Anti-thymic stromal lymphopoietin (tslp) antibodies and uses thereof

Abstract

The present application provides human TSLP antibodies and their medical uses. Also provided are murine antibodies, chimeric antibodies, humanized antibodies comprising the CDR regions of the antibodies, pharmaceutical compositions comprising the human TSLP antibodies, and uses of the antibodies in the manufacture of medicaments for the treatment of TSLP-mediated diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to an anti-TSLP antibody. This invention also relates to the use of this TSLP antibody. Background Technology

[0002] Human thymine stromal lymphopoietin (TSLP) is an IL-7-like, type I cytokine composed of a short chain of four helical bundles linked by three pairs of intrachain disulfide bonds. It is a member of the IL-2 cytokine family, and its encoding gene is located on chromosome 5q22.1. It transduces signals by binding to a heterodimer receptor, which consists of the IL-7Rα subunit and a unique component, TSLPR, homologous to common γ-receptor-like chains. This heterodimer belongs to the hematopoietic factor receptor family, and only when the receptor complex TSLPRα / IL-7Rα works together does it exhibit high affinity. TSLP is expressed by epithelial cells in the thymus, lung, skin, intestine, and tonsils, as well as airway smooth muscle cells, pulmonary fibroblasts, and stromal cells. These cells produce TSLP in response to pro-inflammatory stimuli, and TSLP drives allergic inflammatory responses through its activity on various innate immune cells, including dendritic cells, monocytes, and mast cells.

[0003] TSLP binds to its specific receptors TSLPR and IL-7Rα, forming a ternary complex that initiates signal transduction. For example, through phosphorylation of Janus kinase 1 (JAK1) and JAK2, it activates signal transduction and transcription factors (STAT) 1, STAT3, and STAT5, initiating pro-inflammatory signaling, promoting dendritic cell maturation and activation, and inducing the expression of functional type II helper T cells (Th2), regulatory T cells (Treg), and follicular helper T cells (Tfh), thereby regulating inflammatory processes in the skin, lungs, and intestinal mucosal barriers. TSLPR-deficient mice have lower Th2 immunity but normal or even enhanced Th1 immunity, suggesting that the number of TSLPRs is an important factor affecting the development of allergies.

[0004] TSLP plays a crucial role in the maturation of antigen-presenting cells and hematopoietic cells. TSLP is distributed among various immune cells (including dendritic cells, type II innate lymphocytes (ILC2), T cells, B cells, natural killer T cells (NKT), Treg cells, eosinophils, neutrophils, mast cells, and macrophages) and non-immune cells (platelets and sensory neurons). TSLP acts on various cell lines, particularly myeloid dendritic cells, by forming the TSLP-TSLPR-IL-7Rα complex. TSLP can activate human peripheral blood CD11c+ dendritic cells, upregulate the expression of major histocompatibility complex (MHC) class II, OX40 ligands (OX40L / CD134L, CD252), CD54, CD80, CD83, and CD86, as well as the activation marker DC-LAMP, promoting the differentiation of naive CD4+ T cells (Th0) into Th2 cells.

[0005] In particular, TSLP cannot stimulate myeloid dendritic cells (DCs) to produce Th1 polarizing cytokines IL-12, pro-inflammatory cytokines TNF-α, IL-1β, and IL-6, which is a key characteristic of TSLP in constructing a Th2 immune microenvironment. TSLP-DCs enhance the Th2 immune response by activating Th2 effector memory cells and inhibiting the production of FOXP3+ Tregs. TSLP interacts with CD4+ T cells, CD8+ T cells, and Treg cells, further promoting Th2 cell proliferation and activation. TSLP can also activate mast cells, intrinsic lymphocytes, epithelial cells, and macrophages, and synergistically promote the production of Th2 cytokines (IL-4, IL-5, and IL-13) with epithelial cell chemokines such as IL-25 and IL-33. In addition, TSLP can promote eosinophil activation and chemotaxis. TSLP significantly delayed eosinophil apoptosis in a concentration-dependent manner by upregulating the expression of CD18 and intercellular adhesion 1 (ICAM1), downregulating L-selectin, and inducing the production of IL-6 and chemokines CXCL8 and CXCL1, leading to eosinophil inflammation.

[0006] TSLP can balance innate and adaptive immunity by acting on myeloid and lymphoid cells, playing an important role in initiating and promoting Th2 cell-mediated allergic inflammation.

[0007] TSLP is overexpressed in several autoimmune diseases and inflammatory conditions, such as atopic dermatitis, food allergies, allergic rhinitis, Natherton syndrome, and asthma, indicating an important role for this cytokine in the pathogenesis of these allergic inflammatory diseases. In the animal models described, transgenic overexpression of TSLP in the skin or lungs and removal of the gene target effect of the negative regulator of TSLP lead to allergic inflammatory diseases very similar to human atopic dermatitis or asthma.

[0008] Atopic dermatitis (AD) is a chronic inflammatory skin disease. Defects in the skin-mucosal barrier are the trigger for the allergic process, while TSLP can act as an "alarm" for its development. First, AD lesions provide target sites for allergen invasion, triggering multiple pattern recognition receptors in epithelial cells, leading to the activation of nuclear factor-κB (NF-κB) and the production of reactive oxygen species (ROS). This promotes the release of inflammatory factors such as TSLP, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-25, and IL-33 by producing damage-associated molecular patterns (DAMPs) or pro-inflammatory cytokines (such as IL-1α, high-mobility group box 1 (HMGB1), uric acid, adenosine triphosphate, or S100 protein). TSLP promotes the upregulation of OX40L and Notch ligand expression in immature dendritic cells (DCs), downregulates IL-12 levels, and secretes Th2 cell chemokines (such as CCL17 and CCL22), subsequently stimulating the proliferation of innate lymphocytes and the production of Th2 cytokines. High levels of TSLP can also directly stimulate transient receptor potential kinin 1 (TRPA1)-positive cutaneous sensory neurons, triggering intense itching and exacerbating skin lesions. Thus, they collectively construct a Th2-dominant immune microenvironment that promotes the allergic process.

[0009] In food allergies, TSLP promotes the migration, maturation, and activation of Langerhans cells (LCs), fosters DC polarization, and induces in situ Th2 immunity. Simultaneously, TSLP is highly likely to act as an initiator of the allergic process, triggering a systemic Th2 immune response. Intradermal injection of TSLP induces food allergy in mice, including systemic basophil expansion, increased serum-specific IgE levels, and intestinal mast cell accumulation, promoting gastrointestinal inflammation. Conversely, reducing TSLP levels or removing basophils decreases susceptibility to intestinal food allergy. This suggests that the TSLP-basophil axis may be a potential therapeutic target for food allergy. Furthermore, potential target cells for TSLP-mediated food allergy include DCs, NKT cells, and Treg cells. TSLP stimulates DC maturation and activation, promotes the proliferation and differentiation of CD4+ Th0 cells into Th2 cells, and secretes large amounts of Th2 cytokines in peripheral blood and the esophagus. TSLP binding to NKT cell TSLPR significantly induces NKT cell recruitment of esophageal epithelium and the production of large amounts of IL-13, mediating milk allergy. Meanwhile, TSLP plays an important role in the differentiation of Foxp3+ Treg cells. Moreover, fluctuations in TSLP levels can lead to an imbalance in intestinal immune function, revealing that TSLP is crucial for maintaining intestinal homeostasis.

[0010] Allergic rhinitis is an allergic disease primarily characterized by Th2 immunity in the upper respiratory tract. Epithelial cell-derived histamine-mediated inflammation plays a crucial role, and epithelial cell-derived cytokines (such as IL-33, IL-25, and TSLP) produced by innate lymphocytes (ILC2) are key to initiating and driving the allergic immune response. Studies have shown increased expression of histamine receptor 4 (H4R) in the nasal mucosa of sensitized mice. H4R, as a potential regulator of DC activation and T cell polarization, can induce TSLP production in nasal epithelial cells, activating OX40L signaling on DCs and promoting the Th2 immune response. Th2 cytokines such as IL-4, IL-5, and IL-13 can further activate B cells, eosinophils, and mast cells, constituting systemic Th2 immune dominance and triggering the allergic process. Conversely, blocking H4R to reduce TSLP levels can alleviate allergic inflammation.

[0011] Asthma is a chronic disease characterized by airway inflammation, clinically manifested as recurrent episodes of wheezing, shortness of breath, chest tightness, and cough. Globally, approximately 300 million people suffer from asthma. The global prevalence of asthma has been increasing by about 1% annually over the past two decades. Current treatments include bronchodilators, corticosteroids, combinations of both (such as Seretide and Symbicort), leukotriene modifiers, long-acting cholinergic receptor antagonists (tiotropium bromide), and IgE antibodies, but none can control the condition in all asthma patients.

[0012] Numerous studies have demonstrated that approximately two-thirds of severe asthma cases are characterized by the overexpression of Th2 cytokines, with TSLP being a key factor in inducing this overexpression. The TSLP-TSLPR mechanism primarily operates through the JAK-STAT signaling pathway. Research suggests that upregulation of TSLP binds to TSLPR on dendritic cells (DCs), activating JAK, recruiting the transcription factor STAT5, and inducing downstream signal transduction, ultimately leading to DC activation. DC activation is characterized by upregulation of co-stimulatory molecules (such as CD80, CD40, and CD86) and the secretion of chemokines (TARC / CCL17, MDC / CCL22, and I-309 / CCL1), providing a favorable microenvironment for Th0 cell differentiation into Th2 cells, guiding a Th2-dominated inflammatory response, and accompanied by the release of factors (IL-4, IL-13, and IL-5). TSLP-transgenic mice are susceptible to asthma induced by specific antigens, while TSLP receptor knockout mice show significantly reduced symptoms. Based on the analysis of the mechanisms of asthma and inflammation, anti-cytokine (IL-4, IL-13, IL-5) drugs target only specific inflammatory molecules that drive asthma inflammation, making them suitable only for certain types of severe asthma patients, i.e., subgroups, such as eosinophilic asthma. TSLP targets are significantly different from IL4 and IL5; TSLP is active in the early upstream phase of the inflammatory cascade and may be applicable to a wide range of patients with severe, uncontrolled asthma.

[0013] Researchers have conducted extensive exploration and research on drugs targeting TSLP. In a dust mite-induced mouse asthma model, anti-TSLP monoclonal antibodies effectively blocked TSLP / TSLPR action, reversed airway inflammation, prevented tissue structural changes, and reduced airway hyperresponsiveness (AHR) and TGF-β1 levels. In a serum protein-induced mouse asthma model, anti-TSLP monoclonal antibodies effectively reduced the expression of Th2 factors (IL-4, IL-5, etc.). The safety of anti-TSLP monoclonal antibodies has also been well-established in monkeys. Moreover, anti-TSLP monoclonal antibodies in clinical trials have shown good objective response rates in early clinical trials, effectively alleviating symptoms in test patients.

[0014] Furthermore, researchers have found elevated TSLP levels in many different types of tumors. Tumors recruit immune cells to the tumor, inducing high TSLP expression in tumor-infiltrating bone marrow cells through the expression of IL-1α. TSLP can further induce tumor cells to express the anti-apoptotic protein BCL-2, thereby preventing tumor cell death. Therefore, TSLP is crucial for tumor survival. Compared to TSLP-exposed tumors, breast cancer cells have a higher mortality rate in tumors without TSLP. Further studies have shown that blocking TSLP in a breast cancer model can significantly inhibit the growth of breast tumors and prevent their lung metastasis. In addition, blocking TSLP can prevent not only the growth of breast cancer tumors but also many other tumors with elevated TSLP levels, such as pancreatic cancer, cervical cancer, and multiple myeloma.

