga cccgaaatcc atcttccccg ggctctttga gatacaccaa 840 gggaacttcc aggagtggat cacagacacc cagaacgtgg cccacctcca caagatggca 900 ggtgcagagc aagaaagtgg ccccgaggag cccctggtag tccagttggc caagactgaa 960 gccgagtctc ccaggatgct ggacccacag accgaggaga aagaggcctc tgggggatcc 1020 ctccagcttc cccaccagcc cctccaaggc ggtgatgtgg tcacaatcgg gggcttcacc 1080 tttgtgatga atgaccgctc ctacgtggcg ttgtga 1116 <210> 4 <211> 771 <212> DNA <213> Cynomolgus monkey (Macaca fascicularis) <400> 4 atgggacggt tggttctgct gtggggagct gctgtctttc tgctgggaag ctggatggct 60 ttggggcaag tagcaacagg agaaggacta cagattcaga tcatctactt taatctagaa 120 acggtgcagg tgacatggaa tgccagccac taccccagga gtaacctgag tttccactac 180 aaattcagtc gagatgaggc ctatgaccag tgcaccgtct acattctcca ggaaggtcac 240 acctcggggt gcctcctaga cgcagagcag caagacgata ttctgtattt ctccatcagg 300 aacgggacgc accccgtttt caccgccagt cgctggatct tttattacct gaagcccagt 360 tctccgaagc aggtgagctt ttcgtggcat caggacgcgg tgacagtgac gtgctctgac 420 ctgtcctaca ggggtctcct ctatgaggtt cagtaccgga gccccttcga cacggagtgg 480 cagtccaaac aggaaaatac ctgcaatgtc actatagaag acttggatgc cgagaagtgt 540 tatgctttcc gggcccgggt gaaggccatg gaggatgcgt atgggccaga cacgtacccg 600 agcgactggt cagaggtgac gtgctggcag agaggcaaga ctcgcgactc gtgcccagag 660 cctcgcacgc ctcccaaacc gaagctgtcc aaatttatgt tagtttccag cctggccatc 720 cttctgatgg tgtgtcttct ccttctgtct ttacggaaat tatggaggtg a 771 <210> 5 <211> 209 <212> PRT <213> Homo sapiens <400> 5 Gln Gly Gly Ala Ala Glu Gly Val Gln Ile Gln Ile Ile Tyr Phe Asn 1 5 10 15 Leu Glu Thr Val Gln Val Thr Trp Asn Ala Ser Lys Tyr Ser Arg Thr 20 25 30 Asn Leu Thr Phe His Tyr Arg Phe Asn Gly Asp Glu Ala Tyr Asp Gln 35 40 45 Cys Thr Asn Tyr Leu Leu Gln Glu Gly His Thr Ser Gly Cys Leu Leu 50 55 60 Asp Ala Glu Gln Arg Asp Asp Ile Leu Tyr Phe Ser Ile Arg Asn Gly 65 70 75 80 Thr His Pro Val Phe Thr Ala Ser Arg Trp Met Val Tyr Tyr Leu Lys 85 90 95 Pro Ser Ser Pro Lys His Val Arg Phe Ser Trp His Gln Asp Ala Val 100 105 110 Thr Val Thr Cys Ser Asp Leu Ser Tyr Gly Asp Leu Leu Tyr Glu Val 115 120 125 Gln Tyr Arg Ser Pro Phe Asp Thr Glu Trp Gln Ser Lys Gln Glu Asn 130 135 140 Thr Cys Asn Val Thr Ile Glu Gly Leu Asp Ala Glu Lys Cys Tyr Ser 145 150 155 160 Phe Trp Val Arg Val Lys Ala Met Glu Asp Val Tyr Gly Pro Asp Thr 165 170 175 Tyr Pro Ser Asp Trp Ser Glu Val Thr Cys Trp Gln Arg Gly Glu Ile 180 185 190 Arg Asp Ala Cys Ala Glu Thr Pro Thr Pro Pro Lys Pro Lys Leu Ser 195 200 205 Lys <210> 6 <211> 209 <212> PRT ​​​​Gln Val Ala Thr Gly Glu Gly Leu Gln Ile Gln Ile Ile Tyr Phe Asn 1 5 10 15 Leu Glu Thr Val Gln Val Thr Trp Asn Ala Ser His Tyr Pro Arg Ser 20 25 30 Asn Leu Ser Phe His Tyr Lys Phe Ser Arg Asp Glu Ala Tyr Asp Gln 35 40 45 Cys Thr Val Tyr Ile Leu Gln Glu Gly His Thr Ser Gly Cys Leu Leu 50 55 60 Asp Ala Glu Gln Gln Asp Asp Ile Leu Tyr Phe Ser Ile Arg Asn Gly 65 70 75 80 Thr His Pro Val Phe Thr Ala Ser Arg Trp Ile Phe Tyr Tyr Leu Lys 85 90 95 Pro Ser Ser Pro Lys Gln Val Ser Phe Ser Trp His Gln Asp Ala Val 100 105 110 Thr Val Thr Cys Ser Asp Leu Ser Tyr Arg Gly Leu Leu Tyr Glu Val 115 120 125 Gln Tyr Arg Ser Pro Phe Asp Thr Glu Trp Gln Ser Lys Gln Glu Asn 130 135 140 Thr Cys Asn Val Thr Ile Glu Asp Leu Asp Ala Glu Lys Cys Tyr Ala 145 150 155 160 Phe Arg Ala Arg Val Lys Ala Met Glu Asp Ala Tyr Gly Pro Asp Thr 165 170 175 Tyr Pro Ser Asp Trp Ser Glu Val Thr Cys Trp Gln Arg Gly Lys Thr 180 185 190 Arg Asp Ser Cys Pro Glu Pro Arg Thr Pro Pro Lys Pro Lys Leu Ser 195 200 205 Lys <210> 7 <211> 117 <212> PRT <213> Mouse (Mus musculus) <400> 7 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Met Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Thr Ile Asp Asn Ser Asp Ser Asp Thr Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Val Thr Leu Thr Val Asp Glu Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Val Asp Gly Tyr Leu Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser 115 <210> 8 <211> 6 <212> PRT <213> Mouse (Mus musculus) <400> 8 Thr Asp Tyr Trp Met His 1 5 <210> 9 <211> 17 <212> PRT <213> Mouse (Mus musculus) <400> 9 Thr Ile Asp Asn Ser Asp Ser Asp Thr Thr Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <210> 10 <211> 8 <212> PRT <213> Mouse (Mus musculus) <400> 10 Ser Val Asp Gly Tyr Leu Asp Tyr 1 5 <210> 11 <211> 351 <212> DNA <213> Mouse (Mus musculus) <400> 11 caggttcagc tgcagcagtc tggggctgag cttgtgatgc ctggggcttc agtgaagatg 60 tcctgcaagg cttctggcta cacattcact gactactgga tgcactgggt gaagcagagg 120 tcctgcaagg cttctggcta cacattcact gactactgga tgcactgggt gaagcagagg 120 cctggacaag gccttgagtg gattggaacg attgataatt ctgatagtga tacaacctac 180 cctggacaag gccttgagtg gattggaacg attgataatt ctgatagtga tacaacctac 180 aatcaaaagt tcaagggcaa ggtcacattg actgtagacg aatcctccag cacagcctac 240 aatcaaaagt tcaagggcaa ggtcacattg actgtagacg aatcctccag cacagcctac 240 atgcagctca gcagcctgac atctgaggac tctgcggtct attactgtgc aagatcggtt 300 atgcagctca gcagcctgac atctgaggac tctgcggtct attactgtgc aagatcggtt 300 gatggttacc ttgactactg gggccaaggc accactctca cagtctcctc a 351 gatggttacc ttgactactg gggccaaggc accactctca cagtctcctc a 351 <210> 12<210> 12 <211> 107<211> 107 <212> PRT<212> PRT <213> 小鼠(Mus musculus)<213> Mouse (Mus musculus) <400> 12<400> 12 Glu Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly Glu Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Thr Ser Glu Asn Ile Tyr Ser Tyr Glu Thr Val Thr Ile Thr Cys Arg Thr Ser Glu Asn Ile Tyr Ser Tyr 20 25 30 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 35 40 45 Tyr Phe Ala Lys Thr Leu Thr Asp Gly Val Pro Ser Arg Leu Ser Gly Tyr Phe Ala Lys Thr Leu Thr Asp Gly Val Pro Ser Arg Leu Ser Gly 50 55 60 50 55 60 Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro 65 70 75 80 65 70 75 80 Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 13 <211> 11 <212> PRT <213> Mouse (Mus musculus) <400> 13 Arg Thr Ser Glu Asn Ile Tyr Ser