CN115433275A - anti-Thymic Stromal Lymphopoietin (TSLP) antibodies and uses thereof - Google Patents

Abstract

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

Description

anti-Thymic Stromal Lymphopoietin (TSLP) antibodies and uses thereof

Technical Field

The invention belongs to the field of biomedicine, relates to an anti-TSLP antibody, and also relates to application of the TSLP antibody.

Background

Human Thymic Stromal Lymphopoietin (TSLP) is an IL-7-like, short-chain type I cytokine composed of four helix bundles formed by three pairs of intra-chain disulfide bonds, is a member of the IL-2 cytokine family, and has a coding gene located on the 5q22.1 chromosome. It signals by binding to a heterodimeric receptor consisting of the IL-7R α subunit and a unique component TSLPR with homology to the common γ receptor-like chain, a member of the hematopoietic factor receptor family, and possesses high affinity only when the receptor complex TSLPR α/IL-7R α co-acts. TSLP is expressed by epithelial cells in the thymus, lung, skin, intestine and tonsil as well as airway smooth muscle cells, lung fibroblasts and stromal cells. These cells produce TSLP in response to pro-inflammatory stimuli, and TSLP drives an allergic inflammatory response through its activity on a variety of innate immune cells, including dendritic cells, monocytes, and mast cells.

TSLP binds to its specific receptors TSLPR and IL-7R α, forming a ternary complex, initiating signaling. For example, through phosphorylation of Janus kinase 1 (JAK 1) and JAK2, activation of signal transduction and transcription factor (STAT) 1, STAT3 and STAT5 initiates proinflammatory signals, promotes DC maturation and activation, induces expression of functional type ii helper T cells (Th 2), regulatory T cells (Treg) and follicular helper T cells (Tfh), and regulates inflammatory processes of skin, lung and intestinal mucosal barriers. The Th2 immunity of the TSLPR-deficient mice is low, but the Th1 immunity is normal or even enhanced, which suggests that the TSLPR quantity is an important factor influencing the development of anaphylaxis.

TSLP is critical in antigen presenting cells and hematopoietic cell maturation. TSLP is distributed in a variety of immune cells (including DCs, type ii innate lymphocytes (ILC 2), T cells, B cells, natural killer T cells (NKTs), treg cells, eosinophils, neutrophils, mast cells, and macrophages) and non-immune cells (platelets and sensory neurons). TSLP acts on a variety of cell lines, particularly myeloid DCs, by forming the TSLP-TSLPR-IL-7R α complex. TSLP can activate human peripheral blood CD11c + DC, up-regulate the expression of Major Histocompatibility Complex (MHC) class II, OX40 ligand (OX 40L/CD134L, CD 252), CD54, CD80, CD83 and CD86 and activation marker DC-LAMP, and promote the differentiation of naive CD4+ T cells (Th 0) into Th2 cells.

In particular, TSLP is unable to stimulate myeloid DC to produce the Th1 polarizing cytokines IL-12, the pro-inflammatory cytokines TNF- α, IL-1 β and IL-6, which are key features of TSLP in the construction of Th2 immune microenvironments. TSLP-DC enhances Th2 immune responses by activating Th2 effector memory cells and blocking FOXP3+ Treg production. TSLP interacts with CD4+ T cells, CD8+ T cells, and Treg cells, further promoting Th2 cell proliferation and activation. TSLP can activate mast cells, innate lymphocytes, epithelial cells, macrophages and the like, and together with epithelial cell chemokines such as IL-25 and IL-33, promote the production of Th2 cytokines (IL-4, IL-5 and IL-13). In addition, TSLP can promote eosinophil activation and chemotaxis. TSLP significantly delays eosinophil apoptosis in a concentration-dependent manner, induces production of IL-6 and chemokines CXCL8, CXCL1 by up-regulating CD18 and intercellular adhesion factor 1 (ICAM 1) expression, down-regulating L-selectin, and causes eosinophil inflammation.

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

TSLP is overexpressed in several autoimmune and inflammatory diseases/disorders, such as atopic dermatitis, food allergy, allergic rhinitis, netherton syndrome, asthma, indicating an important role for this cytokine in the pathogenesis of these allergic inflammatory diseases. Transgenic overexpression of TSLP in the skin or lung and removal of the gene targeting effect of the TSLP negative regulator in this animal model resulted in allergic inflammatory diseases very similar to human atopic dermatitis or asthma.

Atopic dermatitis is a chronic inflammatory skin disease. Skin mucosal barrier defects are the fuse of the allergic process, while TSLP can serve as a "warning" of the development of the allergic process. First, AD lesions provide a target site for allergen entry, and multiple pattern recognition receptors in epithelial cells are triggered, causing nuclear factor κ B (NF- κ B) activation, which generates Reactive Oxygen Species (ROS). The release of inflammatory factors such as TSLP, granulocyte macrophage colony stimulating factor (GM-CSF), IL-25, IL-33, etc., is promoted by the production of injury-associated molecular patterns (DAMPs) or proinflammatory cytokines such as IL-1 alpha, high mobility group protein B1 (HMGB 1), uric acid, adenosine triphosphate, or S100 protein. TSLP promotes immature DC up-regulation of OX40L and Notch ligand expression, down-regulation of IL-12 levels, secretion of Th2 cell chemokines (CCL 17 and CCL22, etc.), and subsequent stimulation of innate lymphocyte proliferation and production of Th2 cytokines. High levels of TSLP can also directly stimulate transient receptor potential kinin 1 (TRPA 1) positive cutaneous sensory neurons cells, triggering vigorous itching and aggravating the lesions. Thus, together, a Th2 dominant immune microenvironment promoting allergic progression is constructed.

Food allergy, TSLP favors migration, maturation and activation of Langerhans Cells (LCs), promoting DC polarization, leading to Th2 immunity in situ. At the same time, TSLP is highly likely to act as a promoter of the allergic process, initiating a systemic Th2 immune response. Intradermal injection of TSLP induces food allergy in mice, including systemic basophil expansion, increased serum-specific IgE levels and mast cell intestinal accumulation, promoting inflammatory reactions in the digestive tract. Conversely, decreasing TSLP levels or removing basophils decreases the susceptibility to intestinal food allergy. This suggests that the TSLP-basophil axis may be a potential therapeutic target for food allergy. In addition, potential target cells for TSLP-mediated food allergy are DCs, NKT cells and Treg cells. TSLP stimulates DC maturation and activation, promotes proliferation and differentiation of CD4+ Th0 cells into Th2 cells, and secretes large amounts of Th2 cytokines in peripheral blood and esophagus. TSLP, in combination with NKT cells TSLPR, can significantly induce NKT cells to recruit esophageal epithelium and produce large amounts of IL-13, mediating cow milk allergy. Meanwhile, TSLP plays an important role in Foxp3+ Treg cell differentiation. Moreover, when TSLP levels fluctuate, it is suggested that TSLP is critical to maintain intestinal homeostasis, leading to an imbalance in intestinal immune function.