[0015] Therefore, given the broad applicability of TSLP-binding antibodies, exploring and developing anti-TSLP antibodies based on clinical needs has significant biological and medical implications. Summary of the Invention

[0016] In a first aspect of the invention, an antibody binding to TSLP is provided, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises complementarity-determining regions HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein the amino acid sequence of HCDR1 is as shown in SEQ ID NO:12, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:13, SEQ ID NO:18, or SEQ ID NO:19, the amino acid sequence of HCDR3 is as shown in SEQ ID NO:14, the amino acid sequence of LCDR1 is as shown in SEQ ID NO:15, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:16 or SEQ ID NO:20, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:17. The HCDR and LCDR amino acid sequences are defined according to Kabat.

[0017] In some embodiments, HCDR1, HCDR2 and HCDR3 are shown as SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, respectively, and LCDR1, LCDR2 and LCDR3 are shown as SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17, respectively.

[0018] In some embodiments, HCDR1, HCDR2 and HCDR3 are shown as SEQ ID NO:12, SEQ ID NO:18 and SEQ ID NO:14, respectively, and LCDR1, LCDR2 and LCDR3 are shown as SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17, respectively.

[0019] In some embodiments, HCDR1, HCDR2 and HCDR3 are shown as SEQ ID NO:12, SEQ ID NO:19 and SEQ ID NO:14, respectively, and LCDR1, LCDR2 and LCDR3 are shown as SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17, respectively.

[0020] In some embodiments, HCDR1, HCDR2 and HCDR3 are shown as SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, respectively, and LCDR1, LCDR2 and LCDR3 are shown as SEQ ID NO:15, SEQ ID NO:20 and SEQ ID NO:17, respectively.

[0021] In some embodiments, the amino acid sequence of the heavy chain variable region of the TSLP antibody is as shown in SEQ ID NO: 1, 3, 4, 5, 9 or 10.

[0022] In some embodiments, the amino acid sequence of the light chain variable region of the antibody is as shown in SEQ ID NO:2, 6, 7, 8 or 11.

[0023] In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:2.

[0024] In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:3, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:6.

[0025] In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:3, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:7.

[0026] In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:3, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:8.

[0027] In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:4, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:6.

[0028] In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:4, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:7.

[0029] In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:4, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:8.

[0030] In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:5, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:6.

[0031] In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:5, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:7.

[0032] In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:5, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:8.

[0033] In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:5, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:11.

[0034] In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:9, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:7.

[0035] In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:10, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:7.

[0036] In some implementations, the antibody comprises a heavy chain constant region and a light chain constant region.

[0037] In some embodiments, the antibody includes a heavy chain constant region selected from IgG1, IgG2, or IgG4 subtypes, and the antibody includes a light chain constant region selected from κ or λ subtypes.

[0038] In some embodiments, the antibody comprises an IgG1 subtype heavy chain constant region and the antibody comprises a light chain constant region selected from the κ subtype.

[0039] In some embodiments, the IgG1 subtype constant region sequence is as shown in SEQ ID NO:23 or its mutant SEQ ID NO:22, and the light chain constant region sequence is as shown in SEQ ID NO:21.

[0040] In some implementations, the antibody comprises a full antibody that binds to the TSLP antigen.

[0041] In some implementations, the antibody is an antigen-binding fragment of TSLP.

[0042] In some embodiments, the antibody is a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fv fragment, and a scFv fragment.

[0043] In some embodiments, the antibody is capable of binding to human TSLP (e.g., SEQ ID NO: 28, 29, 34 or 35) and cynomolgus monkey TSLP (e.g., SEQ ID NO: 26 or 27).

[0044] In some implementations, the antibody inhibits the binding of human TSLP to the human TSLP receptor.

[0045] In some embodiments, the antibody inhibits the binding of human TSLP to the human TSLP receptor hTSLPR / IL7Ra (e.g., a single-chain human TSLPR.IL7Ra fusion polypeptide with the amino acid sequences shown in SEQ ID NO:32 and 33, or an hTSLPR / IL7Ra knob / hole dimer formed by fusing the two chains of SEQ ID NO:30 and SEQ ID NO:31).

[0046] In a second aspect of the invention, a nucleic acid molecule is provided that encodes the antibody or antigen-binding fragment thereof described in the first aspect.

[0047] In a third aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0048] (a) the TSLP antibody described in the first aspect of the present invention; and

[0049] (b) Pharmacologically acceptable excipients, diluents or pharmaceutically acceptable carriers.

[0050] In some embodiments, the pharmaceutical composition also contains other drugs for treating cancer (or tumors), autoimmune diseases, or inflammatory diseases.

[0051] In a fourth aspect of the invention, the use of the antibody described in the first aspect or the pharmaceutical composition described in the third aspect in the preparation of a medicament for the prevention or treatment of TSLP-mediated diseases is provided.

[0052] In some implementations, the TSLP-mediated disease is cancer (tumor), autoimmune disease, or inflammatory disease.

[0053] In some implementations, the TSLP-mediated diseases or conditions include breast cancer, pancreatic cancer, gastric cancer, cervical cancer, colorectal cancer, lung cancer, melanoma, B-cell lymphoma, myeloma, asthma, idiopathic pulmonary fibrosis, atopic dermatitis, allergic conjunctivitis, allergic rhinitis, allergic sinusitis, urticaria, Naseton's syndrome, eosinophilic esophagitis, food allergy, allergic diarrhea, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis, allergic fungal sinusitis, chronic pruritus, systemic lupus erythematosus, rheumatoid arthritis, and keratoconjunctivitis. Ron's disease, psoriasis, chronic nephritis, chronic obstructive pulmonary disease, systemic sclerosis, multiple sclerosis, keloid, ulcerative colitis, nasal polyposis, chronic eosinophilic pneumonia, eosinophilic bronchitis, celiac disease, Churg-Strauss syndrome, eosinophilic myalgia syndrome, hypereosinophilic syndrome, eosinophilic granulomatosis with polyangiitis, inflammatory bowel disease, scleroderma, interstitial lung disease, fibrosis caused by chronic hepatitis B or C, radiation-induced fibrosis, and fibrosis caused by wound healing. Attached Figure Description

[0054] Figure 1 ELISA detection of chimeric antibody QP378379 binding to human TSLP protein.

[0055] Figure 2 ELISA was used to identify the chimeric antibody QP378379, which blocks the binding of human TSLP / TSLPR / IL7Ra proteins.

[0056] Figure 3 ELISA was used to identify the chimeric antibody QP378379 binding to cynomolgus monkey TSLP protein.

[0057] Figure 4 ELISA was used to identify the binding of humanized TSLP antibodies to human TSLP protein.

[0058] Figure 5ELISA was used to identify humanized anti-TSLP antibodies that block the binding of human TSLP to TSLPR / IL7Ra.

[0059] Figure 6 ELISA was used to identify the binding of humanized anti-TSLP antibodies to cynomolgus monkey TSLP protein.

[0060] Figure 7 : Reporter gene assay to identify the inhibition of the STAT5 signaling pathway by anti-TSLP antibody.

[0061] Figure 8 ELISA was used to identify the binding of TSLP antibodies to human TSLP protein.

[0062] Figure 9 ELISA was used to identify the blocking effect of anti-TSLP antibodies on the binding of human TSLP to TSLPR / IL7Ra.

[0063] Figure 10 ELISA was used to identify the binding of anti-TSLP antibodies to TSLP protein in cynomolgus monkeys.

[0064] Figure 11 : Reporter gene assay to identify the inhibition of the STAT5 signaling pathway by anti-TSLP antibody. Detailed Implementation

[0065] I. Terminology:

[0066] To facilitate understanding of the present invention, some technical and scientific terms of the present invention will be explained before describing the embodiments.

[0067] Unless otherwise expressly defined elsewhere in this application, all other technical and scientific terms used in this invention have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0068] The amino acid three-letter codes and single-letter codes used in this invention are as described in J. Boil. Chem., 243, p3558 (1968).

[0069] The "antibody" described in this invention includes not only complete antibodies, but also fragments, polypeptide sequences, and their derivatives and analogs that have antigen-binding activity.

[0070] The antigen-binding fragment refers to one or more portions of a full-length antibody that retain the ability to bind to an antigen (e.g., HER2) and competes with the intact antibody for specific binding to the antigen. See also Fundamental Immunology, Ch. 7 (Paul W., ed., 2nd edition, Raven). Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding moieties can be generated by recombinant DNA technology or by enzymatic or chemical fragmentation of intact antibodies. In some cases, antigen-binding moieties include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), and chimeric antibodies containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the peptide. Antigen-binding moieties (e.g., the aforementioned antibody fragments) of antibodies can be obtained from a given antibody (e.g., monoclonal antibody 2E12) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical fragmentation), and the antigen-binding moieties of antibodies can be screened for specificity in the same manner as for intact antibodies. The term "Fd fragment" refers to an antibody fragment consisting of VH and CH1 domains; the term "Fv fragment" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody; the term "dAb fragment" refers to an antibody fragment consisting of the VH domain (Ward et al., Nature). 341:544-546(1989)); The term “Fab fragment” refers to an antibody fragment consisting of VL, VH, CL and CH1 domains; The term “F(ab')2 fragment” refers to an antibody fragment containing two Fab fragments connected by a disulfide bridge on the hinge region.

[0071] As used herein, the terms “fragment,” “derivative,” and “analyte” refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence or secretion sequence, or a sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with a 6His tag). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0072] Both the light and heavy chains are divided into structural homology regions and functional homology regions. The terms "constant" and "variable" are used functionally. In this respect, it should be understood that the variable domains of both the light chain (VL) and heavy chain (VH) portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and heavy chain (CH1, CH2, or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, and complement binding. By convention, the farther the constant domain is from the antibody's antigen-binding site or N-terminus, the higher its number. The N-terminus is the variable region, and the C-terminus is the constant region; the CH3 and CL domains actually contain the carboxyl terms of the heavy and light chains, respectively.

[0073] Chimeric antibody: The term "chimeric antibody" (or its antigen-binding fragment) refers to an antibody molecule (or its antigen-binding fragment) in which (a) the constant region or a portion thereof is altered, substituted, or replaced such that the antigen-binding site (variable region) is linked to a constant region of a different or altered type, effector function, and / or kind, or to a completely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug, etc.) that confers novel properties to the chimeric antibody; or (b) the variable region or a portion thereof is altered, substituted, or replaced with a variable region having a different or altered antigen specificity. For example, a mouse antibody can be modified by replacing its constant region with a constant region derived from a human immunoglobulin. Due to the replacement with a human constant region, the chimeric antibody can retain its antigen recognition specificity while exhibiting reduced antigenicity in humans compared to the original mouse antibody.

[0074] Humanization: The “humanized” form of the term nonhuman (e.g., mouse) antibody is a chimeric antibody containing a minimal sequence derived from a nonhuman immunoglobulin. In most cases, a humanized antibody is a human immunoglobulin (receptor antibody) in which residues from the receptor hypervariable region are replaced by residues from a hypervariable region (donor antibody) of a nonhuman species (such as a mouse, rat, rabbit, or nonhuman primate) with the desired specificity, affinity, and capacity. In some cases, the framework region (FR) residues of a human immunoglobulin are replaced by corresponding nonhuman residues. Furthermore, humanized antibodies may contain residues not found in either the recipient or donor antibody. These modifications are made to further improve antibody performance. Typically, a humanized antibody will contain substantially all of the following: at least one, typically two, variable domains, wherein all or substantially all hypervariable loops correspond to those of nonhuman immunoglobulins, and all or substantially all FRs are those of the human immunoglobulin lo sequence. Humanized antibodies optionally also include an immunoglobulin constant region (Fc), typically at least a portion of the human immunoglobulin constant region. For further details, see Jones et al., 1986, Nature 321:522-525; Riechmann et al., 1988, Nature 332:323-329; and Presta, 1992, Curr. Op. Struct. Biol. 2:593-596. See also the following commentaries and their cited references: Vaswani and Hamilton, 1998, Ann. Allergy, Asthma & Immunol. [Annals of Allergy, Asthma & Immunology] 1:105-115; Harris, 1995, Biochem. Soc. Transactions [Journal of the Biochemist Society] 23:1035-1038; Hurle and Gross, 1994, Curr. Op. Biotech. [Current Perspectives on Biotechnology] 5:428-433.