Tyr Leu Ala 1 5 10 <210> 14 <211> 7 <212> PRT <213> Mouse (Mus musculus) <400> 14 Phe Ala Lys Thr Leu Thr Asp 1 5 <210> 15 <211> 9 <212> PRT <213> Mouse (Mus musculus) <400> 15 Gln His His Tyr Gly Thr Pro Trp Thr 1 5 <210> 16 <211> 321 <212> DNA <213> Mouse (Mus musculus) <400> 16 gaaatccaga tgacccagtc tccagcctcc ctatctgcat ctgtgggaga aactgtcacc 60 atcacatgtc gaacaagtga gaatatttac agttatttag catggtatca gcagaaacag 120 ggaaaatctc ctcagctcct ggtctatttt gcaaaaacct taacagacgg tgtgccatca 180 aggctcagtg gcagtggatc aggcacacag ttttctctga agatcaacag cctgcagcct 240 gaagattttg ggagttatta ctgtcaacat cattatggta ctccgtggac gttcggtgga 300 ggcaccaagc tggaaatcaa a 321 <210> 17 <211> 117 <212> PRT <213> Mouse (Mus musculus) <400> 17 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Met Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr 20 25 30<|0001355|> Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile[[ID=|29]] |> 35 40 45 Gly Thr Ile Asp Asn Ser Asp Ser Asp Thr Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Val Thr Leu Thr Val Asp Glu Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Val Asp Gly Tyr Leu Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser 115 <210> 18 <211> 6 <212> PRT <213> Mouse (Mus musculus) <400> 18 Thr Gly Tyr Trp Met His 1 5 <210> 19 <211> 17 <212> PRT <213> Mouse (Mus musculus) <400> 19 Thr Ile Asp Asn Ser Asp Ser Asp Thr Thr Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <210> 20 <211> 8 <212> PRT <213> Mouse (Mus musculus) <400> 20 Ser Val Asp Gly Tyr Leu Asp Tyr 1 5 <210> 21 <211> 351 <212> DNA <213> Mouse (Mus musculus) <400> 21 caggtccagc tgcagcagtc tggggctgag cttgtgatgc ctggggcttc agtgaagatg 60 caggtccagc tgcagcagtc tggggctgag cttgtgatgc ctggggcttc agtgaagatg 60 tcctgcaagg cttctggcta cacattcact ggctactgga tgcactgggt gaagcagagg 120 tcctgcaagg cttctggcta cacattcact ggctactgga tgcactgggt gaagcagagg 120 cctggacaag gccttgagtg gattggaacg attgataatt ctgatagtga tacaacctac 180 cctggacaag gccttgagtg gattggaacg attgataatt ctgatagtga tacaacctac 180 aatcaaaagt tcaagggcaa ggtcacattg actgtagacg aatcctccag cacagcctac 240 aatcaaaagt tcaagggcaa ggtcacattg actgtagacg aatcctccag cacagcctac 240 atgcagctca gcagcctgac atctgaggac tctgcggtct attactgtgc aagatcggtt 390 atgcagctca gcagcctgac atctgaggac tctgcggtct attactgtgc aagatcggtt 390 gatggttacc ttgactactg gggccaaggc accactctca cagtctcctc a 351 gatggttacc ttgactactg gggccaaggc accactctca cagtctcctc a 351 <210> 22<210> 22 <211> 107<2,11> 107 <212> PRT<212> PRT<000,1403><000,1403><213> 小鼠(Mus musculus)<213> Mouse (Mus musculus) <400> 22<400> 22 <0,001405> Asp Ile Gln Met Ile Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly Asp Ile Gln Met Ile Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr 20 25 30 20 25 30 Leu Ala Trp Tyr Gln Gln Ile Gln Gly Lys Ser Pro Gln Leu Leu Val Leu Ala Trp Tyr Gln Gln Ile Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 35 40 45 Tyr Tyr Ala Lys Thr Leu Pro Glu Gly Val Pro Ser Arg Phe Ser Gly Tyr Tyr Ala Lys Thr Leu Pro Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 50 55 60 <00014,,13> Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 23 <211> 11 <212> PRT <213> Mouse (Mus musculus) <400> 23 Arg Ala Ser Glu Asn Ile Tyr Ser Tyr Leu Ala 1 5 10 <210> 24 <211> 7 <212> PRT <213> Mouse (Mus musculus) <400> 24 Tyr Ala Lys Thr Leu Pro Glu 1 5 <210> 25 <211> 9 <212> PRT <213> Mouse (Mus musculus) <400> 25 Gln His His Tyr Gly Thr Pro Trp Thr 1 5 <210> 26 <211> 321 <212> DNA <213> Mouse (Mus musculus) <400> 26 gacatccaga tgattcagtc tccagcctcc ctatctgcat ctgtgggaga aactgtcacc 60 atcacatgcc gagcaagtga gaatatttac agttatttag catggtatca gcagatacag 120 ggaaaatctc ctcagctcct ggtctattat gcaaaaacct taccagaagg tgtgccatca 180<o001448>aggttcagtg gcagtggatc aggcacacag ttttctctga agatcaacag cctgcagcct 240 gaagattttg ggagttatta ctgtcaacat cattacggta ccccgtggac gttcggtgga 300 ggcaccaagc tggaaatcaa a 321 <210> 27 <211> 117 <212> PRT <213> Mouse (Mus musculus) <400> 27 [[ID=u2]]Gln Val Gln Leu Gln Gln Ser Gly Pro Gln Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 4u Gly Leu Ile Asp Pro Ser Asp Ser Glu Thr Gly Leu Asn Gln Arg Phe 50 55 60 It should be noted that there seems to be an "o001448" in ID=5 which might be a typo. I translated it as "<o001448>" as per the instruction to preserve all tags exactly. If this is incorrect, please correct it in the original text for a more accurate translation.Lys Asp Lys Ala Thr Leu Thr Val Asp Asn Pro Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Asp Gly Tyr Tyr Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser 115 <210> 28 <211> 6 <212> PRT <213> Mouse (Mus musculus) <400> 28 Thr Ser Tyr Trp Met His 1 5 <210> 29 <211> 17 <212> PRT <213> Mouse (Mus musculus) <400> 29 Leu Ile Asp Pro Ser Asp Ser Glu Thr Gly Leu Asn Gln Arg Phe Lys 1 5 10 15 Asp <210> 30 <211> 8 <212> PRT <213> Mouse (Mus musculus) <400> 30 Ser Leu Asp Gly Tyr Tyr Asp Tyr 1 5 <210> 31 <211> 351 <212> DNA <213> Mouse (Mus musculus) <400> 31 caggtccagc tgcagcagtc tgggcctcag ctggttaggc ctggggcttc agtaaagata 60 tcctgcaagg cttctggtta ctcattcacc agctactgga tgcactgggt gaagcagagg 120 cctggacaag gtcttgagtg gattggcttg attgatcctt ccgatagtga aactgggtta 180 aatcagaggt tcaaggacaa ggccacattg actgtagaca atccctccag cacagcctac 240 atgcaactca gcagcccgac atctgaggac tctgcggtct attactgtgc aagatccctt 300 gatggttact atgactactg gggccaaggc accactctca cagtctcctc a 351 <210> 32 <211> 107 <212> PRT <213> Mouse (Mus musculus) <400> 32 Asp Ile Gln Met Ile Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Gly Asn Ile His Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Asn Ala Lys Thr Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Ser Thr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 33 <211> 11 <212> PRT <213> Mouse (Mus musculus) <400> 33 Arg Ala Ser Gly Asn Ile