Allergic rhinitis is an allergic disease mainly caused by Th2 immunity of the upper respiratory tract, wherein inflammation mediated by epithelial cell-derived histamine plays an important role, and epithelial cell-derived cytokines (such as IL-33, IL-25 and TSLP) produced by indigenous lymphocytes ILC2 are key to initiating and driving allergic immune response. The results of the study show that histamine receptor 4 (H4R) expression is increased in the nasal mucosa of sensitized mice, and that H4R, as a potential modulator of DC activation and T cell polarization, induces TSLP in nasal epithelial cells, activates OX40L signaling on DCs, and promotes Th2 immune responses. Th2 cytokines such as IL-4, IL-5 and IL-13 can further activate B cells, eosinophils and mast cells, constitute the whole body Th2 immunodominance and stimulate the allergic process. In contrast, by blocking H4R, TSLP levels are reduced, which can reduce allergic inflammation.

Asthma is a chronic disease characterized by airway inflammation and is characterized clinically by recurrent wheezing, shortness of breath, chest tightness and cough. About 3 million people worldwide suffer from asthma. The global prevalence of asthma has increased approximately 1% annually over the last two decades. Existing therapeutic agents, including bronchodilators, glucocorticoids, combinations of both (sulindac, cibotic), leukotriene modulators, long-acting cholinergic receptor antagonists (tiotropium), igE antibodies, etc., do not control the condition of all asthmatic patients.

A number of studies have demonstrated that about 2/3 of severe asthma is characterized by overexpression of Th2 cytokines, and TSLP is an important factor in causing overexpression of Th2 cytokines. The TSLP-TSLPR action is accomplished primarily through the JAK-STAT signaling pathway. It is thought that upregulation of TSLP, in combination with TSLPR on DC cells, causes JAK activation, recruits the transcription factor STAT5, causes downstream signaling, and ultimately leads to activation of DC cells. DC cell activation shows up-regulation of expression of co-stimulatory molecules (e.g., CD80, CD40, CD 86) and secretion of chemokines (TARC/CCL 17, MDC/CCL22, and I-309/CCL 1), thereby providing a favorable microenvironment for Th0 to Th2 cell differentiation, directing Th2 cell-dominated inflammatory responses, and release of co-factors (IL-4, IL-13, IL-5). TSLP transgenic mice are susceptible to specific antigen-induced asthma, while symptoms are significantly reduced in TSLP receptor knockout mice. From the analysis of the mechanism of asthma and inflammation, anti-cytokine (IL-4, IL-13, IL-5) drugs are only targeted to specific inflammatory molecules that drive asthma inflammation and are only suitable for certain types of severe asthma patients, i.e., a subset of patients, such as eosinophilic asthma. TSLP is significantly different from IL4, IL5 and other targets, and TSLP moves in the early upstream of the inflammatory cascade, and may be suitable for a wide range of severe uncontrolled asthma patients.

Researchers have conducted extensive research and study on drugs targeting TSLP. The anti-TSLP monoclonal antibody can effectively block the effect of TSLP/TSLPR, reverse airway inflammation, prevent the change of tissue structure and reduce the Airway Hyperresponsiveness (AHR) and TGF-beta 1 level in a mite dust induced mouse asthma model. In the serum protein-induced mouse asthma model, the anti-TSLP monoclonal antibody effectively reduced the expression of Th 2-like factors (IL-4, IL-5, etc.). The safety of anti-TSLP monoclonal antibodies is also well documented in monkeys. Furthermore, the anti-TSLP monoclonal antibody which is clinically researched shows good objective response rate in early clinic, and effectively relieves the symptoms of the tested patients.

In addition, researchers have found that TSLP levels are elevated in many different types of tumors. Tumors recruit immune cells to the tumor, inducing tumor-infiltrating myeloid cells to overexpress TSLP by expressing IL-1a factor. And the TSLP can further induce the tumor cells to express the anti-apoptosis protein BCL-2, so that the tumor cells are prevented from dying. Thus, TSLP is critical for tumor survival. Breast cancer cells have a higher mortality rate in tumors without TSLP compared to TSLP-exposed tumors. Further studies have shown that blocking TSLP in breast cancer models can significantly inhibit the growth of breast tumors and prevent their lung metastasis. In addition, blocking TSLP can not only prevent the growth of breast cancer tumors, but also many other tumors with elevated TSLP, such as pancreatic cancer, cervical cancer and multiple myeloma.

Therefore, in view of the wide applicability of antibodies that bind TSLP, the search and development of antibodies against TSLP is of great biological and medical significance based on clinical needs.

Disclosure of Invention

In a first aspect of the invention, there is provided an antibody that binds TSLP, 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 amino acid sequence is set forth in SEQ ID NO. 12, the HCDR2 amino acid sequence is set forth in SEQ ID NO. 13, SEQ ID NO. 18 or SEQ ID NO. 19, and the HCDR3 amino acid sequence is set forth in SEQ ID NO. 14; the LCDR1 amino acid sequence is shown as SEQ ID NO. 15, the LCDR2 amino acid sequence is shown as SEQ ID NO. 16 or SEQ ID NO. 20, and the LCDR3 amino acid sequence is shown as SEQ ID NO. 17. Wherein the HCDR and LCDR amino acid sequences are defined according to Kabat.

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

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

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

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

In some embodiments, the amino acid sequence of the heavy chain variable region of the TSLP antibody is set forth in SEQ ID NO 1, 3, 4, 5, 9, or 10.

In some embodiments, the variable region of the light chain of the antibody has the amino acid sequence set forth in SEQ ID NOs 2, 6, 7, 8, or 11.

In some embodiments, 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.

In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO. 3 and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO. 6.

In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO. 3 and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO. 7.

In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID No. 3 and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID No. 8.

In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO. 4 and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO. 6.

In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO. 4 and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO. 7.

In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO. 4 and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO. 8.

In some embodiments, 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. 6.

In some embodiments, 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.

In some embodiments, 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. 8.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody is set forth in SEQ ID NO. 10 and the amino acid sequence of the light chain variable region of the antibody is set forth in SEQ ID NO. 7.

In some embodiments, the antibody comprises a heavy chain constant region and a light chain constant region.