[0075] In vitro antibody affinity maturation is a process that mimics the in vivo maturation of antibodies in vitro. It involves using a certain antibody as a template to construct a mutant library, screening the mutant library, and obtaining antibodies with significantly improved biological activity.

[0076] The “TSLP” of this invention includes variants, isotypes, homologs, orthologs and paralogs of TSLP.

[0077] The antibodies of this invention include murine antibodies, chimeric antibodies, and humanized antibodies.

[0078] The diseases mediated by TSLP in this invention are not limited, as long as they are TSLP-related diseases. For example, the molecularly induced therapeutic response of this disclosure can be achieved by binding human TSLP and then inhibiting the binding of TSLP to its receptor, or by killing cells that overexpress TSLP.

[0079] As used in this invention, "autoimmune disease" refers to a disease caused by damage to one's own tissues due to an immune response to self-antigens, including but not limited to scleroderma, systemic lupus erythematosus, rheumatoid arthritis, Churg-Strauss syndrome, Wegener's granulomatosis, and pulmonary hemorrhage-nephritis syndrome.

[0080] As used herein, the term "inflammatory disease" refers to a general term for diseases in which inflammation is the primary destructive factor. Inflammation is a biological response of tissues to harmful stimuli; it is a pathological condition accompanied by tissue degeneration, circulatory disturbances, and fluid exudation. Examples of inflammatory diseases include acute and chronic conditions, including but not limited to asthma, allergic pneumonia, atopic dermatitis, rhinitis, Crohn's disease, ankylosing spondylitis, rheumatic fever, fibromyalgia, psoriatic arthritis, chronic nephritis, Sjögren's syndrome, and multiple sclerosis.

[0081] The TSLP-mediated cancers (or tumors) of this invention include breast cancer, pancreatic cancer, gastric cancer, cervical cancer, colorectal cancer, lung cancer, melanoma, B-cell lymphoma, and myeloma. II. Specific Implementation Methods

[0083] Experimental methods not specifying particular conditions in this work are generally performed under standard conditions or as recommended by the raw material or product manufacturer. Examples include molecular cloning, laboratory manuals, Cold Spring Harbor Laboratory, contemporary molecular biology methods, cell biology, etc. Reagents not specifying their origin are commercially available, standard reagents.

[0084] Example 1: Cloning, expression, and purification of TSLP, recombinant TSLP receptor protein, and control antibody Tezepelumab.

[0085] Cloning design: Recombinant proteins including human TSLP, human TSLP (R127A, R130A), cynomolgus monkey TSLP, cynomolgus monkey TSLP (R127A, R130A), human TSLPR, and human IL7Ra were designed, each with different tags such as His, flag, and FC, for purification and identification. See Table 1 for the recombinant protein numbering and sequence number correspondence.

[0086] Table 1: Cloning and design of recombinant TSLP and TSLP receptor proteins

[0087]

[0088]

[0089] According to publicly available information (WHO Drug Information Vol.30, No.1, 2016), the full-length antibody sequence of Amgen's anti-TSLP antibody Tezepelumab (AMG157) was obtained (as shown below). Full-length light and heavy chain expression plasmids QD119 and QD120 were constructed and transiently transfected to produce the protein, designated QP119120, which will be used as a control in subsequent experiments.

[0090] QD119 / pQD-Tezepelumab-Vλ / SEQ ID NO:24:

[0091] SYVLTQPPSVSVAPGQTARITCGGNNLGSKSVHWYQQKPGQAPVLVVYDDSDRPSWIPERFSGSNSGNTATLTISRGEAGDEADYYCQVWDSSSDHVVFGGGTKLTV LGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0092] QD120 / pQDH-Tezepelumab-H(IgG2) / SEQ ID NO:25:

[0093] QMQLVESGGGVVQPGRSLRLSCAASGFTFRTYGMHWVRQAPGKGLEWVAVIWYDGSNKHYADSVKGRFTITRDNSKNTLNLQMNSLRAEDTAVYYCARAPQWELVHEAFDIW GQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKC CVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKT ISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0094] Transient expression: One day before transfection of 293E cells, the cells to be transfected were centrifuged and the medium was changed to adjust the cell density to approximately 0.5 × 10⁻⁶. 6 / ml. Prepare plasmid and transfection reagent PEI. The required amount of plasmid for transfection is 100ug / 100ml of cells, and the mass ratio of PEI to plasmid is 2:1. Mix the plasmid and PEI thoroughly and let stand for 15 min. Slowly add the plasmid and PEI mixture into 293E cells and incubate in a shaker at 37°C, 120 rpm, with 8% CO2. On the fifth day after transfection, collect the cell supernatant by centrifuging at 4700 rpm for 20 min.

[0095] Protein purification:

[0096] Protein A purification: Pass the solution through a column with equilibration buffer at least 3 CV (20 mL actual volume), ensuring the pH and conductivity of the final effluent in the instrument are consistent with the equilibration buffer, at a flow rate of 1 mL / min. Pass 40 mL of the centrifuged culture supernatant through the column at a flow rate of 0.33 mL / min. Pass the solution through the column with equilibration buffer again at at least 3 CV (20 mL actual volume), ensuring the pH and conductivity of the final effluent in the instrument are consistent with the equilibration buffer, at a flow rate of 0.33 mL / min. Pass the solution through the column with elution buffer. Begin collecting the elution peak (PAC-EP) when UV280 rises to 15 mAU and stop collecting when UV280 falls to 15 mAU, at a flow rate of 1 mL / min. After sample collection, adjust the pH of PAC-EP to neutral using pH adjustment buffer.

[0097] Example 2: Construction of a stable BaF3-TSLPR-IL7Ra cell line expressing TSLPR and IL7Ra

[0098] To conduct TSLP-STAT5 luciferase reporter gene assays and screen for antibodies that can block the binding of TSLP to TSLPR / IL7Ra and induce activation of the downstream STAT5 signaling pathway, our laboratory constructed a stable BaF3-TSLPR-IL7Ra cell line simultaneously expressing TSLPR and IL7Ra. BaF3 cells (original mouse B cell line) were purchased from Peking Union Medical College Hospital. The plasmids PCDNA3.1-TSLPR-IL-7Ra (QD1246) and PGL4.52 (promega) were co-transfected into BaF3 cells via electroporation. Forty-eight hours post-transfection, all cells were collected, centrifuged, resuspended in fresh culture medium, and selected using G418 1.2 mg / mL and hygmycin 1 mg / mL. The medium was changed by centrifugation every 2-3 days, and the selection process lasted for 2-3 weeks until cell viability reached over 95%. Single-clone plates were used to select single clones, and BaF3-TSLPR-IL7Ra single-cell lines that stably expressed TSLPR and IL7Ra were detected by FACS.

[0099] Example 3: Hybridoma fusion screening and acquisition of anti-human TSLP monoclonal antibody

[0100] Mouse Immunization: Anti-human TSLP monoclonal antibodies were produced by immunizing mice. Balb / c mice, female, 6 weeks old, were used in the experiment. Housing environment: SPF grade. After purchase, mice were housed in a laboratory environment for one week with a 12 / 12-hour light / dark cycle, temperature 20-25℃, humidity 40-60%. Balb / c mice were immunized initially with 50ug / mouse of recombinant protein QP1005 (human TSLP-his) using Freund's complete adjuvant (CFA). Two weeks later, these mice were alternately immunized with 25ug / mouse of recombinant protein QP1005 (human TSLP-his) plus Freund's incomplete adjuvant (IFA) or recombinant protein QP312 (cyno TSLP-his) plus aluminum salt Alum + CpG ODN 1826.

[0101] Cell fusion: Splenic cells were fused from mice with high antibody titers in their serum. 72 hours prior to fusion, the selected mice were immunized by intraperitoneal sprint injection. Optimized PEG-mediated fusion procedures were used to fuse splenic lymphocytes with myeloma Sp2 / 0 cells to obtain hybridoma cells. The fused hybridoma cells were resuspended in HAT complete medium (IMDM medium containing 20% ​​FBS, 1×HAT, and 1×OPI) and aliquoted into 96-well cell culture plates (1×10⁻⁶ cells / well). 5Cells were cultured at 37°C and 5% CO2, 150 μL / well. On day 5 post-fusion, IMDM medium (containing 2×HAT and 1×OPI) with 20% FBS was added, 50 μL / well, and cultured at 37°C and 5% CO2. On days 7-8 post-fusion, the medium was completely replaced according to cell growth density, and 250 μL / well was cultured in HT complete medium (IMDM medium containing 20% ​​FBS, 1×HT, and 1×OPI), at 37°C and 5% CO2.

[0102] Hybridoma cell screening: Based on cell growth density, ELISA was performed on days 10-14 post-fusion to screen for anti-TSLP antibodies in the hybridoma supernatant. The supernatant from the hybridoma fusion wells was used for initial screening in a 96-well plate using ELISA. Wells where the anti-TSLP antibody in the supernatant could bind to human TSLP recombinant protein QP1328 were considered positive. Wells where the anti-TSLP antibody in the supernatant could block the binding of TSLP / TSLPR / IL7Ra were also considered positive. Further ELISA was performed on the positive wells to detect clones in the supernatant where the anti-TSLP antibody bound to cynomolgus monkey TSLP recombinant protein QP313 and simultaneously blocked the binding of TSLP / TSLPR / IL7Ra; these were considered positive clones for anti-TSLP antibodies. Positive clones were expanded and promptly transferred to 24 / 6-well plates. ELISA was used to re-detect the cell culture supernatant by binding to human TSLP and cynomolgus monkey TSLP. Clones that could block the binding of TSLP / TSLPR / IL7Ra were identified as positive clones for anti-TSLP antibodies, yielding the positive clone mab177A12. The ELISA results are shown in Table 2. The positive clone mab177A12 was subjected to 2-3 rounds of limiting dilution to single-cell clones, and single-cell lines were cryopreserved.

[0103] Table 2: Screening for hybridoma-positive clones using ELISA with and without binding or blocking.

[0104]

[0105] Obtaining antibody sequences by hybridoma monoclonal antibody sequencing: mRNA was extracted from hybridoma-positive monoclonal cell lines, and the mRNA was reverse transcribed into cDNA. The cDNA was then used as a template for PCR amplification. PCR-positive clones were selected for sequencing, and the light and heavy chain variable region sequences of the monoclonal antibody were obtained through sequence analysis.

[0106] SEQ ID NO:1mab177A12 VH

[0107] QVQLQQPGAELVKPGASVKMSCKASGYTFTSYWMHWVKQRPGQGLEWIGVIDPSDTYTTYNQKFKGKATLTVDTSSSTAYMQLSSLTSEDSAVYYCTRSLDGFFDYWGQGTTLTVSS

[0108] SEQ ID NO:2mab177A12 VL

[0109] DIQMTQSPASSLSASVGETVSITCRASENIYSYLAWYQQKQGKSPQLLVYFAKTLAAGVPSRFSGSGSGTQFSLKINSLQPEDFGNYYCQHHYDTPWTFGGGTKLEIK

[0110] SEQ ID NO:1mab177A12 VH corresponds to HCDR1, HCDR2, and HCDR3 sequences SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14, respectively; SEQ ID NO:2mab177A12 VL corresponds to LCDR1, LCDR2, and LCDR3 sequences SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, respectively; the specific HCDR and LCDR amino acid sequences are defined according to the Kabat rules and are shown in Table 3.