His Asn Tyr Leu Ala 1 5 10 <210> 34 <211> 7 <212> PRT <213> Mouse (Mus musculus) <400> 34 Asn Ala Lys Thr Leu Ala Asp 1 5 <210> 35 <211> 9 <212> PRT <213> Mouse (Mus musculus) <400> 35 Gln His Phe Trp Ser Thr Pro Trp Thr 1 5 <210> 36 <211> 321 <212> DNA <213> Mouse (Mus musculus) <400> 36 gacatccaga tgattcagtc tccagcctcc ctatctgcat ctgtgggaga aactgtcacc 60 atcacatgtc gagcaagtgg gaatattcac aattatttag catggtatca gcagaaacag 120 ggaaaatctc ctcagctcct ggtctataat gcaaaaacct tagcagatgg tgtgccatca 180 aggttcagtg gcagtggatc aggaacacaa tattctctca agatcaacag cctgcagcct 240 gaagattttg ggagttatta ctgtcaacat ttttggagta ctccgtggac gttcggtgga 300 ggcaccagac tggaaataaa a 321 <210> 37 <211> 117 <212> PRT <213> Mouse (Mus musculus) <400> 37 Gln Val Gln Leu Lys Gln Ser Gly Pro Gln Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile Asp Pro Ser Asp Ser Glu Thr Thr Leu Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Thr Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Asp Gly Tyr Tyr Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110<了 Leu Thr Val Ser Ser 115 <210> 38 <211> 6 <212> PRT <213> Mouse (Mus musculus) <400> 38 Thr Ser Tyr Trp Met His ]>1 5 <210> 39 <211> 17 <212> PRT <213> Mouse (Mus musculus) <400> 39 Met Ile Asp Pro Ser Asp Ser Glu Thr Thr Leu Asn Gln Lys Phe Lys 1 5 10 15 Asp <210> 40 <211> 8 <212> PRT <213> Mouse (Mus musculus) <400> 40 Ser Leu Asp Gly Tyr Tyr Asp Tyr 1 5 <210> 41 <211> 351 It should be noted that there seems to be a misspelling in the tag "<了 " which is likely meant to be " ". This has been left as is in the translation to maintain consistency with the original text.<212> DNA <213> Mouse (Mus musculus) <400> 41 caggtccagc tgaagcagtc tgggcctcag ttggttaggc ctggggcttc agtgaagata 60 tcctgcaagg cttctggtta ctcattcacc agctactgga tgcactgggt gaagcagagg 120 cctggtcaag gtcttgagtg gattggcatg attgatcctt ccgatagtga aactacgtta 180 aatcagaagt tcaaggacaa ggccacattg actgtagaca aatcctccac cacagcctac 240 atgcaactca gcagcccgac atctgaggac tctgcggtct attactgtgc aagatccctt 300 gatggttact acgactactg gggccaaggc accactctca cagtctcctc a 351 <210> 42 <211> 107 <212> PRT <213> Mouse (Mus musculus) <400> 42 Asp Val Gln Met Ile Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Thr Ser Glu Asn Ile Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Phe Ala Lys Thr Leu Thr Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 43 <211> 11 <212> PRT <213> Mouse (Mus musculus) <400> 43 Arg Thr Ser Glu Asn Ile Tyr Ser Tyr Leu Ala 1 5 10 <210> 44 <211> 7 <212> PRT <213> Mouse (Mus musculus) <400> 44 Phe Ala Lys Thr Leu Thr Asp 1 5 <210> 45 <211> 9 <212> PRT <213> Mouse (Mus musculus) <400> 45 Gln His His Tyr Gly Thr Pro Trp Thr 1 5 <210> 46 <211> 321 <212> DNA <213> Mouse (Mus musculus) <400> 46 gatgtccaga tgattcagtc tccagcctcc ctatctgcat ctgtgggaga aactgtcacc 60 atcacatgtc gaacaagtga gaatatttac agttatttag catggtatca gcagaaacag 120 ggaaaatctc ctcagctcct ggtctatttt gcaaaaacct taacagacgg tgtgccatca 180 aggttcagtg gcagtggatc aggcacacag ttttctctga agatcaacag cctgcagcct 24(0 gaagattttg ggagttatta ctgtcaacat cattatggta ctccgtggac gttcggtgga 300 ggcaccaagc tggaaatcaa a 321 <210> 47 <211> 117 <212> PRT <213> Mouse (Mus musculus) <400> 47<l Gln Val Gln Leu Gln Gln Ser Gly Pro Gln Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Asp Pro Ser Asp Ser Glu Thr Ile Leu Asn Gln Lys Phe It should be noted that there seems to be a minor error in the original text where "24(0" is likely a typo and should probably be "240". This has been corrected in the translation as much as possible while maintaining the integrity of the original text structure.50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Phe Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Asp Gly Tyr Tyr Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser 115 <210> 48 <211> 6 <212> PRT <213> Mouse (Mus musculus) <400> 48 Thr Ser Tyr Trp Met His 1 5 <210> 49 <211> 17 <212> PRT <213> Mouse (Mus musculus) <400> 49 Val Ile Asp Pro Ser Asp Ser Glu Thr Ile Leu Asn Gln Lys Phe Lys 1 5 10 15 Asp <210> 50[[ID=...]] <211> 8 <212> PRT <213> Mouse (Mus musculus) <400> 50 Ser Leu Asp Gly Tyr Tyr Asp Tyr 1 5 <210> 51 <211> 351 <212> DNA <213> Mouse (Mus musculus) <400> 51 caggttcagc tgcagcagtc tgggcctcag ctggttaggc ctggggcttc agtgaagata 60 tcctgcaagg cttctggtta ctcattcacc agctactgga tgcactgggt gaagcagagg 120 cctggacaag gtcttgagtg gattggcgtg attgatcctt ccgatagtga gactatatta 180 aatcagaagt tcaaggacaa ggccacattg actgtagaca aatcctccag tacagcctac 240 atgcaattca gcagcccgac atctgaggac tctgcggtct attactgtgc aagatccctt 300 gatggttact acgactactg gggccaaggc accactctca cagtctcctc a 351 <210> 52 <211> 107 <212> PRT <213> Mouse (Mus musculus) <400> 52 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Gly Glu Asn Ile Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Ala Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Gly Thr Pro Trp 85 90 95 Ala Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 53 <211> 11 <212> PRT <213> Mouse (Mus musculus) <400> 53 Arg Ala Gly Glu Asn Ile Tyr Ser Tyr Leu Ala 1 5 10 <210> 54 <211> 7 <212> PRT <213> Mouse (Mus musculus) <400> 54 Asn Ala Lys Thr Leu Ala Glu 1 5 <210> 55 <211> 9 <212> PRT <213> Mouse (Mus musculus) <400> 55 Gln His Phe Trp Gly Thr Pro Trp Ala 1 5 <210> 56 <211> 321 <212> DNA <213> Mouse (Mus musculus) <400> 56 gacatccaga tgacacagtc tccagcctcc ctatctgcat ctgtgggaga aactgtcacc 60 atcacatgtc gagcaggtga gaatatttac agttatttag catggtatca gcagaaacag 120 ggaaaatctc ctcagctcct ggtctataat gcaaaaacct tagcagaagg tgtgccatca 180 aggttcagtg gcagtgcatc aggcacacag ttttctctga agatcaacag cctgcagcct 240 gaagattttg ggagttatta ctgtcaacat ttttggggta ctccgtgggc gttcggtgga 300 ggcaccagac tggaaataaa a 321 <210> 57 <211> 117 <212> PRT <213> Mouse (Mus musculus) <400> 57 Gln Val Gln Leu Gln Gln Ser Gly Pro