In some embodiments, the antibody comprises a heavy chain constant region selected from an IgG1 subtype, an IgG2 subtype, or an IgG4 subtype, and the antibody comprises a light chain constant region selected from a kappa subtype or a lambda subtype.

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

In some embodiments, the IgG1 subtype constant region sequence is set forth in SEQ ID NO 23 or mutant thereof SEQ ID NO 22, and the light chain constant region sequence is set forth in SEQ ID NO 21.

In some embodiments, the antibody comprises a whole antibody that binds the TSLP antigen.

In some embodiments, the antibody is an antigen-binding fragment of TSLP.

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

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

In some embodiments, the antibody inhibits binding of human TSLP to the human TSLP receptor.

In some embodiments, the antibody inhibits binding of human TSLP to human TSLP receptor hTSPR/IL 7Ra (e.g., a single chain human TSLPR.IL7Ra fusion polypeptide of the amino acid sequences set forth in SEQ ID NOS: 32, 33 or an hTSPR/IL 7Ra knob/hole dimer fused to both chains of SEQ ID NO:30 and SEQ ID NO: 31).

In a second aspect of the invention, there is provided a nucleic acid molecule encoding an antibody or antigen-binding fragment thereof according to the first aspect.

In a third aspect of the present invention, there is provided a pharmaceutical composition comprising:

(a) A TSLP antibody according to the first aspect of the invention; and

(b) A pharmaceutically acceptable excipient, diluent or pharmaceutically acceptable carrier.

In some embodiments, the pharmaceutical composition further comprises other drugs for treating cancer (or tumor), autoimmune diseases, or inflammatory diseases.

In a fourth aspect of the invention, there is provided the use of an antibody according to the first aspect or a pharmaceutical composition according to the third aspect in the manufacture of a medicament for the prevention or treatment of a TSLP-mediated disease.

In some embodiments, the TSLP-mediated disease is a cancer (tumor), autoimmune disease, or inflammatory disease.

In some embodiments, the TSLP-mediated disease or disorder comprises breast cancer, pancreatic cancer, gastric cancer, cervical cancer, colorectal cancer, lung cancer, melanoma, B-cell lymphoma, myeloma, asthma, idiopathic pulmonary fibrosis, atopic dermatitis, allergic conjunctivitis, allergic rhinitis, allergic rhinosinusitis, urticaria, endoxetan syndrome, eosinophilic esophagitis, food allergy, allergic diarrhea, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis, allergic fungal rhinosinusitis, chronic pruritus, systemic lupus erythematosus, rheumatoid arthritis, crohn'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, eosinophilic syndrome, eosinophilic granulomatosis with granuloangiitis, inflammatory bowel disease, scleroderma, interstitial lung disease, B-or C-type hepatitis, fibrosis induced by radiation and wound healing.

Drawings

FIG. 1: ELISA detects that chimeric antibody QP378379 binds to human TSLP protein.

FIG. 2: ELISA identifies that the chimeric antibody QP378379 blocks human TSLP/TSLPR/IL7Ra protein binding.

FIG. 3: ELISA identified chimeric antibody QP378379 bound to cynomolgus monkey TSLP protein.

FIG. 4: the ELISA identified that the humanized TSLP antibody bound to human TSLP protein.

FIG. 5: the ELISA identified that humanized anti-TSLP antibodies blocked the binding of human TSLP to TSLPR/IL7 Ra.

FIG. 6: ELISA identified humanized anti-TSLP antibodies bound to cynomolgus TSLP protein.

FIG. 7: reporter experiments identify anti-TSLP antibodies inhibit STAT5 signaling pathway experiments.

FIG. 8: ELISA identified TSLP antibodies bound to human TSLP protein.

FIG. 9: ELISA identified that anti-TSLP antibodies blocked the binding of human TSLP to TSLPR/IL7 Ra.

FIG. 10: ELISA identified that anti-TSLP antibodies bound to cynomolgus TSLP protein.

FIG. 11: reporter gene experiments identified anti-TSLP antibodies inhibited STAT5 signaling pathway experiments.

Detailed Description

1. The terms:

in order that the present invention may be more readily understood, some technical and scientific terms of the present invention are described before describing the embodiments.

Unless clearly defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The three letter codes and the one letter codes for amino acids used in the present invention are as described in j.boil.chem.,243, p3558 (1968).

The "antibody" of the present invention includes not only intact antibodies, but also fragments, polypeptide sequences, and derivatives and analogs thereof having antigen binding activity.

The antigen binding fragment refers to one or more portions of a full-length antibody that retain the ability to bind an antigen (e.g., HER 2) and compete with the intact antibody for specific binding to the antigen. In some cases, the antigen binding portion includes Fab, fab ', F (ab ') 2, fd, fv, dAb, and Complementarity Determining Region (CDR) fragments, single chain antibodies (e.g., scFv), chimeric antibodies comprising at least a portion of an antibody sufficient to confer specific antigen binding capacity to the polypeptide, single chain antibodies (e.g., scFv), chimeric antibodies comprising an antibody fragment sufficient to confer specific antigen binding capacity to the polypeptide, conventional techniques known to those of skill in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation) can be used to obtain the antigen binding portion of an antibody (e.g., an antibody fragment described above) from a given antibody (e.g., monoclonal antibody 2E 12), and the antibody binding portion can be specifically screened for specificity in the same manner as for an intact antibody. The term "Fv fragment" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody, the term "dAb fragment" means an antibody fragment consisting of the VH domain (Ward et al, nature 341 544-546 (1989)), the term "Fab fragment" means an antibody fragment consisting of the VL, VH, CL and CH1 domains, and the term "F (ab ') 2 fragment" means an antibody fragment comprising two Fab fragments connected by a disulfide bridge at the hinge region.

As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that retains substantially the same biological function or activity as an antibody of the invention. A polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide 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) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which the mature polypeptide is fused to another compound, such as a compound that extends the half-life of the polypeptide, e.g. polyethylene glycol, or (iv) a polypeptide in which an additional amino acid sequence is fused to the sequence of the polypeptide (e.g. a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with a 6His tag). Such fragments, derivatives and analogs are well within the skill of those in the art in light of the teachings herein.

Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it is understood that the variable domains of both the light chain (VL) and heavy chain (VH) portions determine antigen recognition and specificity. In contrast, the constant domains of the light (CL) and heavy (CH 1, CH2 or CH 3) chains confer important biological properties such as secretion, transplacental mobility, fc receptor binding, complement fixation, etc. By convention, the farther a constant region domain is from the antigen binding site or amino terminus of an antibody, the greater its numbering. The N-terminus is a variable region and at the C-terminus is a constant region; the CH3 domain and CL domain actually comprise the carboxy-termini of the heavy and light chains, respectively.