[0111] Table 3: CDR sequence and numbering of the variable region of mab177A12 light and heavy chains

[0112]

[0113] Example 3: Construction of anti-human TSLP monoclonal chimeric antibody

[0114] Cloning: Using the mouse monoclonal antibody sequence obtained from the monoclonal antibody sequencing in Example 3 as a template, primers were designed to amplify the antibody VH / VK gene fragments by PCR. Expression vector pQD (fragment with signal peptide and constant region gene (CH1-FC / CL)) was constructed and digested using restriction enzymes. Utilizing the unique characteristics of restriction endonucleases such as BsmBI, which recognize sequences different from their digestion sites, expression vector pQD (fragment with signal peptide and constant region gene (CH1-FC / CL)) was designed and constructed. The vector was digested with BsmBI and the gel was recovered for later use. The PCR-amplified antibody VH / VK gene fragments and the BsmBI-digested and recovered expression vector pQD (fragment with signal peptide and constant region gene (CH1-FC / CL)) were added to DH5H competent cells at a 3:1 ratio. The cells were incubated at 0°C on ice for 30 min, followed by a heat shock at 42°C for 90 s. Five volumes of LB medium were added, and the cells were incubated at 37°C for 45 min. The cells were then plated on LB-Amp plates and cultured overnight at 37°C. Single clones were picked and sent for sequencing to obtain the target clones. The sequences and protein expression numbers of the variable regions of the light and heavy chains of each clone are shown in Table 4. In this table, all antibody light chain variable regions are fused with the kappa light chain constant region CL to form the full-length chimeric antibody light chain, and antibody heavy chain variable regions are fused with the human IgG1 constant region to form the full-length chimeric antibody heavy chain. The sequences of CL and human IgG1 constant region are shown below:

[0115] CL SEQ ID NO:21

[0116] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0117] Human IgG-Fc SEQ ID NO:23

[0118] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0119] Table 4: Clone numbers and protein composition of chimeric antibody construction

[0120]

[0121] Transient expression: HEK293E was used to adjust cell density to 1×10⁻⁶ cells / cells. 6 Prepare plasmid and PEI transfection reagent. The required plasmid quantity for transfection is 100ug / 100ml of cells, using a PEI to plasmid mass ratio of 2:1. Mix the plasmid and PEI thoroughly and let stand for 15 min. Slowly add the plasmid and PEI mixture into HEK293E cells and incubate in a shaker at 37°C, 120 rpm, with 8% CO2. On the fifth day after transfection, collect the cell supernatant by centrifuging at 4700 rpm for 20 min.

[0122] Protein purification: Protein A purification: Connect a purification column packed with 1 ml of Protein A packing material to the SCG protein purification system; pass the column through equilibration buffer (1×PBS, pH 7.4) at least 3 CV (actual volume 20 ml), ensuring the pH and conductivity of the final effluent from the instrument are consistent with the equilibration buffer, at a flow rate of 1 ml / min; after centrifuging the cell culture medium at 4°C using a refrigerated centrifuge, pass the supernatant through the column at a loading volume of 40 ml, at a flow rate of 0.33 ml / min; after loading, pass the column through equilibration buffer (1×PBS, pH 7.4) at least 3 CV (actual volume 20 ml), ensuring the pH and conductivity of the final effluent from the instrument are consistent with the equilibration buffer, at a flow rate of 0.33 ml / min; pass the column through elution buffer (50 mM / L citric acid, pH 3.5), starting to collect the elution peak (PAC-EP) when UV280 rises to 15 mAU, and stopping collection when UV280 falls to 15 mAU, at a flow rate of 1 ml / min. After sample collection, PAC-EP was adjusted to neutral pH using 8.02M / L Tris-HCl solution. The buffer was replaced with PBS by centrifugation in the ultrafiltration tube. Protein purity was identified by SDS-PAGE and HPLC-SEC. Proteins with a purity greater than 95% were reserved for use.

[0123] ELISA identification of chimeric antibody binding to human TSLP protein: Coating: Dilute protein QP1328 (FC fusion of human TSLP) to 1 μg / ml with PBS, add 60 μl / well to the ELISA plate, and incubate at 4°C for 18-24 h. Blocking: Wash the incubated ELISA plate using a Biotek automated plate washer, washing twice with PBS. After washing, add 200 μl / well of 3% BSA blocking buffer and block at room temperature for 1 h. Antibody incubation: Incubate different concentrations of human TSLP chimeric antibody at room temperature for 1 h. Wash the incubated ELISA plate using a Biotek automated plate washer, washing three times with PBST. Incubation of enzyme-labeled antibody: The enzyme-labeled antibody GoatAnti-Fab (HRP) needs to be diluted 1:5000 with blocking buffer, 60 μl / well, and incubated at room temperature for 1 h; TMB color development: Wash the incubated ELISA plate using a Biotek automatic plate washer, wash 5 times with PBST, then add 100 μl / well of substrate color development solution TMB, and incubate at room temperature in the dark for 10 min; Termination: After color development, quickly add 100 μL / well of stop solution 1M H2SO4 to terminate the reaction. Reading: Measure the OD value at a wavelength of 450 nm using a microplate reader. Result analysis: Data entry and analysis were performed using Graphpad Prism software. Results are as follows: Figure 1 As shown, the chimeric antibody QP378379 binds to recombinant human TSLP protein.

[0124] ELISA identification of chimeric antibody blocking human TSLP / TSLPR / IL7Ra protein binding: Coating: Dilute protein QP1171 (FC fusion protein of human TSLPR / IL7Ra) to 2 μg / ml with PBS, add 60 μl / well to ELISA plates, and incubate at 4℃ for 18-24 h. Blocking: Wash the incubated ELISA plates with PBS twice using a Biotek automated plate washer. After washing, add 200 μl / well of 3% BSA blocking buffer and block at room temperature for 1 h. Antibody incubation: Mix different concentrations of human TSLP chimeric antibody and 0.02 μg / ml Biotin-QP1005 (biotinylated human TSLP FC fusion protein) at a 1:1 ratio, add 60 μl / well to the blocked ELISA plates, and incubate at room temperature for 1 h. Wash the incubated ELISA plates with PBST three times using a Biotek automated plate washer. For incubation, the enzyme-labeled streptavidin (HRP) was first diluted 5000-fold with blocking buffer, 60 μl / well, and incubated at room temperature for 1 h. For TMB color development: the ELISA plates were washed with a Biotek automated plate washer after incubation, washed 5 times with PBST, and then 100 μl / well of TMB substrate solution was added, incubated at room temperature in the dark for 10 min. For termination: after color development, 100 μl / well of 1M H2SO4 stop solution was quickly added to terminate the reaction. Reading: The OD value was measured at 450 nm using a microplate reader. Results analysis: Data entry and analysis were performed using Graphpad Prism software. Results are as follows... Figure 2 As shown, the chimeric antibody QP378379 can block the binding of human TSLP to TSLPR / IL7Ra.

[0125] ELISA identification of chimeric antibody binding to cynomolgus monkey TSLP protein: Coating: Dilute protein QP313 (FC fusion of cynomolgus monkey TSLP) to 1 μg / ml with PBS, add 60 μl / well to ELISA plates, and incubate at 4°C for 18-24 h. Blocking: Wash the incubated ELISA plates twice with PBS using a Biotek automated plate washer. After washing, add 200 μl / well of 3% BSA blocking buffer and block at room temperature for 1 h. Antibody incubation: Incubate different concentrations of human TSLP chimeric antibody at room temperature for 1 h. Wash the incubated ELISA plates three times with PBST using a Biotek automated plate washer. Incubation of enzyme-labeled antibody: The enzyme-labeled antibody Goat Anti-Fab (HRP) needs to be diluted 1:5000 with blocking buffer, 60 μl / well, and incubated at room temperature for 1 h; TMB color development: Wash the incubated ELISA plate using a Biotek automatic plate washer, wash 5 times with PBST, then add 100 μl / well of substrate color development solution TMB, and incubate at room temperature in the dark for 10 min; Termination: After color development, quickly add 1M H2SO4 stop solution at 100 μL / well to terminate the reaction. Reading: Measure the OD value at a wavelength of 450 nm using a microplate reader. Result analysis: Data entry and analysis were performed using Graphpad Prism software. Results are as follows: Figure 3 As shown, the chimeric antibody QP378379 binds to the TSLP protein of cynomolgus monkeys.

[0126] Example 4: Humanization of anti-human TSLP monoclonal antibody

[0127] By comparing the germline gene database of heavy and light chain variable regions of human antibodies with the IMGT human antibody heavy and light chain variable regions and using MOE software, germline genes of heavy and light chain variable regions with high homology to QP378379 were selected as templates. The CDRs of the murine antibody were transplanted into the corresponding human templates to form variable region sequences in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Then, some important amino acid residues were selected for reverse mutation combinations. The amino acid residues were identified and annotated using the Kabat numbering system.

[0128] 1. Humanized molecular cloning of anti-TSLP antibodies

[0129] Primers were designed and PCR was used to construct the VH / VK gene fragments of each humanized antibody. Then, homologous recombination was performed with the expression vector pQD (a fragment containing a signal peptide and a constant region gene (CH1-FC / CL)) to construct the full-length antibody expression vector VH-CH1-FC-pQD / VK-CL-pQD.

[0130] Multiple primers were designed using the online software DNAWorks (v3.2.4) (http: / / helixweb.nih.gov / dnaworks / ) to synthesize the VH / VK gene fragment containing the required recombination gene: 5'-30bp signal peptide + VH / VK + 30bp CH1 / CL-3'. Following the instructions for TaKaRa Primer STAR GXL DNA polymerase, the designed primers were used for two-step PCR amplification to obtain the VH / VK gene fragment containing the required recombination gene. The expression vector pQD (containing the signal peptide and constant region gene (CH1-FC / CL) fragment) was constructed and digested using specific restriction endonucleases, such as BsmBI, which recognize sequences different from their digestion sites. The vector was digested with BsmBI, and the gel was recovered for later use. The recombinant expression vectors VH-CH1-FC-pQD / VK-CL-pQD were constructed. VH / VK contained the gene fragments required for recombination. The expression vector pQD (containing the signal peptide and constant region gene (CH1-FC / CL) fragments) was recovered by BsmBI restriction enzyme digestion and added to DH5H competent cells at a ratio of 3:1. The cells were incubated at 0°C on ice for 30 min, followed by heat shock at 42°C for 90 s. Five volumes of LB medium were added, and the cells were incubated at 37°C for 45 min. The cells were then plated on LB-Amp plates and cultured overnight at 37°C. Single clones were picked and sent for sequencing to obtain the target clones.

[0131] Table 5 shows the sequences and protein expression numbers of the light and heavy chain variable regions of each clone's humanized design. In this table, all antibody light chain variable regions are fused with the kappa light chain constant region (CL) to form the full-length light chain, and antibody heavy chain variable regions are fused with the human IgG1 constant region to form the full-length heavy chain.

[0132] Table 5: Clone number and protein of QP378379 antibody humanization construction.