Gln Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Thr Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asp Pro Ser Asp Gly Glu Ala Gly Leu Asn Gln Asn Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Ile Ser Ser Arg Thr Ala Tyr 65 70 75 80 Met Gln Ile Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Ser Leu Asp Gly Tyr Tyr Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser 115 <210> 58 <211> 6 <212> PRT <213> Mouse (Mus musculus) <400> 58 Thr Thr Tyr Trp Met His 1 5 <210> 59 <211> 17 <212> PRT <213> Mouse (Mus musculus) <400> 59 Leu Ile Asp Pro Ser Asp Gly Glu Ala Gly Leu Asn Gln Asn Phe Lys 1 5 10 15 Asp <210> 60 <211> 8 <212> PRT <213> Mouse (Mus musculus) <400> 60 Ser Leu Asp Gly Tyr Tyr Asp Tyr 1 5 <210> 61 <211> 351 <212> DNA <213> Mouse (Mus musculus) <400> 61 caggtccagc tgcagcagtc tgggcctcag ctggttaggc ctggggcttc agtgaagata 60 tcctgcaagg cttctggtta ctcattcacc acctactgga tgcactgggt gaagcagagg 120 cctggacaag gtcttgagtg gattggcttg attgatcctt ccgatggtga agctgggtta 180 aatcagaact tcaaggacaa ggccacattg actgtagaca tttcctccag aacagcctac 240 atgcaaatca gcagcccgac atctgaggac tctgcggttt atttctgtgc aagatccctt 300 gatggttact acgactactg gggccaaggc accactctca cagtctcctc a 351 <210> 62 <211> 107 <212> PRT <213> Mouse (Mus musculus) <400> 62 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asp Ile Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Asn Ala Lys Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Ser Tyr Tyr Cys Gln His Phe Trp Ser Thr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 63 <211> 11 <212> PRT <213> Mouse (Mus musculus) <400> 63 Arg Ala Ser Glu Asp Ile Tyr Ser Tyr Leu Ala 1 5 10 <210> 64 <211> 7 <212> PRT <213> Mouse (Mus musculus) <400> 64 Asn Ala Lys Thr Leu Ala Glu 1 5 <210> 65 <211> 9 <212> PRT <21�> Mouse (Mus musculus) <400> 65 Gln His Phe Trp Ser Thr Pro Trp Thr 1 5 <210> 66 <211> 321 <212> DNA <213> Mouse (Mus musculus) <400> 66 gacatccaga tgacccagtc tccagcctcc ctgtctgcat ctgtgggaga aactgtcacc 60 atcacatgtc gagcaagtga ggatatttac agttatttag catggtatca gcagaaacag 120 ggaaaatctc ctcagctcct ggtctataat gcaaaaacct tagcagaagg tgtgccatca 180 aggttcagtg gcagtggatc aggcacacag ttttctctga agatcaacag cctgcagcct 240 gaagattttg ggagttatta ctgtcaacat ttttggagta ctccgtggac gttcggtgga 300 ggcaccaagc tggagctgaa a 321 <210> 67 <211> 117 <212> PRT <213> Mouse (Mus musculus) <400> 67 Gln Val Gln Leu Leu Gln Ser Gly Pro Gln Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Ile Ser Tyr 20 25 30 Trp Ile His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Ile Ile Asp Pro Ser Asp Ser Asp Thr Ser Leu Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Thr Thr Ala Tyr 65 70 75 80 Ile Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Asp Gly Tyr Tyr Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser 115 <210> 68 <211> 6 <212> PRT <213> Mouse (Mus musculus) <400> 68 Ile Ser Tyr Trp Ile His 1 5 <210> 69 <211> 17 <212> PRT <213> Mouse (Mus musculus) <400> 69 Ile Ile Asp Pro Ser Asp Ser Asp Thr Ser Leu Asn Gln Lys Phe Lys 1 5 10 15 Asp <210> 70 <211> 8 <212> PRT <213> Mouse (Mus musculus) <400> 70 Ser Leu Asp Gly Tyr Tyr Asp Tyr 1 5 <210> 71 <211> 351 <212> DNA <213> Mouse (Mus musculus) <400> 71 caggtgcaac tactgcagtc tgggcctcag ctggttaggc ctggggcttc agtgaagata 60 tcctgcaagg cttctggtta ctcattcatt agttactgga tacactgggt gaagcagagg 120 cctggacaag gtcttgagtg gattggcatt attgatcctt ccgatagtga cactagctta 180 aatcagaagt tcaaggacaa ggccacattg actgtggaca aatcctccac cacagcctac 240 attcaactca gcagcccgac atctgaggac tctgcggtct attactgtgc aagatccctt 300 gatggttact acgactactg gggccaaggc accactctca cagtctcctc a 351 <210> 72 <211> 107 <212> PRT <213> Mouse (Mus musculus) <400> 72 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Gln Gly Lys Ser Pro Gln Leu Leu Val 35 40 45 Tyr Asn Ala Arg Thr Leu Pro Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Phe Ser Leu Lys Ile Asn Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Gly Ile Tyr Tyr Cys Gln His His Tyr Thr Thr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 73 <211> 11 <212> PRT <213> Mouse (Mus musculus) <400> 73 Arg Ala Ser Glu Asn Ile Tyr Ser Tyr Leu Ala 1 5 10 <210> 74 <211> 7 <212> PRT <213> Mouse (Mus musculus) <400> 74 Asn Ala Arg Thr Leu Pro Glu 1 5 <210> 75 <211> 9 <212> PRT <213> Mouse (Mus musculus) <400> 75 Gln His His Tyr Thr Thr Pro Trp Thr 1 5 <210> 76 <211> 321 <212> DNA <213> Mouse (Mus musculus) <400> 76 gacatccaga tgactcagtc tccagcctcc ctatctgcat ctgtgggaga aactgtcacc 60 atcacatgtc gagcaagtga gaatatttac agttatttag catggtatca gcagaaacag 120 ggaaaatctc ctcaactcct ggtctataat gcaagaacct taccagaagg tgtgccatca 180 cggttcagtg gcagtggatc aggcacacag ttttctctga agatcaacag cctgcagcct 240 gaagattttg ggatttatta ctgtcaacat cattatacta ctccgtggac gttcggtgga 300 ggcaccaagc tggaaatcaa a 321 <210> 77 <211> 117 <212> PRT <213> Human (Homo sapiens) <400> 77 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Ile Ser Tyr 20 25 30 Trp Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Asp Pro Ser Asp Ser Asp Thr Ser Leu Asn Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Val Asp Lys Ser Ser Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Asp Gly Tyr Tyr Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 78 <211> 6 <212> PRT <213> Homo sapiens <400> 78 Ile Ser Tyr Trp Ile His 1 5 <210> 79 <211> 17 <212> PRT <213> Homo sapiens <400> 79 Ile Ile Asp Pro Ser Asp Ser Asp Thr Ser Leu Asn Gln Lys Phe Gln 1 5 10 15 Gly <210> 80 <211> 8 <212> PRT <213> Homo sapiens[[ID=X]] <400> 80<00020XZ>Ser Leu Asp Gly Tyr Tyr Asp Tyr 1 5 <210> 81 <211> 351 <212> DNA <213> Homo sapiens <400> 81 caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcagcag cgtgaaggtg 60 tcctgcaagg ctagcggcta cagcttcatc agctactgga tccactgggt ccgacaggcc 120 cctggacaag gacttgagtg gatgggcatc atcgacccca gcgacagcga