Chimeric antibody: the term "chimeric antibody" (or antigen-binding fragment thereof) is an antibody molecule (or antigen-binding fragment thereof) in which (a) the constant regions or portions thereof are altered, replaced, or exchanged such that the antigen-binding site (variable region) is linked to a constant region of a different or altered type, effector function, and/or species, or to an entirely different molecule (e.g., an enzyme, toxin, hormone, growth factor, drug, etc.) that confers new properties to the chimeric antibody; or (b) the variable region or a portion thereof is altered, replaced or exchanged for 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 from a human immunoglobulin. The chimeric antibody can retain its specificity of recognizing an antigen while having reduced antigenicity in humans compared to the original mouse antibody due to the replacement by human constant regions.

Humanization: the term "humanized" form of a non-human (e.g., murine) antibody is a chimeric antibody containing minimal sequences derived from non-human immunoglobulins. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) with the desired specificity, affinity, and capacity, e.g., mouse, rat, rabbit, or non-human primate. In some cases, framework Region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may contain residues that are not found in the recipient antibody or the donor antibody. These modifications were made to further improve antibody performance. Typically, a humanized antibody will comprise substantially all of the following: at least one, typically two, variable domains, wherein all or substantially all hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all FRs are those of a human immunoglobulin lo sequence. The humanized antibody optionally further comprises an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin constant region. For further details, see Jones et al, 1986, nature [ Nature ] 321; riechmann et al, 1988, nature [ Nature ] 332; and Presta,1992, curr. Op. Struct.biol. [ current research status of structural biology ] 2. See also the following review articles and references cited therein: vaswani and Hamilton,1998, ann. Allergy, asthma &Immunol. [ allergy, asthma and annual Immunity ] 1; harris,1995, biochem. Soc. Transactions [ journal of society of biochemistry ] 23; hurle and Gross,1994, curr. Op. Biotech [ current biotech view ] 5.

The in vitro affinity maturation of the antibody is a process for simulating in vitro maturation of the antibody, namely, the antibody with greatly improved bioactivity is obtained by constructing a mutant library by taking a certain antibody as a modified template and screening the mutant library.

"TSLP" in the context of the present invention includes variants, isoforms, homologs, orthologs, and paralogs of TSLP.

The antibodies of the present invention include murine, chimeric and humanized antibodies.

TSLP-mediated disease of the invention is not limited as long as it is a disease associated with TSLP, e.g., a therapeutic response induced using the molecules of the disclosure can be achieved by binding human TSLP, then blocking TSLP binding to its receptor, or killing cells that overexpress TSLP.

The term "autoimmune disease" as used herein refers to a disease caused by an autoimmune response from the body to an autoantigen resulting in damage to the tissues of the body, including, but not limited to, scleroderma, systemic lupus erythematosus, rheumatoid arthritis, churg-Strauss syndrome, wegener's granulomatosis, and goodpasture's syndrome.

The term "inflammatory disease" as used herein refers to the general term for diseases in which inflammation is the major damaging factor. Inflammation is the biological response of tissue to noxious stimuli, a condition that is accompanied by tissue degeneration, circulatory disturbances, and fluid exudation. Examples of inflammatory diseases include acute and chronic diseases including, but not limited to, asthma, allergic pneumonia, atopic dermatitis, rhinitis, crohn's disease, ankylosing spondylitis, rheumatic fever, fibromyalgia, psoriatic arthritis, chronic nephritis, sjogren's syndrome, and multiple sclerosis, among others.

The TSLP mediated cancer (or tumor) comprises breast cancer, pancreatic cancer, gastric cancer, cervical cancer, colorectal cancer, lung cancer, melanoma, B cell lymphoma and myeloma.

2. Detailed description of the preferred embodiments

The experimental method in which the specific conditions are not specified in the present experiment is generally performed under the conventional conditions or the conditions recommended by the manufacturers of the raw materials or the commercial products. Such as molecular cloning, a laboratory manual, cold spring harbor laboratory, contemporary methods of molecular biology, cell biology, and the like. Reagents of specific sources are not indicated, and conventional reagents are purchased in the market.

Example 1: cloning, expression and purification of TSLP and TSLP receptor recombinant protein and control antibody Tezepelumab

Cloning design: recombinant proteins such as human TSLP, human TSLP (R127A, R130A), cynomolgus TSLP (R127A, R130A), human TSLPR, human IL7Ra and the like are designed respectively, and tags such as His, flag, FC and the like are not the same for purification and identification. The recombinant protein numbers and the sequence numbers are shown in table 1:

table 1: cloning design construction of TSLP and TSLP receptor recombinant protein

According to the published Information (WHO Drug Information Vol.30, no.1,2016), the full-length antibody sequence (shown below) of the anti-TSLP antibody Tezepelumab (AMG 157) of an ann company is inquired, full-length light heavy chain expression plasmids QD119 and QD120 are respectively constructed, transient expression is carried out to produce protein, the protein number is QP119120, and the full-length light heavy chain expression plasmids are used for later experiment control:

QD119/pQD-Tezepelumab-Vλ/SEQ ID NO:24:

SYVLTQPPSVSVAPGQTARITCGGNNLGSKSVHWYQQKPGQAPVLVVYDDSDRPSWIPERFSGSNSGNTATLTISRGEAGDEADYYCQVWDSSSDHVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

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

QMQLVESGGGVVQPGRSLRLSCAASGFTFRTYGMHWVRQAPGKGLEWVAVIWYDGSNKHYADSVKGRFTITRDNSKNTLNLQMNSLRAEDTAVYYCARAPQWELVHEAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

transient expression: the day before 293E cell transfection, the cells to be transfected are centrifuged and the cell density is adjusted to about 0.5X 10 ⁶ And (4) the concentration is/ml. Plasmid and transfection reagent PEI are prepared, the amount of plasmid to be transfected is 100ug/100ml of cells, and the mass ratio of PEI to plasmid is 2. The plasmid and PEI are mixed evenly and kept stand for 15min. The plasmid and PEI mixture was slowly added to 293E cells, incubated in a shaker at 37 ℃ 8% CO2, 120rpm, and the fifth day of transfection, and centrifuged at 4700rpm for 20min in a horizontal centrifuge to collect the cell supernatant.