[0133]

[0134] 2. Expression of humanized anti-TSLP antibody protein

[0135] HEK293E cell density was adjusted to 1×10⁻⁶. 6 Prepare plasmid and PEI transfection reagent. The required plasmid quantity for transfection is 100ug / 100ml of cells, using a PEI to plasmid mass ratio of 2:1. Mix the plasmid and PEI thoroughly and let stand for 15 min. Slowly add the plasmid and PEI mixture into HEK293E cells and incubate in a shaker at 37°C, 120 rpm, with 8% CO2. On the fifth day after transfection, collect the cell supernatant by centrifuging at 4700 rpm for 20 min.

[0136] 3. Protein purification, Protein A purification

[0137] Connect a purification column packed with 1 ml of Protein A packing material to the SCG protein purification system; pass the column through equilibration buffer (1×PBS, pH 7.4) at a flow rate of at least 3 CV (20 ml), ensuring the pH and conductivity of the final effluent from the instrument are consistent with the equilibration buffer, at a flow rate of 1 ml / min; after centrifuging the cell culture medium at 4°C using a refrigerated centrifuge, pass the supernatant through the column at a flow rate of 0.33 ml / min; after loading, pass the column through equilibration buffer (1×PBS, pH 7.4) at a flow rate of at least 3 CV (20 ml), ensuring the pH and conductivity of the final effluent from the instrument are consistent with the equilibration buffer, at a flow rate of 0.33 ml / min; pass the column through elution buffer (50 mM / L citric acid, pH 3.5), starting to collect the elution peak (PAC-EP) when UV280 rises to 15 mAU, and stopping collection when UV280 falls to 15 mAU, at a flow rate of 1 ml / min. After sample collection, PAC-EP was adjusted to neutral pH using 2M / L Tris-HCl solution (pH 8.0). The buffer was replaced with PBS by centrifugation in the ultrafiltration tube. Protein purity was identified by SDS-PAGE and HPLC-SEC. Proteins with a purity greater than 95% were reserved for use.

[0138] 4. Identification of humanized TSLP antibody activity (Binding-ELISA)

[0139] ELISA identification of humanized TSLP antibody binding to human TSLP protein: Coating: Dilute protein QP1328 (FC fusion of human TSLP) to 1 μg / ml with PBS, add 60 μl / well to ELISA plates, and incubate at 4°C for 18-24 h. Blocking: Wash the incubated ELISA plates twice with PBS using a Biotek automated plate washer. After washing, add 200 μl / well of 3% BSA blocking buffer and block at room temperature for 1 h. Antibody incubation: Incubate with different concentrations of humanized anti-TSLP antibody at room temperature for 1 h. Wash the incubated ELISA plates three times with PBST using a Biotek automated plate washer. Incubation of enzyme-labeled antibody: The enzyme-labeled antibody Goat Anti-Fab (HRP) needs to be diluted 5000 times with blocking buffer, 60 μl / well, and incubated at room temperature for 1 hour; TMB color development: Wash the incubated ELISA plate using a Biotek automatic plate washer, wash 5 times with PBST, and then add 100 μl / well of substrate color development solution TMB, incubating at room temperature in the dark for 10 minutes; Termination: After color development, quickly add 1M H2SO4 stop solution at 100 μL / well to terminate the reaction. Reading: Measure the OD value at a wavelength of 450 nm using a microplate reader. Result analysis: Data entry and analysis were performed using Graphpad Prism software. Results are as follows: Figure 4As shown, different variants of humanized anti-TSLP antibodies can all bind to recombinant human TSLP protein.

[0140] 5. Identification of humanized TSLP antibody activity (Blocking-ELISA)

[0141] Experimental Procedure: Dilute protein QP1171 (the FC fusion protein of human TSLPR / IL7Ra) to 2 μg / ml with PBS, and add 60 μl / well to the ELISA plate. Incubate at 4°C for 18-24 h. Blocking: Wash the incubated ELISA plate twice with PBS using a Biotek automated plate washer. After washing, add 200 μl / well of 3% BSA blocking buffer and block at room temperature for 1 h. Antibody Incubation: Mix different concentrations of human TSLP chimeric antibody and 0.02 μg / ml Biotin-QP1006 (the FC fusion protein of biotinylated human TSLP) at a 1:1 ratio, add 60 μl / well to the blocked ELISA plate, and incubate at room temperature for 1 h. Wash the incubated ELISA plate three times with PBST using a Biotek automated plate washer. For incubation, the enzyme-labeled streptavidin (HRP) was first diluted 5000-fold with blocking buffer, 60 μl / well, and incubated at room temperature for 1 h. For TMB color development: the ELISA plate was washed with a Biotek automated plate washer after incubation, washed 5 times with PBST, and then 100 μl / well of TMB substrate solution was added, incubated at room temperature in the dark for 10 min. For termination: after color development, 100 μl / well of stop solution (1M H₂SO₄) was quickly added to terminate the reaction. Reading: The OD value was measured at 450 nm using a microplate reader. Results analysis: Data entry and analysis were performed using Graphpad Prism software. Results are as follows... Figure 5 As shown, different variants of humanized anti-TSLP antibodies can block the binding of human TSLP to TSLPR / IL7Ra.

[0142] 6. ELISA identification of chimeric antibody binding to cynomolgus monkey TSLP protein

[0143] Coating: Dilute protein QP313 (FC fusion of cynomolgus monkey TSLP) to 1 μg / ml with PBS, add 60 μl / well to the ELISA plate, and incubate at 4°C for 18-24 h. Blocking: Wash the incubated ELISA plate twice with PBS using a Biotek automated plate washer. After washing, add 200 μl / well of 3% BSA blocking buffer and block at room temperature for 1 h. Antibody Incubation: Incubate with different concentrations of human TSLP chimeric antibody at room temperature for 1 h. Wash the incubated ELISA plate three times with PBST using a Biotek automated plate washer. Incubation of enzyme-labeled antibody: The enzyme-labeled antibody Goat Anti-Fab (HRP) needs to be diluted 1:5000 with blocking buffer, 60 μl / well, and incubated at room temperature for 1 h; TMB color development: Wash the incubated ELISA plate using a Biotek automatic plate washer, wash 5 times with PBST, then add 100 μl / well of substrate color development solution TMB, and incubate at room temperature in the dark for 10 min; Termination: After color development, quickly add 1M H2SO4 stop solution at 100 μL / well to terminate the reaction. Reading: Measure the OD value at a wavelength of 450 nm using a microplate reader. Result analysis: Data entry and analysis were performed using Graphpad Prism software. Results are as follows: Figure 6 As shown, all variants of the humanized anti-TSLP antibody bind to the TSLP protein in cynomolgus monkeys.

[0144] 7. Biacore assay to detect the binding affinity and kinetics between anti-TSLP antibody and recombinant human TSLP protein.

[0145] The affinity of anti-TSLP antibody for human TSLP was determined using a Biacore S200 analyzer. The experimental procedure is as follows:

[0146] Experimental procedure: A certain amount of the antibody to be tested was captured by affinity using a Protein A chip, and then human TSLP protein (QP1005) was passed through the chip surface. The reaction signal was detected in real time using Biacore to obtain the binding and dissociation curves.

[0147] The experimental results are shown in Table 6. The humanized anti-TSLP antibodies QP419423, QP420421, QP420422, QP420423 and the chimeric antibody QP378379 before humanization all bound to human TSLP protein.

[0148] Table 6: Affinity results of anti-TSLP antibodies binding to human TSLP

[0149]

[0150] 8. Reporter gene assay for inhibiting the STAT5 signaling pathway with anti-TSLP antibody

[0151] Experimental Procedure: Stable BaF3-TSLPR-IL-7Ra cell lines were washed twice with PBS and then plated in 4E4 wells onto opaque reaction plates (2E6 / ml, 20 μL / well). The culture medium was basal medium without IL-3. Different concentrations of anti-TSLP antibody (100 μg / mL, 4-fold serial dilution) were added and incubated for 20 min. Then, 20 μL / well of TSLP recombinant protein (QP1005) at a final concentration of 2 ng / mL was added. After 6 h of incubation, the results were detected using the One-Glo Luciferase Assay System (promega, E6120) kit. 60 μL / well of reagent was added, and the Bio-TECK instrument was used to read and record the results.

[0152] The results are as follows Figure 7 As shown, the humanized anti-TSLP antibodies QP419423, QP420421, QP420422, QP420423 and the pre-humanized chimeric antibody QP378379 can all inhibit the downstream STAT5 signaling pathway caused by TSLP and its receptor binding.

[0153] Example 5: Affinity maturation of humanized anti-TSLP antibody QP420422

[0154] 1. Construction of affinity maturation antibodies

[0155] Humanized phage vectors were constructed: Humanized QP420422 was constructed into phage vectors in scFv mode (VH-(GGGGS)3-VL) as wild-type sequences (i.e., mutant sequences obtained relative to affinity maturation screening). VH, the (GGGGS)3 adapter, and VL were spliced ​​using overlap PCR, and then ligated into the phage vectors using NcoI and NotI restriction enzyme sites.

[0156] Construction of phage display libraries: Using the constructed wild-type scFv as a template, a mutation primer was designed for each CDR. Degenerate primers were used to introduce mutations into all CDR regions to construct a mutant library. PCR fragments were digested with NcoI and NotI enzymes, ligated into phage vectors, and finally electroporated into E. coli TG1. A separate library was constructed using degenerate primers for each CDR.

[0157] Library panning: After the library is phage-packaged to extract phage particles for panning, liquid chromatography panning is performed using biotinylated QP1005 (human TSLP recombinant protein) antigen and streptavidin magnetic beads. The first round of panning uses a concentration of 5 nM, and each subsequent round reduces the antigen concentration by 10 times compared to the previous round. After three rounds of panning, 250 clones are selected for phage ELISA to detect binding activity, and positive clones are sequenced.

[0158] ELISA assay: After alignment analysis of the sequenced clones and removal of redundant sequences, the non-redundant sequences were converted into full-length IG (hIgG1,κ) for expression in mammalian cells 293E. The affinity-purified full-length IG was then subjected to ELISA assay.

[0159] Three mutant antibodies were finally obtained: clones QD3611 (SEQ ID NO:9) and QD3616 (SEQ ID NO:10) with heavy chain CDR2 mutation based on QD420 (SEQ ID NO:5) as shown in Table 7, and clone QD3625 (SEQ ID NO:11) with light chain CDR2 mutation based on QD422 (SEQ ID NO:7) as shown in Table 8.

[0160] Table 7: CDR2 mutations in the heavy chain of the maturation molecule for TSLP antibody affinity

[0161]

[0162] The light chain CDR2 mutations of QD422 as the parent gene are shown in Table 7 for clone QD3625:

[0163] Table 8: CDR2 mutation in the light chain of the maturation molecule for TSLP antibody affinity

[0164]

[0165] 2. TSLP antibody activity identification (Binding-ELISA)

[0166] ELISA identification of antibody binding to human TSLP protein: Coating: Dilute protein QP1328 (FC fusion of human TSLP) to 1 μg / ml with PBS, add 60 μl / well to the ELISA plate, and incubate at 4°C for 18-24 h. Blocking: Wash the incubated ELISA plate using a Biotek automated plate washer, washing twice with PBS. After washing, add 200 μl / well of 3% BSA blocking buffer and block at room temperature for 1 h. Antibody incubation: Incubate with different concentrations of anti-TSLP antibody at room temperature for 1 h. Wash the incubated ELISA plate using a Biotek automated plate washer, washing three times with PBST. Incubation of enzyme-labeled antibody: The enzyme-labeled antibody Goat Anti-Fab (HRP) needs to be diluted 5000 times with blocking buffer, 60 μl / well, and incubated at room temperature for 1 hour; TMB color development: Wash the incubated ELISA plate using a Biotek automatic plate washer, wash 5 times with PBST, and then add 100 μl / well of substrate color development solution TMB, incubating at room temperature in the dark for 10 minutes; Termination: After color development, quickly add 1M H2SO4 stop solution at 100 μL / well to terminate the reaction. Reading: Measure the OD value at a wavelength of 450 nm using a microplate reader. Result analysis: Data entry and analysis were performed using Graphpad Prism software.