cacaagcctg 180 aaccagaaat tccagggcag agtgaccctg accgtggaca agtctagcac caccgcctac 240 atggaactga gcagcctgag aagcgaggac accgccgtgt actactgtgc cagatctctg 300 gacggctact acgactactg gggccaggga acactggtca ccgttagctc t 351 <210> 82 <211> 117 <212> PRT <213> Homo sapiens <400> 82 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Ile Ser Tyr 20 25 30 Trp Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Asp Pro Ser Asp Ser Asp Thr Ser Leu Asn Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Asp Gly Tyr Tyr Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110<​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ 1 5 <210> 86 <211> 351 <212> DNA <213> Homo sapiens <400> 86 caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcagcag cgtgaaggtg 60 tcctgcaagg ctagcggcta cagcttcatc agctactgga tccactgggt ccgacaggcc 120 cctggacaag gacttgagtg gatgggcatc atcgacccca gcgacagcga cacaagcctg 180 aaccagaaat tccagggcag agtgaccatc accgccgacg agtctaccag caccgcctac 240 atggaactga gcagcctgag aagcgaggac accgccgtgt actactgtgc cagatctctg 300 gacggctact acgattactg gggccaggga accctggtca ccgtttcttc t 351 <210> 87 <211> 117 <212> PRT <213> Homo sapiens <400> 87 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Arg Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Ile Ser Tyr 20 25 30 Trp Ile His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Asp Pro Ser Asp Ser Asp Thr Ser Leu Asn Gln Ser Phe 50 55 60 Gln Gly His Val Thr Leu Ser Val Asp Lys Ser Ser Thr Thr Ala Tyr 35 40 45 Gly Ile Ile Asp Pro Ser Asp Ser Asp Thr Ser Leu Asn Gln Ser Phe 50 55 60 Gln Gly His Val Thr Leu Ser Val Asp Lys Ser Ser Thr Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Asp Gly Tyr Tyr Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 88 <211> 6 <212> PRT <213> Homo sapiens <400> 88 Ile Ser Tyr Trp Ile His 1 5 <210> 89 <211> 17 <212> PRT <213> Homo sapiens <400> 89 Ile Ile Asp Pro Ser Asp Ser Asp Thr Ser Leu Asn Gln Ser Phe Gln 1 5 10 15 Gly <210> 90 <211> 8 <212> PRT <213> Homo sapiens <400> 90 Ser Leu Asp Gly Tyr Tyr Asp Tyr 1 5 <210> 91 [[ID=ll]]<211> 351 <212> DNA <213> Homo sapiens <400> 91 gaagtgcagc tggttcagtc tggcgccgaa gtgaagaagc ctggcgagag cctgagaatc 60 agctgcaagg ccagcggcta cagcttcatc agctactgga tccactgggt ccgacagatg 120 cctggcaaag gcctggaatg gatgggcatc atcgacccca gcgacagcga cacaagcctg 180 aaccagtctt tccagggcca cgtgacactg agcgtggaca agtctagcac caccgcctac 240 ctgcagtggt ctagcctgaa ggcctctgac accgccatgt actactgcgc cagatctctg 300 gacggctact acgattactg gggccaggga accctggtca ccgtttcttc t 351 <210> 92 <211> 117 <Zl2> PRT[[ID=Z6]] <213> Homo sapiens <400> 92 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Arg Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Ile Ser Tyr 20 25 30 Trp Ile His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Asp Pro Ser Asp Ser Asp Thr Ser Leu Asn Gln Ser Phe 50 55 60 Gln Gly His Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Asp Gly Tyr Tyr Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 93 <211> 6 <212> PRT <213> Homo sapiens <400> 93 Ile Ser Tyr Trp Ile His 1 5 <210> 94 <211> 17 <212> PRT <213> Homo sapiens <400> 94 Ile Ile Asp Pro Ser Asp Ser Asp Thr Ser Leu Asn Gln Ser Phe Gln 1 5 10 15 Gly <210> 95 <211> 8 <212> PRT <213> Homo sapiens <400> 95 Ser Leu Asp Gly Tyr Tyr Asp Tyr 1 5 <210> 96 <211> 351 <212> DNA <213> Homo sapiens <400> 96 gaagtgcagc tggttcagtc tggcgccgaa gtgaagaagc ctggcgagag cctgagaatc 60 agctgcaagg ccagcggcta cagcttcatc agctactgga tccactgggt ccgacagatg 120 cctggcaaag gcctggaatg gatgggcatc atcgacccca gcgacagcga cacaagcctg 180 aaccagtctt tccagggcca cgtgacaatc agcgccgaca agagcatcag caccgcctac 240 ctgcagtggt ctagcctgaa ggcctctgac accgccatgt actactgcgc cagatctctg 300 gacggctact acgactactg gggccaggga acactggtca ccgttagctc t 351 <210> 97 <211> 117 <212> PRT <213> Homo sapiens <400> 97 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Asp Pro Ser Asp Ser Asp Thr Ser Leu Asn Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Asp Gly Tyr Tyr Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 98 <211> 6 <212> PRT <213> Homo sapiens <400> 98 Thr Asp Tyr Trp Met His 1 5 <210> 99 <211> 17 <212> PRT <213> Homo sapiens <400> 99 Ile Ile Asp Pro Ser Asp Ser Asp Thr Ser Leu Asn Gln Lys Phe Gln 1 5 10 15 Gly <210> 100 <211> 8 <212> PRT[[ID=1'4]] <213> Homo sapiens <400> 100 Ser Leu Asp Gly Tyr Tyr Asp Tyr 1 5 <210> 101 <211> 351 <212> DNA <213> Homo sapiens <400> 101 caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcagcag cgtgaaggtg 60 tcctgcaagg ctagcggcta cacattcacc gactactgga tgcactgggt ccgacaggct 120 ccaggacagg gacttgagtg gatgggcatc atcgacccca gcgacagcga cacaagcctg 180 aaccagaaat tccagggcag agtgaccctg accgtggaca cctctacaag caccgcctac 240 atggaactga gcagcctgag aagcgaggac accgccgtgt actactgtgc cagatctctg 300 gacggctact acgattactg gggccaggga accctggtca ccgtttcttc t 351 <210> 102 <211> 117 <212> PRT <213> Homo sapiens <400> 102 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Thr Ile Asp Asn Ser Asp Ser Asp Thr Ser Leu Asn Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr<00​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ <212> PRT <213> Homo sapiens <400> 104 Thr Ile Asp Asn Ser Asp Ser Asp Thr Ser Leu Asn Gln Lys Phe Gln 1 5 10 15 Gly <210> 105 <211> 8 <212> PRT <213> Homo sapiens <400> 105 Ser Leu Asp Gly Tyr Tyr Asp Tyr[[ID=2�]] 1 5 <210> 106 <211> 351 <212> DNA <213> Homo sapiens <400> 106 caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcagcag cgtgaaggtg 60 tcctgcaagg ctagcggcta cacattcacc gactactgga tgcactgggt ccgacaggct 120 ccaggacagg gacttgagtg gatgggcacc atcgacaaca gcgacagcga cacaagcctg 180 aaccagaaat tccagggcag agtgaccctg accgtggaca cctctacaag caccgcctac 240 atggaactga gcagcctgag aagcgaggac accgccgtgt actactgtgc cagatctctg 300 gacggctact acgattactg gggccaggga accctggtca ccgtttcttc t 351 <210> 