Protein purification:

protein A purification: passing the balance liquid through the column with at least 3CV and actual volume of 20ml to ensure that the pH and the electric conductivity of the solution flowing out of the final instrument are consistent with those of the balance liquid and the flow rate is 1ml/min; passing the centrifuged culture solution supernatant through a column, and loading 40ml of the culture solution at a flow rate of 0.33ml/min; passing through the column with at least 3CV, with the actual volume of 20ml, to ensure the pH and conductance of the solution flowing out of the final instrument to be consistent with those of the balance solution, and the flow rate of 0.33ml/min; and (4) passing the eluent through the column, starting to collect an elution peak (PAC-EP) when the UV280 rises to 15mAU, and stopping collecting when the UV280 drops to 15mAU at a flow rate of 1ml/min. After the sample collection was completed, PAC-EP was adjusted to neutral with pH adjusting solution.

Example 2: construction of BaF3-TSLPR-IL7Ra Stable Trans-cell Strain stably expressing TSLPR and IL7Ra

In order to develop a TSLP-STAT5 luciferase reporter gene experiment and screen an antibody which can block the combination of TSLP and TSLPR/IL7Ra and cause the activation of a downstream STAT5 signal pathway, a BaF3-TSLPR-IL7Ra stable transfer cell strain which can simultaneously express TSLPR and IL7Ra is constructed in the laboratory. BaF3 cells (mouse pro-B cell strain) are purchased from Beijing cooperative hospital, plasmids PCDNA3.1-TSLPR-IL-7Ra (QD 1246) and PGL4.52 (promega) are co-transfected into the BaF3 cells in an electrotransfection mode, all the cells are collected and centrifuged after 48 hours after transfection, fresh culture medium is added for resuspension, and G418 1.2mg/mL is added for screening, hygmycin 1mg/mL; the liquid is changed by centrifugation every 2-3 days, and the total screening is carried out for 2-3 weeks, so that the cell activity reaches more than 95%. Monoclonal plates were used, and single clones were selected and tested by FACS for single cell strains of BaF3-TSLPR-IL7Ra stably expressing TSLPR and IL7 Ra.

Example 3: hybridoma fusion screening and anti-human TSLP monoclonal antibody acquisition

Mouse immunization: anti-human TSLP monoclonal antibodies were generated by immunizing mice. Balb/c white mice, female, 6 weeks old were used for the experiments. A breeding environment: SPF grade. After the mice are purchased, the mice are raised in a laboratory environment for 1 week, and the light/dark period is adjusted at 12/12 hours, and the temperature is 20-25 ℃; the humidity is 40-60%. Balb/c mice were immunized, after two weeks for the first time, with Freund's complete adjuvant (CFA) to immunize the recombinant protein QP1005 (human TSLP-his) at 50 ug/mouse, and thereafter, with the recombinant protein QP1005 (human TSLP-his) plus Freund's incomplete adjuvant (IFA) or the recombinant protein QP312 (cyno TSLP-his) plus aluminum salt Alum + CpG ODN 1826 to immunize alternately at 25 ug/mouse.

Cell fusion: mice with high antibody titers in serum were selected for splenocyte fusion. Selected mice were immunized by intraperitoneal injection of a sprint 72 hours prior to fusion. Splenic lymphocytes were fused with myeloma Sp2/0 cells using an optimized PEG-mediated fusion procedure to give hybridoma cells. HAT complete fusion of hybridoma cellsThe medium (20% FBS, 1 XHAT and 1 XPPI in IMDM medium) was resuspended and aliquoted into 96-well cell culture plates (1X 10) ⁵ 150 ul/well), 37 ℃ 5% ₂ And (5) culturing. Adding 20% of FBS-containing IMDM medium (containing 2 XHAT and 1 XPI) on day 5 after the fusion, 50 ul/well, 37 ℃,5% ₂ And (4) culturing. Day 7-8 after fusion, based on cell growth density, the medium was changed to HT complete medium (IMDM medium containing 20% FBS, 1 XHT and 1 XPPI), 250 ul/well, 37 ℃,5% CO ₂ And (4) culturing.

Screening hybridoma cells: based on cell growth density, anti-TSLP antibodies were screened in hybridoma supernatants by ELISA assays 10-14 days after fusion. And (3) taking the supernatant of the hybridoma fusion hole, carrying out whole-plate primary screening on a 96-well plate by ELISA, and detecting that the anti-TSLP antibody in the supernatant can be combined with human TSLP recombinant protein QP1328, thus obtaining a primary screening positive hole. And detecting that the anti-TSLP antibody in the supernatant can block the combination of TSLP/TSLPR/IL7Ra, namely, the supernatant is a primary screening positive hole. And then, detecting the clone which is used for detecting the combination of the anti-TSLP antibody in the supernatant and the TSLP recombinant protein QP313 of the cynomolgus monkey and can block the combination of TSLP/TSLPR/IL7Ra by ELISA (enzyme-Linked immuno sorbent assay) of the primary screening positive hole, namely the anti-TSLP antibody positive clone hole. And (3) expanding the positive clone, transferring the amplified positive clone to a 24/6 pore plate in time, detecting cell culture supernatant to be combined with human TSLP and cynomolgus monkey TSLP again through ELISA, and simultaneously, blocking the combined clone hole of TSLP/TSLPR/IL7Ra, namely, obtaining the positive clone mab177A12. The screening ELISA results are shown in table 2. The positive clone mab177A12 was subjected to 2-3 rounds of limiting dilution to obtain single cell clones, and the single cell strains were cryopreserved.

Table 2: binding and blocking ELISA screening of hybridoma positive clones

Sequencing the hybridoma monoclonal antibody to obtain an antibody sequence: taking hybridoma positive monoclonal cell strain to extract mRNA, carrying out reverse transcription on the mRNA to obtain cDNA, carrying out PCR amplification by taking the cDNA as a template, selecting PCR positive clone, carrying out sequencing, and carrying out sequence analysis to obtain the light and heavy chain variable region sequence of the monoclonal antibody.

SEQ ID NO:1mab177A12 VH

QVQLQQPGAELVKPGASVKMSCKASGYTFTSYWMHWVKQRPGQGLEWIGVIDPSDTYTTYNQKFKGKATLTVDTSSSTAYMQLSSLTSEDSAVYYCTRSLDGFFDYWGQGTTLTVSS

SEQ ID NO:2mab177A12 VL

DIQMTQSPASLSASVGETVSITCRASENIYSYLAWYQQKQGKSPQLLVYFAKTLAAGVPSRFSGSGSGTQFSLKINSLQPEDFGNYYCQHHYDTPWTFGGGTKLEIK

The sequences of HCDR1, HCDR2 and HCDR3 corresponding to the VH of SEQ ID NO. 1mab177A12 are SEQ ID NO. 12, SEQ ID NO. 13 and SEQ ID NO. 14, respectively; LCDR1, LCDR2 and LCDR3 sequences corresponding to SEQ ID NO. 2mab177A12 VL are SEQ ID NO. 15, SEQ ID NO. 16 and SEQ ID NO. 17, respectively; the HCDR and LCDR amino acid sequences are specifically defined according to Kabat rules in table 3.