[0167] The results are as follows Figure 8 As shown, different variants of the anti-TSLP antibody can bind to recombinant human TSLP protein, and are superior to the control antibody QP119120 (AMG157).

[0168] 3. Identification of humanized TSLP antibody activity (Blocking-ELISA)

[0169] Experimental Procedure: Dilute protein QP1171 (the FC fusion protein of human TSLPR / IL7Ra) to 2 μg / ml with PBS, and add 60 μl / well to the ELISA plate. Incubate at 4°C for 18-24 h. Blocking: Wash the incubated ELISA plate twice with PBS using a Biotek automated plate washer. After washing, add 200 μl / well of 3% BSA blocking buffer and block at room temperature for 1 h. Antibody Incubation: Mix different concentrations of human TSLP chimeric antibody and 0.02 μg / ml Biotin-QP1006 (the FC fusion protein of biotinylated human TSLP) at a 1:1 ratio, add 60 μl / well to the blocked ELISA plate, and incubate at room temperature for 1 h. Wash the incubated ELISA plate three times with PBST using a Biotek automated plate washer. For incubation, the enzyme-labeled streptavidin (HRP) was first diluted 5000-fold with blocking buffer, 60 μl / well, and incubated at room temperature for 1 h. For TMB color development: the ELISA plate was washed with a Biotek automated plate washer after incubation, washed 5 times with PBST, and then 100 μl / well of TMB substrate solution was added, incubated at room temperature in the dark for 10 min. For termination: after color development, 100 μl / well of stop solution (1M H₂SO₄) was quickly added to terminate the reaction. Reading: The OD value was measured at 450 nm using a microplate reader. Results analysis: Data entry and analysis were performed using Graphpad Prism software. Results are as follows... Figure 9 As shown, different variants of anti-TSLP antibodies can block the binding of human TSLP to TSLPR / IL7Ra.

[0170] 4. ELISA identification of antibody binding to cynomolgus monkey TSLP protein:

[0171] Coating: Dilute protein QP313 (FC fusion of cynomolgus monkey TSLP) to 1 μg / ml with PBS, add 60 μl / well to the ELISA plate, and incubate at 4°C for 18-24 h. Blocking: Wash the incubated ELISA plate twice with PBS using a Biotek automated plate washer. After washing, add 200 μl / well of 3% BSA blocking buffer and block at room temperature for 1 h. Antibody Incubation: Incubate with different concentrations of human TSLP chimeric antibody at room temperature for 1 h. Wash the incubated ELISA plate three times with PBST using a Biotek automated plate washer. Incubation of enzyme-labeled antibody: The enzyme-labeled antibody Goat Anti-Fab (HRP) needs to be diluted 1:5000 with blocking buffer, 60 μl / well, and incubated at room temperature for 1 h; TMB color development: Wash the incubated ELISA plate using a Biotek automatic plate washer, wash 5 times with PBST, then add 100 μl / well of substrate color development solution TMB, and incubate at room temperature in the dark for 10 min; Termination: After color development, quickly add 1M H2SO4 stop solution at 100 μL / well to terminate the reaction. Reading: Measure the OD value at a wavelength of 450 nm using a microplate reader. Result analysis: Data entry and analysis were performed using Graphpad Prism software. Results are as follows: Figure 10 As shown, all variants of the anti-TSLP antibody bind to the TSLP protein in cynomolgus monkeys.

[0172] 5. Biacore assay to detect the binding affinity and kinetics between anti-TSLP antibody and recombinant human TSLP protein.

[0173] The affinity of anti-TSLP antibody for human TSLP was determined using Biacore 8K. The experimental procedure is as follows:

[0174] Experimental procedure: A certain amount of the antibody to be tested was captured by affinity using a Protein A chip, and then human TSLP protein (QP1005) was passed through the chip surface. The reaction signal was detected in real time using Biacore to obtain the binding and dissociation curves.

[0175] The experimental results are shown in Table 9. The anti-TSLP antibodies QP36110422, QP36160422, and QP04203625 showed better affinity for human TSLP protein than the control antibody AMG157.

[0176] Table 9: Affinity results of anti-TSLP antibodies binding to human TSLP

[0177]

[0178] 6. Reporter gene assay for inhibiting the STAT5 signaling pathway with anti-TSLP antibody

[0179] Experimental Procedure: Stable BaF3-TSLPR-IL-7Ra cell lines were washed twice with PBS and then plated in 4E4 wells onto opaque reaction plates (2E6 / ml, 20 μL / well). The culture medium was basal medium without IL-3. Different concentrations of anti-TSLP antibody (100 μg / mL, 4-fold serial dilution) were added and incubated for 20 min. Then, 20 μL / well of TSLP recombinant protein (QP1005) at a final concentration of 2 ng / mL was added. After 6 h of incubation, the results were detected using the One-Glo Luciferase Assay System (promega, E6120) kit. 60 μL / well of reagent was added, and the Bio-TECK instrument was used to read and record the results.