107 <211> 117 <212> PRT <213> Homo sapiens <400> 107 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Thr Ile Asp Asn Ser Asp Ser Asp Thr Ser Leu Asn Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Val Asp Glu Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Asp Gly Tyr Tyr Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 108 <211> 6 <212> PRT <213> Homo sapiens <400> 108 Thr Asp Tyr Trp Met His 1 5 <210> 109 <211> 17 <212> PRT <213> Homo sapiens <400> 109 Thr Ile Asp Asn Ser Asp Ser Asp Thr Ser Leu Asn Gln Lys Phe Gln 1 5 10 15 Gly <210> 110 <211> 8 <212> PRT <213> Homo sapiens <400> 110 Ser Leu Asp Gly Tyr Tyr Asp Tyr 1 5 <210> 111 <211> 351 <212> DNA <213> Homo sapiens <400> 111 caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcagcag cgtgaaggtg 60 tcctgcaagg ctagcggcta cacattcacc gactactgga tgcactgggt ccgacaggct 120 ccaggacagg gacttgagtg gatgggcacc atcgacaaca gcgacagcga cacaagcctg 180 aaccagaaat tccagggcag agtgaccctg accgtggaca cctctacaag caccgcctac 240 atggaactga gcagcctgag aagcgaggac accgccgtgt actactgtgc cagatctctg 300 gacggctact acgattactg gggccaggga accctggtca ccgtttcttc t 351 <210> 112 <211> 117 [[ID=,6]]<212> PRT <213> Homo sapiens <400> 112 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Trp Met His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met[[ID=#21]] 35 40 45 Gly Ile Ile Asp Pro Ser Asp Ser Asp Thr Ser Leu Asn Gln Ser Phe 50 55 60 Gln Gly His Val Thr Leu Ser Val Asp Lys Ser Ser Thr Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Asp Gly Tyr Tyr Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 113 <211> 6 <212> PRT <213> Homo sapiens <400> 113 Thr Asp Tyr Trp Met His 1 5 <210> 114 <211> 17 <212> PRT <213> Homo sapiens <400> 114 Ile Ile Pro Asp Ser Asp Ser Asp Thr Ser Leu Asn Gln Ser Phe Gln 1 5 10 15 Gly <210> 115 <211> 8 <212> PRT <213> Homo sapiens <400> 115 Ser Leu Asp Gly Tyr Tyr Asp Tyr 1 5 <210> 116 <211> 351 <212> DNA <213> Homo sapiens <400> 116 gaagtgcagc tggttcagtc tggcgccgaa gtgaagaagc ctggcgagag cctgaagagatc 60 agctgcagg ccagcggcta caccttcacc gactactgga tgcactgggt ccgacagatg 120 cctggcaaag gcctggaatg gatggggcatc atcgacccca gcgacagcga cacaagcctg 180 aaccagtctt tccagggcca cgtgacactg agcgtggaca agtctagcac caccgcctac 240 ctgcagtggt ctagcctgaa ggcctctgac accgccatgt actactgcgc cagatctctg 300 gacggctact acgattactg gggccaggga accctggtca ccgtttcttc t 351 <210> 117 <211> 117 <212> PRT <213> Homo sapiens <400> 117 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Trp Met His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Thr Ile Asp Asn Ser Asp Ser Asp Thr Ser Leu Asn Gln Ser Phe 50 55 60 Gln Gly His Val Thr Leu Ser Val Asp Lys Ser Ser Thr Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Asp Gly Tyr Tyr Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 118 <211> 6 <212> PRT <213> Homo sapiens <400> 118 Thr Asp Tyr Trp Met His 1 5 <210> 119 <211> 17 <212> PRT<0​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​aaccagagct ttcagggcca cgtgacactg agcgtggaca agtctagcac caccgcctac 240 ctgcagtggt ctagcctgaa ggcctctgac accgccatgt actactgcgc cagatctctg 300 gacggctact acgattactg gggccaggga accctggtca ccgtttcttc t 351 <210> 122 <211> 117 <212> PRT <213> Homo sapiens <400> 122 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Asp Pro Ser Asp Ser Asp Thr Ser Leu Asn Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Asp Gly Tyr Tyr Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 123 <211> 6 <212> PRT <213> Homo sapiens <400> 123 Thr Asp Tyr Trp Met His 1 5 <210> 124 <211> 17 <212> PRT <213> Homo sapiens <400> 124 Ile Ile Asp Pro Ser Asp Ser Asp Thr Ser Leu Asn Gln Lys Phe Gln 1 5 10 15 Gly <210> 125 <211> 8 <212> PRT <213> Homo sapiens <400> 125 Ser Leu Asp Gly Tyr Tyr Asp Tyr 1 5 <210> 126 <211> 351 <212> DNA <213> Homo sapiens <400> 126 caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcagcag cgtgaaggtg 60 tcctgcagg ctagcggcta cacattcacc gactactgga tgcactgggt ccgacaggct 120 ccaggacagg gacttgagtg gatggggcatc atcgacccca gcgacagcga cacaagcctg 180 aaccagaaat tccagggcag agtgaccatc accgccgaca cctctacaag caccgcctac 240 atggaactga gcagcctgag aagcgaggac accgccgtgt actactgtgc cagatctctg 300 gacggctact acgattactg gggccaggga accctgtca ccgtttcttc t 351 <210> 127 <211> 117 <212> PRT <213> Homo sapiens <400> 127 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Thr Ile Asp Asn Ser Asp Ser Asp Thr Ser Leu Asn Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Only Asp Is Thr Thr On Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Asp Gly Tyr Tyr Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 128 <211> 6 <212> PRT <213> Homo sapiens <400> 128 Thr Asp Tyr Trp Met His 1 5 <210> 129 <211> 17 <212> PRT <213> Homo sapiens <400> 129 Thr Ile Asp Asn Ser Asp Ser Asp Thr Ser Leu Asn Gln Lys Phe Gln 1 5 10 15 Gly <210> 130 <211> 8 <2​​​​​​​​​​​​​​​​​​​caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcagcag cgtgaaggtg 60 tcctgcagg ctagcggcta cacattcacc gactactgga tgcactgggt ccgacaggct 120 ccaggacagg gacttgagtg gatggggcacc atcgacaaca gcgacagcga cacaagcctg 180 aaccagaaat tccagggcag agtgaccatc accgccgaca cctctacaag caccgcctac 240 atggaactga gcagcctgag aagcgaggac accgccgtgt actactgtgc cagatctctg 300 gacggctact acgattactg gggccaggga accctgtca ccgtttcttc t 351 <210> 132 <211> 117 <212> PRT <213> Homo sapiens <400> 132 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Thr Ile Asp Asn Ser Asp Ser Asp Thr Ser Leu Asn Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Asp Gly Tyr Tyr Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 133 <211> 6 <212> PRT <213> Homo sapiens <400> 133 Thr Asp Tyr Trp Met His 1 5 <210> 134 <211> 17 <212> PRT <213> Homo sapiens <400> 134 Thr Ile Asp Asn Ser Asp Ser Asp Thr Ser Leu Asn Gln Lys Phe Gln 1 5 10 15 Gly <210> 135 <211> 8* <212> PRT <213> Homo sapiens <400> 135 Ser Leu Asp Gly Tyr Tyr Asp Tyr 1 5 <210> 136 <211> 351 <212> DNA * Note: The original text has a typo in the line break in the middle of the translation. It should be "8" instead of "8*". The corrected translation is provided above.