Table 3: mab177A12 light and heavy chain variable region CDR sequences and numbering

Example 3: construction of anti-human TSLP monoclonal chimeric antibody

Cloning and constructing: the mouse monoclonal antibody sequence obtained by sequencing the monoclonal antibody of example 3 is used as a template, and primers are respectively designed to amplify the VH/VK gene segments of the antibody. Constructing and enzyme cutting an expression vector pQD (a signal peptide and constant region gene (CH 1-FC/CL) fragment), and designing and constructing the expression vector pQD (the signal peptide and constant region gene (CH 1-FC/CL) fragment) by utilizing the characteristics of a plurality of special restriction endonucleases, such as BsmBI, of a recognition sequence different from an enzyme cutting site. BsmBI enzyme cuts the carrier, cuts the gel and recycles for standby. The antibody VH/VK gene fragment amplified by PCR and BsmBI enzyme digestion recovery expression vector pQD (signal peptide and constant region gene (CH 1-FC/CL) fragment) are respectively added into DH5H competent cells according to the proportion of 3. The light chain variable region sequences and protein expression numbers of each clone are shown in Table 4, in which all antibody light chain variable regions are fused with kappa light chain constant region CL to form the full-length light chain of the chimeric antibody, the antibody heavy chain variable region is fused with human IgG1 constant region to form the full-length heavy chain of the chimeric antibody, and the sequences of CL and human IgG1 constant regions are shown as follows:

CL SEQ ID NO:21

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Human IgG-Fc SEQ ID NO:23

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

table 4: chimeric antibody construction clone number and protein

Transient expression: HEK293E adjusted cell Density to 1 × 10 ⁶ The plasmid and PEI transfection reagent were prepared per ml, the amount of plasmid to be transfected was 100ug/100ml of cells, and the mass ratio of PEI to plasmid was 2. And mixing the plasmid and PEI evenly, and standing for 15min. Slowly adding the plasmid and PEI mixture to HEK293E cells, adding 8% CO ₂ Culturing in a shaker at 120rpm and 37 deg.C, and collecting cell supernatant by centrifuging at 4700rpm for 20min in a horizontal centrifuge on the fifth day of transfection.

Protein purification: protein A purification: connecting a purification column filled with 1ml of ProteinA filler in an SCG protein purification instrument system; passing the balance solution through a column with the pH value of 1 × PBS (phosphate buffer solution) of 7.4 at least 3CV and the actual volume of 20ml, ensuring that the pH value and the conductance of the solution flowing out of the final instrument are consistent with those of the balance solution and the flow rate is 1ml/min; centrifuging the cell culture solution by a refrigerated centrifuge at 4 ℃, and passing the supernatant through a column, wherein the sample loading volume is 40ml, and the flow rate is 0.33ml/min; after the sample loading is finished, the solution is subjected to column chromatography by using a balance solution of 1 × PBS PH7.4 with at least 3CV and the actual volume of 20ml, so that the pH and the conductance of the solution flowing out of the final instrument are consistent with those of the balance solution, and the flow rate is 0.33ml/min; the column was run with 50mM/L citric acid pH3.5, and the collection of the elution peak (PAC-EP) was started when UV280 rose to 15mAU and stopped when UV280 dropped to 15mAU, at a flow rate of 1ml/min. After the sample is collected, the PAC-EP is adjusted to be neutral by using a Tris-HCl adjusting solution with the pH value of 8.02M/L, and the buffer is replaced by PBS, SDS-PAGE and HPLC-SEC by the centrifugation of an ultrafiltration tube to identify the protein purity, wherein the protein purity is more than 95 percent for standby.

ELISA identification of chimeric antibody binding to human TSLP protein: protein QP1328 (FC fusion of human TSLP) was diluted to 1. Mu.g/ml with PBS and added to the ELISA plates at 60. Mu.l/well and incubated at 4 ℃ for 18-24h. And (3) sealing: the incubated ELISA plates were washed with a Biotek automatic plate washer, 2 times with PBS, and after washing, 3% BSA blocking solution was added to 200. Mu.l/well and blocked at room temperature for 1h. Incubation of the antibody: human TSLP chimeric antibody at different concentrations was incubated for 1h at room temperature, and incubated ELISA plates were washed with Biotek automatic plate washer and washed 3 times with PBST. Incubation of enzyme-labeled antibodies: the enzyme-labeled antibody, goat Anti-Fab (HRP), was prepared by first blocking the mixture with a 1: diluting by 5000 times, and incubating for 1h at normal temperature at 60 μ l/hole; TMB color development: washing the incubated ELISA plate with a Biotek automatic plate washing machine, washing with PBST for 5 times, adding substrate color development liquid TMB according to the dosage of 100 mul/hole, and developing for 10min at normal temperature in a dark place; and (4) terminating: after the color development is finished, stop solution 1M H2SO4 is added rapidly according to the dosage of 100 mu L/hole to stop the reaction. Reading: the OD value at a wavelength of 450nm was measured on a microplate reader. Results analysis, data entry and analysis were performed using Graphpad Prism software analysis. The results are shown in figure 1, chimeric antibody QP378379 binds to human TSLP recombinant protein.

ELISA the chimeric antibody was identified to block human TSLP/TSLPR/IL7Ra protein binding: protein QP1171 (FC fusion protein of human TSLPR/IL7 Ra) was diluted to 2. Mu.g/ml with PBS, added to the ELISA plate at 60. Mu.l/well and incubated at 4 ℃ for 18-24h. And (3) sealing: the incubated ELISA plates were washed with a Biotek automatic plate washer, 2 times with PBS, and after washing, 3% BSA blocking solution was added to 200. Mu.l/well and blocked at room temperature for 1h. Incubation of the antibody: various concentrations of human TSLP chimeric antibody and 0.02ug/ml Biotin-QP1005 (FC fusion protein of biotinylated human TSLP) were mixed as follows at 1: mixing at a ratio of 1, adding into the well-sealed ELISA plate at a concentration of 60. Mu.l/well at room temperature for 1h, and performing incubation on the ELISA plate by using a Biotek automatic plate washing machineWash with PBST 3 times. Incubating enzyme-labeled Streptavidin (HRP) and diluting by 5000 times with a confining liquid, wherein each hole is 60 mu l, and incubating for 1h at normal temperature; TMB color development: washing the incubated ELISA plate with a Biotek automatic plate washing machine, washing with PBST for 5 times, adding substrate color development liquid TMB according to the dosage of 100 mul/hole, and developing for 10min at normal temperature in a dark place; and (4) terminating: after the color development is finished, the stop solution 1 MH is rapidly added according to the dosage of 100 mu L/hole ₂ SO ₄ The reaction was terminated. Reading: the OD value at a wavelength of 450nm was measured on a microplate reader. Results analysis, data entry and analysis were performed using Graphpad Prism software analysis. The results are shown in FIG. 2, where the chimeric antibody QP378379 blocked the binding of human TSLP to TSLPR/IL7 Ra.