[0180] The results are as follows Figure 11 As shown, anti-TSLP antibodies QP04203625, QP36110422, and QP36160422 can inhibit the downstream STAT5 signaling pathway caused by TSLP and its receptor binding, and their inhibition IC50 values ​​are superior to those of the control antibody QP119120 (AMG157). sequence list <110> Qiyu Biotechnology (Shanghai) Co., Ltd. <120> Anti-thymocyte stromal lymphopoietin (TSLP) antibodies and their uses <130> Q202115P <160> 35 <170> SIPOSequenceListing 1.0 <210> 1 <211> 117 <212> PRT <213> Artificial Sequence <400> 1 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr 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 Thr Tyr Thr Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Thr 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 Thr Arg Ser Leu Asp Gly Phe Phe Asp Tyr Trp Gly Gln Gly Thr Thr 100 105 110 Leu Thr Val Ser Ser 115 <210> 2 <211> 107 <212> PRT <213> Artificial Sequence <400> 2 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Ser 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 Phe Ala Lys Thr Leu Ala Ala 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 Asn Tyr Tyr Cys Gln His His Tyr Asp Thr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 3 <211> 117 <212> PRT <213> Artificial Sequence <400> 3 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Val Ile Asp Pro Ser Asp Thr Tyr Thr Thr Tyr Asn Gln Lys Phe 50 55 60 Lys 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 Thr Arg Ser Leu Asp Gly Phe Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 4 <211> 117 <212> PRT <213> Artificial Sequence <400> 4 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Asp Pro Ser Asp Thr Tyr Thr Thr Tyr Asn Gln Lys Phe 50 55 60 Lys 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 Thr Arg Ser Leu Asp Gly Phe Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 5 <211> 117 <212> PRT <213> Artificial Sequence <400> 5 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Asp Pro Ser Asp Thr Tyr Thr Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala 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 Thr Arg Ser Leu Asp Gly Phe Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 6 <211> 107 <212> PRT <213> Artificial Sequence <400> 6 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 Phe Ala Lys Thr Leu Ala Ala 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 Asp Thr Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 7 <211> 107 <212> PRT <213> Artificial Sequence <400> 7 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 Ser Pro Lys Leu Leu Val 35 40 45 Tyr Phe Ala Lys Thr Leu Ala Ala 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 Asp Thr Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 8 <211> 107 <212> PRT <213> Artificial Sequence <400> 8 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 Gln Gly Lys Ser Pro Lys Leu Leu Val 35 40 45 Tyr Phe Ala Lys Thr Leu Ala Ala Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln 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 Asp Thr Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 9 <211> 117 <212> PRT <213> Artificial Sequence <400> 9 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Asp Pro Tyr Asp Ala Asp Thr Thr Tyr Asn Gln Arg Phe 50 55 60 Lys Gly Lys Ala 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 Thr Arg Ser Leu Asp Gly Phe Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 10 <211> 117 <212> PRT <213> Artificial Sequence <400> 10 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Val Ile Asp Pro Tyr Asp Thr Asp Thr Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala 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 Thr Arg Ser Leu Asp Gly Phe Phe Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 11 <211> 107 <212> PRT <213> Artificial Sequence <400> 11 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 Ser Pro Lys Leu Leu Val 35 40 45 Tyr Phe Ala Arg Arg Pro Ala Ala 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 Asp Thr Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 12 <211> 5 <212> PRT <213> Artificial Sequence <400> 12 Ser Tyr Trp Met His 1 5 <210> 13 <211> 17 <212> PRT <213> Artificial Sequence <400> 13 Val Ile Asp Pro Ser Asp Thr Tyr Thr Thr Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <210> 14 <211> 8 <212> PRT <213> Artificial Sequence <400> 14 Ser Leu Asp Gly Phe Phe Asp Tyr 1 5 <210> 15 <211> 11 <212> PRT <213> Artificial Sequence <400> 15 Arg Ala Ser Glu Asn Ile Tyr Ser Tyr Leu Ala 1 5 10 <210> 16 <211> 7 <212> PRT <213> Artificial Sequence <400> 16 Phe Ala Lys Thr Leu Ala Ala 1 5 <210> 17 <211> 9 <212> PRT <213> Artificial Sequence <400> 17 Gln His His Tyr Asp Thr Pro Trp Thr 1 5 <210> 18 <211> 17 <212> PRT <213> Artificial Sequence <400> 18 Val Ile Asp Pro Tyr Asp Ala Asp Thr Thr Tyr Asn Gln Arg Phe Lys 1 5 10 15 Gly <210> 19 <211> 17 <212> PRT <213> Artificial Sequence <400> 19 Val Ile Asp Pro Tyr Asp Thr Asp Thr Thr Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <210> 20 <211> 7 <212> PRT <213> Artificial Sequence <400> 20 Phe Ala Arg Arg Pro Ala Ala 1 5 <210> twenty one <211> 107 <212> PRT <213> Artificial Sequence <400> twenty one Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 22 <211> 330 <212> PRT <213> Artificial Sequence <400> 22 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 23 <211> 330 <212> PRT <213> Artificial Sequence <400> 23 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 24 <211> 214 <212> PRT <213> Artificial Sequence <400> 24 Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Leu Gly Ser Lys Ser Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr 35 40 45 Asp Asp Ser Asp Arg Pro Ser Trp Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Gly Glu Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His 85 90 95 Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys 100 105 110 Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln 115 120 125 Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly 130 135 140 Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly 145 150 155 160 Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala 165 170 175 Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser 180 185 190 Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val 195 200 205 Ala Pro Thr Glu Cys Ser 210 <210> 25 <211> 448 <212> PRT <213> Artificial Sequence <400> 25 Gln Met Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Thr Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Thr Arg Asp Asn Ser Lys Asn Thr Leu Asn 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Pro Gln Trp Glu Leu Val His Glu Ala Phe Asp Ile Trp 100 105 110 Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys 210 215 220 Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val 290 295 300 Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 26 <211> 145 <212> PRT <213> Artificial Sequence <400> 26 Tyr Asp Phe Thr Asn Cys Asp Phe Gln Lys Ile Glu Ala Asp Tyr Leu 1 5 10 15 Arg Thr Ile Ser Lys Asp Leu Ile Thr Tyr Met Ser Gly Thr Lys Ser 20 25 30 Thr Asp Phe Asn Asn Thr Val Ser Cys Ser Asn Arg Pro His Cys Leu 35 40 45 Thr Glu Ile Gln Ser Leu Thr Phe Asn Pro Thr Pro Arg Cys Ala Ser 50 55 60 Leu Ala Lys Glu Met Phe Ala Arg Lys Thr Lys Ala Thr Leu Ala Leu 65 70 75 80 Trp Cys Pro Gly Tyr Ser Glu Thr Gln Ile Asn Ala Thr Gln Ala Met 85 90 95 Lys Lys Ala Arg Lys Ala Lys Val Thr Thr Asn Lys Cys Leu Glu Gln 100 105 110 Val Ser Gln Leu Leu Gly Leu Trp Arg Arg Phe Ile Arg Thr Leu Leu 115 120 125 Lys Lys Gln Asp Tyr Lys Asp Asp Asp Asp Lys His His His His His 130 135 140 His 145 <210> 27 <211> 363 <212> PRT <213> Artificial Sequence <400> 27 Tyr Asp Phe Thr Asn Cys Asp Phe Gln Lys Ile Glu Ala Asp Tyr Leu 1 5 10 15 Arg Thr Ile Ser Lys Asp Leu Ile Thr Tyr Met Ser Gly Thr Lys Ser 20 25 30 Thr Asp Phe Asn Asn Thr Val Ser Cys Ser Asn Arg Pro His Cys Leu 35 40 45 Thr Glu Ile Gln Ser Leu Thr Phe Asn Pro Thr Pro Arg Cys Ala Ser 50 55 60 Leu Ala Lys Glu Met Phe Ala Arg Lys Thr Lys Ala Thr Leu Ala Leu 65 70 75 80 Trp Cys Pro Gly Tyr Ser Glu Thr Gln Ile Asn Ala Thr Gln Ala Met 85 90 95 Lys Lys Ala Arg Lys Ala Lys Val Thr Thr Asn Lys Cys Leu Glu Gln 100 105 110 Val Ser Gln Leu Leu Gly Leu Trp Arg Arg Phe Ile Arg Thr Leu Leu 115 120 125 Lys Lys Gln Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro 130 135 140 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 145 150 155 160 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 165 170 175 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 180 185 190 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 195 200 205 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 210 215 220 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 225 230 235 240 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 245 250 255 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 260 265 270 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 275 280 285 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 290 295 300 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 305 310 315 320 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 325 330 335 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 340 345 350 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 355 360 <210> 28 <211> 164 <212> PRT <213> Artificial Sequence <400> 28 Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly 1 5 10 15 Val Gln Cys Tyr Asp Phe Thr Asn Cys Asp Phe Glu Lys Ile Lys Ala 20 25 30 Ala Tyr Leu Ser Thr Ile Ser Lys Asp Leu Ile Thr Tyr Met Ser Gly 35 40 45 Thr Lys Ser Thr Glu Phe Asn Asn Thr Val Ser Cys Ser Asn Arg Pro 50 55 60 His Cys Leu Thr Glu Ile Gln Ser Leu Thr Phe Asn Pro Thr Ala Gly 65 70 75 80 Cys Ala Ser Leu Ala Lys Glu Met Phe Ala Met Lys Thr Lys Ala Ala 85 90 95 Leu Ala Ile Trp Cys Pro Gly Tyr Ser Glu Thr Gln Ile Asn Ala Thr 100 105 110 Gln Ala Met Lys Lys Arg Arg Lys Arg Lys Val Thr Thr Asn Lys Cys 115 120 125 Leu Glu Gln Val Ser Gln Leu Gln Gly Leu Trp Arg Arg Phe Asn Arg 130 135 140 Pro Leu Leu Lys Gln Gln Asp Tyr Lys Asp Asp Asp Asp Lys His His 145 150 155 160 His His His His <210> 29 <211> 385 <212> PRT <213> Artificial Sequence <400> 29 Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly 1 5 10 15 Val Gln Cys Tyr Asp Phe Thr Asn Cys Asp Phe Glu Lys Ile Lys Ala 20 25 30 Ala Tyr Leu Ser Thr Ile Ser Lys Asp Leu Ile Thr Tyr Met Ser Gly 35 40 45 Thr Lys Ser Thr Glu Phe Asn Asn Thr Val Ser Cys Ser Asn Arg Pro 50 55 60 His Cys Leu Thr Glu Ile Gln Ser Leu Thr Phe Asn Pro Thr Ala Gly 65 70 75 80 Cys Ala Ser Leu Ala Lys Glu Met Phe Ala Met Lys Thr Lys Ala Ala 85 90 95 Leu Ala Ile Trp Cys Pro Gly Tyr Ser Glu Thr Gln Ile Asn Ala Thr 100 105 110 Gln Ala Met Lys Lys Arg Arg Lys Arg Lys Val Thr Thr Asn Lys Cys 115 120 125 Leu Glu Gln Val Ser Gln Leu Gln Gly Leu Trp Arg Arg Phe Asn Arg 130 135 140 Pro Leu Leu Lys Gln Gln Asp Lys Thr His Thr Cys Pro Pro Cys Pro 145 150 155 160 Ala Pro Glu Leu Leu Gly Gly Pro Glu Leu Pro Gly Gly Pro Ser Val 165 170 175 Phe Val Phe Pro Pro Lys Pro Lys Asp Val Leu Ser Ile Thr Leu Thr 180 185 190 Pro Lys Val Thr Cys Val Val Val Asp Val Gly Lys Glu Asp Pro Glu 195 200 205 Ile Glu Phe Ser Trp Ser Val Gly Asp Lys Glu Val His Thr Ala Glu 210 215 220 Thr Lys Pro Lys Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 225 230 235 240 Val Leu Pro Ile Gln His Gln Asp Trp Leu Thr Gly Lys Glu Phe Lys 245 250 255 Cys Lys Val Asn Asn Lys Ala Leu Pro Ala Pro Ile Glu Arg Thr Ile 260 265 270 Ser Lys Ala Lys Gly Gln Thr Gln Glu Pro Gln Val Tyr Thr Leu Ala 275 280 285 Pro His Arg Glu Glu Leu Ala Lys Asp Thr Val Ser Val Thr Cys Leu 290 295 300 Val Lys Gly Phe Tyr Pro Pro Asp Ile Asn Val Glu Trp Gln Arg Asn 305 310 315 320 Gly Gln Pro Glu Ser Glu Gly Ala Tyr Ala Thr Thr Leu Pro Gln Gln 325 330 335 Asp Asn Asp Gly Thr Tyr Phe Leu Tyr Ser Lys Leu Ser Val Gly Lys 340 345 350 Asn Thr Trp Gln Arg Gly Glu Thr Phe Thr Cys Val Val Met His Glu 355 360 365 Ala Leu His Asn His Ser Thr Gln Lys Ser Ile Thr Gln Ser Ser Gly 370 375 380 Lys 385 <210> 30 <211> 460 <212> PRT <213> Artificial Sequence <400> 30 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 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Pro Lys Ser Ser 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys Gly Ser Ser His His His His His His 450 455 460 <210> 31 <211> 472 <212> PRT <213> Artificial Sequence <400> 31 Glu Ser Gly Tyr Ala Gln Asn Gly Asp Leu Glu Asp Ala Glu Leu Asp 1 5 10 15 Asp Tyr Ser Phe Ser Cys Tyr Ser Gln Leu Glu Val Asn Gly Ser Gln 20 25 30 His Ser Leu Thr Cys Ala Phe Glu Asp Pro Asp Val Asn Thr Thr Asn 35 40 45 Leu Glu Phe Glu Ile Cys Gly Ala Leu Val Glu Val Lys Cys Leu Asn 50 55 60 Phe Arg Lys Leu Gln Glu Ile Tyr Phe Ile Glu Thr Lys Lys Phe Leu 65 70 75 80 Leu Ile Gly Lys Ser Asn Ile Cys Val Lys Val Gly Glu Lys Ser Leu 85 90 95 Thr Cys Lys Lys Ile Asp Leu Thr Thr Ile Val Lys Pro Glu Ala Pro 100 105 110 Phe Asp Leu Ser Val Ile Tyr Arg Glu Gly Ala Asn Asp Phe Val Val 115 120 125 Thr Phe Asn Thr Ser His Leu Gln Lys Lys Tyr Val Lys Val Leu Met 130 135 140 His Asp Val Ala Tyr Arg Gln Glu Lys Asp Glu Asn Lys Trp Thr His 145 150 155 160 Val Asn Leu Ser Ser Thr Lys Leu Thr Leu Leu Gln Arg Lys Leu Gln 165 170 175 Pro Ala Ala Met Tyr Glu Ile Lys Val Arg Ser Ile Pro Asp His Tyr 180 185 190 Phe Lys Gly Phe Trp Ser Glu Trp Ser Pro Ser Tyr Tyr Phe Arg Thr 195 200 205 Pro Glu Ile Asn Asn Ser Ser Gly Glu Met Asp Gly Gly Gly Gly Ser 210 215 220 Gly Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys 225 230 235 240 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 245 250 255 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 260 265 270 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 275 280 285 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 290 295 300 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 305 310 315 320 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 325 330 335 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 340 345 350 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 355 360 365 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys 370 375 380 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 385 390 395 400 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 405 410 415 Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 420 425 430 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 435 440 445 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Ser Ser 450 455 460 Asp Tyr Lys Asp Asp Asp Asp Lys 465 470 <210> 32 <211> 749 <212> PRT <213> Artificial Sequence <400> 32 Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Gly Gly Ala Ala Glu Gly Val Gln Ile Gln Ile Ile 20 25 30 Tyr Phe Asn Leu Glu Thr Val Gln Val Thr Trp Asn Ala Ser Lys Tyr 35 40 45 Ser Arg Thr Asn Leu Thr Phe His Tyr Arg Phe Asn Gly Asp Glu Ala 50 55 60 Tyr Asp Gln Cys Thr Asn Tyr Leu Leu Gln Glu Gly His Thr Ser Gly 65 70 75 80 Cys Leu Leu Asp Ala Glu Gln Arg Asp Asp Ile Leu Tyr Phe Ser Ile 85 90 95 Arg Asn Gly Thr His Pro Val Phe Thr Ala Ser Arg Trp Met Val Tyr 100 105 110 Tyr Leu Lys Pro Ser Ser Pro Lys His Val Arg Phe Ser Trp His Gln 115 120 125 Asp Ala Val Thr Val Thr Cys Ser Asp Leu Ser Tyr Gly Asp Leu Leu 130 135 140 Tyr Glu Val Gln Tyr Arg Ser Pro Phe Asp Thr Glu Trp Gln Ser Lys 145 150 155 160 Gln Glu Asn Thr Cys Asn Val Thr Ile Glu Gly Leu Asp Ala Glu Lys 165 170 175 Cys Tyr Ser Phe Trp Val Arg Val Lys Ala Met Glu Asp Val Tyr Gly 180 185 190 Pro Asp Thr Tyr Pro Ser Asp Trp Ser Glu Val Thr Cys Trp Gln Arg 195 200 205 Gly Glu Ile Arg Asp Ala Cys Ala Glu Thr Pro Thr Pro Pro Lys Pro 210 215 220 Lys Leu Ser Lys Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser 225 230 235 240 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly 245 250 255 Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly 260 265 270 Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser 275 280 285 Glu Ser Gly Tyr Ala Gln Asn Gly Asp Leu Glu Asp Ala Glu Leu Asp 290 295 300 Asp Tyr Ser Phe Ser Cys Tyr Ser Gln Leu Glu Val Asn Gly Ser Gln 305 310 315 320 His Ser Leu Thr Cys Ala Phe Glu Asp Pro Asp Val Asn Thr Thr Asn 325 330 335 Leu Glu Phe Glu Ile Cys Gly Ala Leu Val Glu Val Lys Cys Leu Asn 340 345 350 Phe Arg Lys Phe Leu Gln Glu Ile Tyr Phe Ile Glu Thr Lys Lys Phe Leu 355 360 365 Leu Ile Gly Lys Ser Asn Ile Cys Val Lys Val Gly Glu Lys Ser Leu 370 375 380 Thr Cys Lys Ile Asp Leu Thr Thr Ile Val Lys Pro Glu Ala Pro 385 390 395 400 Phe Asp Leu Ser Val Ile Tyr Arg Glu Gly Ala Asn Asp Phe Val Val 405 410 415 Thr Phe Asn Thr Ser His Leu Gln Lys Lys Tyr Val Lys Val Leu Met 420 425 430 His Asp Has Only Tyr Arg Glu Lys Asp Glu Asn Lys Thr Thr His 435 440 445 Val Asn Leu Ser Ser Thr Lys Leu Thr Leu Leu Gln Arg Lys Leu Gln 450 455 460 Pro Wing Wing Met Tyr Glu Ile Lys Val Arg Ser Ile Pro Asp His Tyr 465 470 475 480 Phe Lys Gly Phe Trp Ser Glu Trp Ser Pro Ser Tyr Tyr Phe Arg Thr 485 490 495 Pro Glu Ile Asn Asn Ser Ser Gly Glu Met Asp Gly Gly Gly Gly Ser 500 505 510 Gly Gly Gly Gly Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys 515 520 525 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 530 535 540 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 545 550 555 560 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 565 570 575 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 580 585 590 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 595 600 605 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 610 615 620 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 625 630 635 640 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 645 650 655 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 660 665 670 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 675 680 685 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 690 695 700 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 705 710 715 720 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 725 730 735 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 740 745 <210> 33 <211> 526 <212> PRT <213> Artificial Sequence <400> 33 Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Gly Gly Ala Ala Glu Gly Val Gln Ile Gln Ile Ile 20 25 30 Tyr Phe Asn Leu Glu Thr Val Gln Val Thr Trp Asn Ala Ser Lys Tyr 35 40 45 Ser Arg Thr Asn Leu Thr Phe His Tyr Arg Phe Asn Gly Asp Glu Ala 50 55 60 Tyr Asp Gln Cys Thr Asn Tyr Leu Leu Gln Glu Gly His Thr Ser Gly 65 70 75 80 Cys Leu Leu Asp Ala Glu Gln Arg Asp Asp Ile Leu Tyr Phe Ser Ile 85 90 95 Arg Asn Gly Thr His Pro Val Phe Thr Ala Ser Arg Trp Met Val Tyr 100 105 110 Tyr Leu Lys Pro Ser Ser Pro Lys His Val Arg Phe Ser Trp His Gln 115 120 125 Asp Ala Val Thr Val Thr Cys Ser Asp Leu Ser Tyr Gly Asp Leu Leu 130 135 140 Tyr Glu Val Gln Tyr Arg Ser Pro Phe Asp Thr Glu Trp Gln Ser Lys 145 150 155 160 Gln Glu Asn Thr Cys Asn Val Thr Ile Glu Gly Leu Asp Ala Glu Lys 165 170 175 Cys Tyr Ser Phe Trp Val Arg Val Lys Ala Met Glu Asp Val Tyr Gly 180 185 190 Pro Asp Thr Tyr Pro Ser Asp Trp Ser Glu Val Thr Cys Trp Gln Arg 195 200 205 Gly Glu Ile Arg Asp Ala Cys Ala Glu Thr Pro Thr Pro Pro Lys Pro 210 215 220 Lys Leu Ser Lys Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser 225 230 235 240 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly 245 250 255 Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly 260 265 270 Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser 275 280 285 Glu Ser Gly Tyr Ala Gln Asn Gly Asp Leu Glu Asp Ala Glu Leu Asp 290 295 300 Asp Tyr Ser Phe Ser Cys Tyr Ser Gln Leu Glu Val Asn Gly Ser Gln 305 310 315 320 His Ser Leu Thr Cys Ala Phe Glu Asp Pro Asp Val Asn Thr Thr Asn 325 330 335 Leu Glu Phe Glu Ile Cys Gly Ala Leu Val Glu Val Lys Cys Leu Asn 340 345 350 Phe Arg Lys Phe Leu Gln Glu Ile Tyr Phe Ile Glu Thr Lys Lys Phe Leu 355 360 365 Leu Ile Gly Lys Ser Asn Ile Cys Val Lys Val Gly Glu Lys Ser Leu 370 375 380 Thr Cys Lys Ile Asp Leu Thr Thr Ile Val Lys Pro Glu Ala Pro 385 390 395 400 Phe Asp Leu Ser Val Ile Tyr Arg Glu Gly Ala Asn Asp Phe Val Val 405 410 415 Thr Phe Asn Thr Ser His Leu Gln Lys Lys Tyr Val Lys Val Leu Met 420 425 430 His Asp Has Only Tyr Arg Glu Lys Asp Glu Asn Lys Thr Thr His 435 440 445 Val Asn Leu Ser Ser Thr Lys Leu Thr Leu Leu Gln Arg Lys Leu Gln 450 455 460 Pro Wing Wing Met Tyr Glu Ile Lys Val Arg Ser Ile Pro Asp His Tyr 465 470 475 480 Phe Lys Gly Phe Trp Ser Glu Trp Ser Pro Ser Tyr Tyr Phe Arg Thr 485,490,495 Pro Glu Ile Asn Asn Ser Ser Gly Glu Met Asp Gly Gly Gly Gly Ser 500 505 510 Asp Tyr Lys Asp Asp Asp Asp Lys His His His His His His 515 520 525 <210> 34 <211> 382 <212> PRT <213> Artificial Sequence <400> 34 Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly 1 5 10 15 Val Gln Cys Tyr Asp Phe Thr Asn Cys Asp Phe Glu Lys Ile Lys Ala 20 25 30 Ala Tyr Leu Ser Thr Ile Ser Lys Asp Leu Ile Thr Tyr Met Ser Gly 35 40 45 Thr Lys Ser Thr Glu Phe Asn Asn Thr Val Ser Cys Ser Asn Arg Pro 50 55 60 His Cys Leu Thr Glu Ile Gln Ser Leu Thr Phe Asn Pro Thr Ala Gly 65 70 75 80 Cys Ala Ser Leu Ala Lys Glu Met Phe Ala Met Lys Thr Lys Ala Ala 85 90 95 Leu Ala Ile Trp Cys Pro Gly Tyr Ser Glu Thr Gln Ile Asn Ala Thr 100 105 110 Gln Ala Met Lys Lys Arg Arg Lys Arg Lys Val Thr Thr Asn Lys Cys 115 120 125 Leu Glu Gln Val Ser Gln Leu Gln Gly Leu Trp Arg Arg Phe Asn Arg 130 135 140 Pro Leu Leu Lys Gln Gln Glu Pro Lys Ser Ser Asp Lys Thr His Thr 145 150 155 160 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 165 170 175 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 180 185 190 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 195 200 205 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 210 215 220 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 225 230 235 240 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 245 250 255 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 260 265 270 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 275 280 285 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 290 295 300 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 305 310 315 320 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 325 330 335 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 340 345 350 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 355 360 365 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380 <210> 35 <211> 382 <212> PRT <213> Artificial Sequence <400> 35 Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly 1 5 10 15 Val Gln Cys Tyr Asp Phe Thr Asn Cys Asp Phe Glu Lys Ile Lys Ala 20 25 30 Ala Tyr Leu Ser Thr Ile Ser Lys Asp Leu Ile Thr Tyr Met Ser Gly 35 40 45 Thr Lys Ser Thr Glu Phe Asn Asn Thr Val Ser Cys Ser Asn Arg Pro 50 55 60 His Cys Leu Thr Glu Ile Gln Ser Leu Thr Phe Asn Pro Thr Ala Gly 65 70 75 80 Cys Ala Ser Leu Ala Lys Glu Met Phe Ala Met Lys Thr Lys Ala Ala 85 90 95 Leu Ala Ile Trp Cys Pro Gly Tyr Ser Glu Thr Gln Ile Asn Ala Thr 100 105 110 Gln Ala Met Lys Lys Ala Arg Lys Ala Lys Val Thr Thr Asn Lys Cys 115 120 125 Leu Glu Gln Val Ser Gln Leu Gln Gly Leu Trp Arg Arg Phe Asn Arg 130 135 140 Pro Leu Leu Lys Gln Gln Glu Pro Lys Ser Ser Asp Lys Thr His Thr 145 150 155 160 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 165 170 175 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 180 185 190 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 195 200 205 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 210 215 220 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 225 230 235 240 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 245 250 255 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 260 265 270 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 275 280 285 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 290 295 300 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 305 310 315 320 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 325 330 335 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 340 345 350 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 355 360 365 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380