<213> Homo sapiens <400> 136 caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcagcag cgtgaaggtg 60 tcctgcaagg ctagcggcta cacattcacc gactactgga tgcactgggt ccgacaggct 120 ccaggacagg gacttgagtg gatgggcacc atcgacaaca gcgacagcga cacaagcctg 180 aaccagaaat tccagggcag agtgaccatc accgccgacg agtctagcag caccgcctac 240 atggaactga gcagcctgag aagcgaggac accgccgtgt actactgtgc cagatctctg 300 gacggctact acgattactg gggccaggga accctggtca ccgtttcttc t 351 <210> 137 <211> 117 <212> PRT <213> Homo sapiens <400> 137 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Trp Met His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Asp Pro Ser Asp Ser Asp Thr Ser Leu Asn Gln Ser Phe 50 55 60 Gln Gly His Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Asp Gly Tyr Tyr Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <2__10> 138 <2__11> 6 <2__12> PRT <2__13> Homo sapiens <4__00> 138 Thr Asp Tyr Trp Met His 1 5 <2__10> 139 <2__11> 17 <2__12> PRT <2__13> Homo sapiens <4__00> 139 Ile Ile Asp Pro Ser Asp Ser Asp Thr Ser Leu Asn Gln Ser Phe Gln 1 5 10 15 Gly <2__10> 140 <2__11> 8 <2__12> PRT <2__13> Homo sapiens <4__00> 140 Ser Leu Asp Gly Tyr Tyr Asp Tyr 1 5 <210> 141 <211> 351 <212> DNA <213> Homo sapiens <400> 141 gaagtgcagc tggttcagtc tggcgccgaa gtgaagaagc ctggcgagag cctgaagagatc 60 agctgcagg ccagcggcta caccttcacc gactactgga tgcactgggt ccgacagatg 120 cctggcaaag gcctggaatg gatggggcatc atcgacccca gcgacagcga cacaagcctg 180 aaccagtctt tccaggcca cgtgacaatc agcgccgaca agagcatcag caccgcctac 240 ctgcagtggt ctagcctgaa ggcctctgac accgccatgt actactgcgc cagatctctg 300 gacggctact acgactactg gggccaggga acactgtca ccgttagctc t 351 <210> 142 <211> 117 <212> PRT <213> Homo sapiens <400> 142 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Trp Met His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Thr Ile Asp Asn Ser Asp Ser Asp Thr Ser Leu Asn Gln Ser Phe 50 55 60 Gln Gly His Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Asp Gly Tyr Tyr Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 143 <211> 6 <212> PRT <213> Homo sapiens <400> 143 Thr Asp Tyr Trp Met His 1 5 <210> 144 <211> 17 <212> PRT <213> Homo sapiens <400> 144 Thr Ile Asp Asn Ser Asp Ser Asp Thr Ser Leu Asn Gln Ser Phe Gln 1 5 10 15 Gly <210> 145 <211> 8 <212> PRT <213> Homo sapiens <400> 145 Ser Leu Asp Gly Tyr Tyr Asp Tyr 1 5 <210> 146 <211> 351 <212> DNA <213> Homo sapiens <400> 146 gaagtgcagc tggttcagtc tggcgccgaa gtgaagaagc ctggcgagag cctgaagatc 60 agctgcaagg ccagcggcta caccttcacc gactactgga tgcactgggt ccgacagatg 120 cctggcaaag gcctggaatg gatgggcacc atcgacaaca gcgacagcga cacaagcctg 180 aaccagagct ttcagggcca cgtgacaatc agcgccgaca agtctatcag caccgcctac 240 ctgcagtggt ctagcctgaa ggcctctgac accgccatgt actactgcgc cagatctctg 300 gacggctact acgactactg gggccaggga acactggtca ccgttagctc t 351 <210> 147 <211> 117 <212> PRT <213> Homo sapiens <400> 147 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Trp Met His Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Thr Ile Asp Asn Ser Asp Ser Asp Thr Ser Leu Asn Gln Ser Phe 50 55 60 Gln Gly His Val Thr Ile Ser Ala Asp Lys Ser Ser Thr Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Asp Gly Tyr Tyr Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 148<�002754><211> 6 <212> PRT <213> Homo sapiens <400> 148 Thr Asp Tyr Trp Met His 1 5 <210> 149 <211> 17 <212> PRT <213> Homo sapiens <400> 149 Thr Ile Asp Asn Ser Asp Ser Asp Thr Ser Leu Asn Gln Ser Phe Gln 1 5 10 15 Gly <210> 150 <211> 8 <212> PRT <213> Homo sapiens <400> 150 Ser Leu Asp Gly Tyr Tyr Asp Tyr 1 5 <210> 151 <211> 351 <212> DNA <213> Homo sapiens <400> 151 gaagtgcagc tggttcagtc tggcgccgaa gtgaagaagc ctggcgagag cctgaagatc 60 agctgcaagg ccagcggcta caccttcacc gactactgga tgcactgggt ccgacagatg 120 cctggcaaag gcctggaatg gatgggcacc atcgacaaca gcgacagcga cacaagcctg 180 aaccagagct ttcagggcca cgtgacaatc agcgccgaca agtctagcac caccgcctac 240 ctgcagtggt ctagcctgaa ggcctctgac accgccatgt actactgcgc cagatctctg 300 gacggctact acgattactg gggccaggga accctggtca ccgtttcttc t 351 <210> 152 <211> 117 <212> PRT <213> Homo sapiens <400> 152 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Thr Ile Asp Asn Ser Asp Ser Asp Thr Thr Tyr Asn Pro Ser Phe 50 55 60 Gln Gly His Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Ser Val Asp Gly Tyr Leu Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 153 <211> 6 <212> PRT <213> Homo sapiens <400> 153 Thr Asp Tyr Trp Met His 1 5 <210> 154 <211> 17 <212> PRT <213> Homo sapiens <400> 154 Thr Ile Asp Asn Ser Asp Ser Asp Thr Thr Tyr Asn Pro Ser Phe Gln 1 5 10 15 Gly <210> 155 <211> 8 <212> PRT <213> Homo sapiens <400> 155 Ser Val Asp Gly Tyr Leu Asp Tyr 1 5 <210> 156 <211> 351 <212> DNA <213> Homo sapiens <400> 156 caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcagcag cgtgaaggtg 60 tcctgcaagg ctagcggcta cacattcacc gactactgga tgcactgggt ccgacaggct 120 ccaggacagg gacttgagtg gatgggcacc atcgacaaca gcgacagcga cacaacctac 180 aacccatctt tccagggcca cgtgacaatc agcgccgaca agagcatcag caccgcctac 240 ctgcagtggt ctagcctgaa ggcctctgac accgccatgt actactgtgc cagatctgtg 300 gacggctacc tggattactg gggccaggga accctggtca ccgtttcttc t 351 <210> 157 <211> 117 <212> PRT <213> Homo sapiens <400> 157 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Thr Ile Asp Asn Ser Asp Ser Asp Thr Thr Tyr Asn Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Val Asp Gly Tyr Leu Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 158 <211> 6 <212> PRT <213> Homo sapiens <400> 158 Thr Asp Tyr Trp Met His 1 5 <210> 159 <211> 17 <212> PRT <213> Homo sapiens <400> 159 Thr Ile