ELISA identification of chimeric antibody binding to cynomolgus TSLP protein coating: protein QP313 (FC fusion from cynomolgus TSLP) was diluted to 1. Mu.g/ml in PBS and added to the ELISA plates at 60. Mu.l/well and incubated at 4 ℃ for 18-24h. And (3) sealing: the incubated ELISA plates were washed with a Biotek automatic plate washer, 2 times with PBS, and after washing, 3% BSA blocking solution was added to 200. Mu.l/well and blocked at room temperature for 1h. Incubation of antibody: human TSLP chimeric antibody at different concentrations was incubated for 1h at room temperature, and incubated ELISA plates were washed with Biotek automatic plate washer and washed 3 times with PBST. Incubation of enzyme-labeled antibody: the enzyme-labeled antibody, goat Anti-Fab (HRP), was prepared by first blocking the mixture with a 1: diluting by 5000 times, performing incubation for 1h at normal temperature, wherein the concentration is 60 mu l/hole; TMB color development: washing the incubated ELISA plate with a Biotek automatic plate washing machine, washing with PBST for 5 times, adding substrate color development liquid TMB according to the dosage of 100 mul/hole, and developing for 10min at normal temperature in a dark place; and (4) terminating: after the color development is finished, stop solution 1M H2SO4 is added rapidly according to the dosage of 100 mu L/hole to stop the reaction. Reading: the OD value at a wavelength of 450nm was measured on a microplate reader. Results analysis, data entry and analysis were performed using Graphpad Prism software analysis. The results are shown in figure 3, chimeric antibody QP378379 binds to cynomolgus monkey TSLP protein.

Example 4: humanization of anti-human TSLP monoclonal antibodies

By comparing an IMGT human antibody heavy and light chain variable region germline gene database with MOE software, respectively selecting heavy and light chain variable region germline genes with high homology with QP378379 as templates, respectively transplanting CDRs of a murine antibody into corresponding human templates to form variable region sequences in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4. And selecting some important amino acid residues for back mutation combination. Wherein the amino acid residues are determined and annotated by the Kabat numbering system.

1. Humanized molecular cloning of anti-TSLP antibody

Designing primers, carrying out PCR (polymerase chain reaction) to build various humanized antibody VH/VK gene fragments, and carrying out homologous recombination on the humanized antibody VH/VK gene fragments and an expression vector pQD (signal peptide and constant region gene (CH 1-FC/CL) fragment) to construct an antibody full-length expression vector VH-CH1-FC-pQD/VK-CL-pQD.

The online software DNAworks (v3.2.4) (http:// helix web. Nih. Gov/DNAWorks /) was used to design multiple primers to synthesize VH/VK gene fragments containing recombination: 5'-30bp signal peptide + VH/VK +30bp CH1/CL-3', according to the operating instruction of Primer STAR GXL DNA polymerase of TaKaRa company, a plurality of primers are designed and amplified by two-step PCR to obtain a gene fragment containing the recombination of VH/VK. Constructing and enzyme cutting an expression vector pQD (signal peptide and constant region gene (CH 1-FC/CL) fragment), and designing and constructing the expression vector pQD (signal peptide and constant region gene (CH 1-FC/CL) fragment) by utilizing the characteristics of some special restriction enzymes such as BsmBI (restriction endonuclease) and different recognition sequences from enzyme cutting sites. BsmBI enzyme cuts the carrier, cuts the gel and recycles for standby. Recombinant construction of expression vector VH-CH1-FC-pQD/VK-CL-pQD.VH/VK contains gene fragment required by recombination and BsmBI enzyme digestion recovery expression vector pQD (signal peptide and constant region gene (CH 1-FC/CL) fragment) is added into DH5H competent cells according to the proportion of 3.

The humanized design light and heavy chain variable region sequences and protein expression numbers for each clone are shown in table 5, where all antibody light chain variable regions fused to kappa light chain constant region CL make up the full length light chain and the antibody heavy chain variable region fused to human IgG1 constant region make up the full length heavy chain:

table 5: QP378379 antibody humanization constructs clone numbers and proteins.

2. Humanized anti-TSLP antibody protein expression

HEK293E adjusted cell density to 1 × 10 ⁶ The plasmid and PEI transfection reagent were prepared per ml, the amount of plasmid to be transfected was 100ug/100ml of cells, and the mass ratio of PEI to plasmid was 2. The plasmid and PEI are mixed evenly and kept stand for 15min. Slowly adding the plasmid and PEI mixture to HEK293E cells at 8% CO ₂ Culturing in a shaker at 120rpm and 37 deg.C, and collecting cell supernatant by centrifuging at 4700rpm for 20min in a horizontal centrifuge on the fifth day of transfection.

3. Protein purification, protein A purification

Connecting a purification column filled with 1ml of ProteinA filler in an SCG protein purification instrument system; passing the balance solution through a column with the pH value of 1 × PBS (phosphate buffer solution) of 7.4 at least 3CV and the actual volume of 20ml, ensuring that the pH value and the conductance of the solution flowing out of the final instrument are consistent with those of the balance solution and the flow rate is 1ml/min; centrifuging the cell culture solution by a refrigerated centrifuge at 4 ℃, and passing the supernatant through a column, wherein the sample loading volume is 40ml, and the flow rate is 0.33ml/min; after the sample loading is finished, the solution passes through a column by using a balance solution of 1 × PBS PH7.4 with at least 3CV and the actual volume of 20ml, so that the pH and the conductance of the solution flowing out of the final instrument are consistent with those of the balance solution, and the flow rate is 0.33ml/min; the column was run with 50mM/L citric acid pH3.5 as eluent, and the collection of the elution peak (PAC-EP) was started when UV280 rose to 15mAU, and stopped when UV280 dropped to 15mAU, at a flow rate of 1ml/min. After the sample is collected, the PAC-EP is adjusted to be neutral by using a pH8.0M/L Tris-HCl adjusting solution, and the buffer is replaced by PBS, SDS-PAGE and HPLC-SEC by ultrafiltration tube centrifugation to identify the protein purity, wherein the protein purity is more than 95 percent for standby.