Claims

1. An anti-TSLP antibody comprising a heavy chain variable region comprising complementarity determining regions HCDR1, HCDR2, and HCDR3, and a light chain variable region comprising complementarity determining regions LCDR1, LCDR2, and LCDR3, wherein: the HCDR1, HCDR2, and HCDR3 are as set forth in SEQ ID NO: 12, SEQ ID NO: 18, and SEQ ID NO: 14, respectively, and the LCDR1, LCDR2, and LCDR3 are as set forth in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively; or the HCDR1, HCDR2, and HCDR3 are as set forth in SEQ ID NO: 12, SEQ ID NO: 19, and SEQ ID NO: 14, respectively, and the LCDR1, LCDR2, and LCDR3 are as set forth in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively; or the HCDR1, HCDR2, and HCDR3 are as set forth in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively, and the LCDR1, LCDR2, and LCDR3 are as set forth in SEQ ID NO: 15, SEQ ID NO: 20, and SEQ ID NO: 17, respectively; or the HCDR1, HCDR2, and HCDR3 are as set forth in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively, and the LCDR1, LCDR2, and LCDR3 are as set forth in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively.

2. The antibody of claim 1, wherein the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 1, 3, 4, 5, 9, or 10.

3. The antibody of claim 1, wherein the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 2, 6, 7, 8, or 11.

4. The antibody of claim 1, wherein the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 2; or the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 7; or the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 11; or the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO: 7; or the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO:

11. ​ ​ ​ ​ ​ ​ ​ ​ The amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO: 10, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO:

7.

5. The antibody of claim 1, wherein the antibody comprises a whole antibody, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, and a sc-Fv against TSLP antigen.

6. The antibody of claim 1, wherein the antibody comprises a heavy chain constant region selected from the group consisting of an IgGl subtype, an IgG2 subtype, an IgG3 subtype, or an IgG4 subtype and a conventional variant thereof, and the antibody comprises a light chain constant region selected from the group consisting of a kappa subtype or a lambda subtype and a conventional variant thereof.

7. An isolated polynucleotide, wherein the polynucleotide encodes the TSLP antibody of any one of claims 1-6.

8. A pharmaceutical composition comprising the TSLP antibody of any one of claims 1-6 and a pharmaceutically acceptable excipient or carrier.

9. Use of the antibody of any one of claims 1-6 or the pharmaceutical composition of claim 8 in the manufacture of a medicament for preventing or treating a TSLP-mediated disease, wherein the TSLP-mediated disease is selected from the group consisting of asthma, atopic dermatitis, allergic conjunctivitis, allergic rhinitis, food allergy.

10. Use of the antibody of any one of claims 1-6 or the pharmaceutical composition of claim 8 in the manufacture of a medicament for treating a TSLP-mediated disease, wherein the TSLP-mediated disease is selected from the group consisting of breast cancer, eosinophilic esophagitis, eosinophilic gastroenteritis, chronic eosinophilic pneumonia, eosinophilic bronchitis, eosinophilic myalgia syndrome, hypereosinophilic syndrome, eosinophilic granulomatosis with polyangiitis, chronic obstructive pulmonary disease, urticaria, nasal polyposis, ulcerative colitis.

Images