Asp Asn Ser Asp Ser Asp Thr Thr Tyr Asn Gln Lys Phe Gln 1 5 10 15 Gly <210> 160 <211> 8 <212> PRT <213> Homo sapiens <400> 160 Ser Val Asp Gly Tyr Leu Asp Tyr 1 5 <210> 161 <211> 351 <212> DNA <​​​​​​​​​​​​​​​​ <211> 117 <212> PRT <213> Homo sapiens <400> 162 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Thr Ile Asp Asn Ser Asp Ser Asp Thr Thr Tyr Asn Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Val Asp Gly Tyr Leu Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 163 <211> 6 <212> PRT <213> Homo sapiens <400> 163 Thr Asp Tyr Trp Met His 1 5 <210> 164 <211> 17 <212> PRT <213> Homo sapiens <400> 164 Thr Ile Asp Asn Ser Asp Ser Asp Thr Thr Tyr Asn Gln Lys Phe Gln 1 5 10 15 Gly <210> 165 <211> 8 <212> PRT <213> Homo sapiens <400> 165 Ser Val Asp Gly Tyr Leu Asp Tyr 1 5 <210> 166 <211> 351 <212> DNA <213> Homo sapiens <400> 166 caggttcagc tggttcagtc tggcgccgaa gtgaagaaac ctggcagcag cgtgaaggtg 60 tcctgcaagg ctagcggcta cacattcacc gactactgga tgcactgggt ccgacaggct 120 ccaggacagg gacttgagtg gatgggcacc atcgacaaca gcgacagcga cacaacctac 180 aaccagaaat tccagggcag agtgaccatg accagagaca cctctacaag caccgtctac 240 atggaactga gcagcctgag aagcgaggac accgccgtgt actactgtgc cagatctgtg 300 gacggctacc tggattactg gggccaggga accctggtca ccgtttcttc t 351 <210> 167 <211> 107 <212> PRT <213> Homo sapiens <400> 167 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asn Ala Arg Thr Leu Pro Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Thr Thr Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 168 <211> 11 <212> PRT <213> Homo sapiens <400> 168 Arg Ala Ser Glu Asn Ile Tyr Ser Tyr Leu Ala 1 5 10 <210> 169 <211> 7 <212> PRT <213> Homo sapiens <400> 169 Asn Ala Arg Thr Leu Pro Glu 1 5 <210> 170 <211> 9 <212> PRT <213> Homo sapiens <400> 170 Gln His His Tyr Thr Thr Pro Trp Thr 1 5 <210> 171 <211> 321 <212> DNA <213> Homo sapiens <400> 171 gacatccaga tgacacagag ccctagcagc ctgtctgcca gcgtgggaga cagagtgacc 60 atcacctgta gagccagcga gaacatctac agctacctgg cctggtatca gcagaagcct 120 ggcaaggctc ccaagctgct gatctacaac gccagaacac tgcctgaggg cgtgccctct 180 agattcagcg gatctggctc tggcaccgac ttcaccctga caatctctag cctgcagcct 240 gaggacttcg ccacctacta ctgccagcac cactacacca caccttggac attcggccag 300 ggcaccaagg tggaaatcaa g 321 <210> 172 <211> 107 <212> PRT <213> Homo sapiens <400> 172 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Glu Asn Ile Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Phe Ala Lys Thr Leu Thr Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro [[ID=***]]65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His His Tyr Gly Thr Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 173 <211> 11 <212> PRT <213> Homo sapiens <400> 173 Arg Thr Ser Glu Asn Ile Tyr Ser Tyr Leu Ala 1 5 10 <210> 174 <211> 7 <212> PRT <213> Homo sapiens <400> 174 Phe Ala Lys Thr Leu Thr Asp 1 5 <210> 175 <211> 9 <212> PRT <213> Homo sapiens <400> 175 Gln His His Tyr Gly Thr Pro Trp Thr 1 5 <210> 176 <211> 321 <212> DNA <213> Homo sapiens <400> 176 gacatccaga tgacacagag ccctagcagc ctgtctgcca gcgtgggaga cagagtgacc 60 atcacctgta gaaccagcga gaacatctac agctacctgg cctggtatca gcagaagcct 120 ggcaaggctc ccaagctgct gatctacttc gccaagacac tgaccgacgg cgtgccctct 180 agattcagcg gatctggctc tggcaccgac ttcaccctga caatctctag cctgcagcct 240 gaggacttcg ccacctacta ctgccagcac cactacggca caccttggac attcggccag 300 ggcaccaagg tggaaatcaa g 321

Claims

1. Use of an antibody or antigen-binding moiety thereof conjugated to TSLP in the preparation of a medicament for the treatment of asthma or chronic obstructive pulmonary disease, wherein said antibody or antigen-binding moiety comprises a combination of CDRs selected from the following heavy and light chains: (1) Each contains the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 78, 79 and 80 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 173, 174 and 175 respectively; (2) Each contains the heavy chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 98, 79 and 80 respectively, and the light chain CDR1, CDR2 and CDR3 sequences shown in SEQ ID NO: 173, 174 and 175 respectively; The antibody is a humanized antibody.

2. Use of a nucleic acid molecule encoding the antibody or its antigen-binding portion as described in claim 1 in the preparation of a medicament for treating asthma or chronic obstructive pulmonary disease.

3. Use of a carrier containing the nucleic acid molecule of claim 2 in the preparation of a medicament for treating asthma or chronic obstructive pulmonary disease.

4. Use of cells containing the nucleic acid molecule of claim 2 or the carrier of claim 3 in the preparation of a medicament for treating asthma or chronic obstructive pulmonary disease.

5. Use of a composition in the preparation of a medicament for treating asthma or chronic obstructive pulmonary disease, said composition comprising the antibody or antigen-binding portion thereof as claimed in claim 1, the nucleic acid molecule as claimed in claim 2, the carrier as claimed in claim 3, and / or the cell as claimed in claim 4.

6. Use of a kit in the preparation of a medicament for treating asthma or chronic obstructive pulmonary disease, the kit comprising the antibody or antigen-binding portion thereof as claimed in claim 1, the nucleic acid molecule as claimed in claim 2, the carrier as claimed in claim 3, the cell as claimed in claim 4, and / or the composition as claimed in claim 5.

7. Use of the antibody or antigen-binding portion thereof as claimed in claim 1, the nucleic acid molecule as claimed in claim 2, the carrier as claimed in claim 3, the cell as claimed in claim 4, and / or the composition as claimed in claim 5 in the preparation of a kit for treating asthma or chronic obstructive pulmonary disease.

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