4. Humanized TSLP antibody Activity identification (Binding-ELISA)

ELISA identification of humanized TSLP antibodies binding to human TSLP protein: protein QP1328 (FC fusion of human TSLP) was diluted to 1. Mu.g/ml with PBS and added to the ELISA plates at 60. Mu.l/well and incubated at 4 ℃ for 18-24h. And (3) sealing: the incubated ELISA plates were washed with a Biotek automatic plate washer, 2 times with PBS, and after washing, 3% BSA blocking solution was added to 200. Mu.l/well and blocked at room temperature for 1h. Incubation of the antibody: humanized anti-TSLP antibodies at different concentrations were incubated for 1h at room temperature, and incubated ELISA plates were washed using a Biotek automated plate washer 3 times with PBST. Incubation of enzyme-labeled antibodies: an enzyme-labeled antibody Goat Anti-Fab (HRP) needs to be diluted by 5000 times of a sealing solution, 60 mu l/hole, and incubated for 1h at normal temperature; TMB color development: washing the incubated ELISA plate by using a Biotek automatic plate washing machine, washing the plate by using PBST for 5 times, adding a substrate color development solution TMB according to the dosage of 100 mu l/hole, and developing for 10min in a dark place at normal temperature; and (4) terminating: after the color development is finished, stop solution 1M H2SO4 is added rapidly according to the dosage of 100 mu L/hole to stop the reaction. Reading: the OD value at a wavelength of 450nm was measured on a microplate reader. Results analysis, data entry and analysis were performed using Graphpad Prism software analysis. The results are shown in FIG. 4, where different variants of the humanized anti-TSLP antibody were able to bind to human TSLP recombinant protein.

5. Humanized TSLP antibody Activity identification (Blocking-ELISA)

The experimental steps are as follows: protein QP1171 (FC fusion protein of human TSLPR/IL7 Ra) was diluted to 2. Mu.g/ml with PBS, added to the ELISA plate at 60. Mu.l/well and incubated at 4 ℃ for 18-24h. And (3) sealing: the incubated ELISA plates were washed with a Biotek automatic plate washer, 2 times with PBS, and after washing, 3% BSA blocking solution was added to 200. Mu.l/well and blocked at room temperature for 1h. Incubation of antibody: different concentrations of chimeric human TSLP antibody and 0.02ug/ml Biotin-QP1006 (FC fusion protein of biotinylated human TSLP) were mixed according to 1: mix at a ratio of 1, add to the well-sealed ELISA plates at 60. Mu.l/well for 1h at room temperature, wash the incubated ELISA plates using a Biotek automatic plate washer, and wash 3 times with PBST. Incubating enzyme-labeled Streptavidin (HRP) and diluting by 5000 times with a confining liquid, wherein each hole is 60 mu l, and incubating for 1h at normal temperature; TMB color development: washing the incubated ELISA plate with a Biotek automatic plate washing machine, washing with PBST for 5 times, adding substrate color development liquid TMB according to the dosage of 100 mul/hole, and developing for 10min at normal temperature in a dark place; and (4) terminating: after the color development is finished, adding 1 MH stop solution into the mixture according to the dosage of 100 mu L/hole ₂ SO ₄ The reaction was terminated. Reading: the OD value at a wavelength of 450nm was measured on a microplate reader. Results analysis, data entry and analysis were performed using Graphpad Prism software analysis. The results are shown in FIG. 5, where different variants of the humanized anti-TSLP antibody blocked the binding of human TSLP to TSLPR/IL7RaAnd (6) mixing.

ELISA identification of chimeric antibody binding to cynomolgus monkey TSLP protein

Coating: protein QP313 (FC fusion of cynomolgus TSLP) was diluted to 1. Mu.g/ml in PBS and added to the ELISA plates at 60. Mu.l/well and incubated for 18-24h at 4 ℃. And (3) sealing: the incubated ELISA plates were washed with a Biotek automatic plate washer, 2 times with PBS, and after washing, 3% BSA blocking solution was added to 200. Mu.l/well and blocked at room temperature for 1h. Incubation of the antibody: human TSLP chimeric antibody at different concentrations was incubated for 1h at room temperature, and incubated ELISA plates were washed with Biotek automatic plate washer and washed 3 times with PBST. Incubation of enzyme-labeled antibody: the enzyme-labeled antibody, goat Anti-Fab (HRP), was prepared by first blocking the mixture with a 1: diluting by 5000 times, and incubating for 1h at normal temperature at 60 μ l/hole; TMB color development: washing the incubated ELISA plate with a Biotek automatic plate washing machine, washing with PBST for 5 times, adding substrate color development liquid TMB according to the dosage of 100 mul/hole, and developing for 10min at normal temperature in a dark place; and (4) terminating: after the color development is finished, stop solution 1M H2SO4 is added rapidly according to the dosage of 100 mu L/hole to stop the reaction. Reading: OD at a wavelength of 450nm was measured on a microplate reader. Results analysis, data entry and analysis were performed using Graphpad Prism software analysis. Results as shown in figure 6, each variant of the humanized anti-TSLP antibody bound to cynomolgus TSLP protein.

Biacore test for binding affinity and kinetics of anti-TSLP antibody to human TSLP recombinant protein

The affinity of anti-TSLP antibodies to human TSLP was determined by Biacore S200, the experimental procedure was as follows:

the experimental steps are as follows: a certain amount of antibody to be detected is subjected to affinity capture by a Protein A chip, then human TSLP Protein (QP 1005) flows on the surface of the chip, and a reaction signal is detected in real time by using Biacore, so that an association and dissociation curve is obtained.

The results of the experiments are shown in table 6, and the humanized anti-TSLP antibody QP419423, QP420421, QP420422, QP420423 and the chimeric antibody QP378379 before humanization all bind to human TSLP protein.

Table 6: binding affinity results of anti-TSLP antibodies to human TSLP

8. anti-TSLP antibody inhibition STAT5 signaling pathway reporter gene experiment

The experimental steps are as follows: taking a stable transformed BaF3-TSLPR-IL-7Ra cell strain, washing the cells for 2 times by using PBS, then paving 4E4/well on an opaque reaction plate, namely 2E6/mL and 20 ul/hole, wherein the culture medium is a basic culture medium without IL-3, adding anti-TSLP antibodies with different concentrations (the concentration in the antibodies is 100ug/mL and 4 times of gradient dilution), and incubating in an incubator for 20 min; adding TSLP recombinant protein (QP 1005) 20 ul/well with a final concentration of 2 ng/mL; after 6h of incubator culture, detection was performed by One-Glo Luciferase Assay System (promega, E6120) kit, 60 ul/well reagent was added, instrument Bio-TECK reading was taken, and the results were recorded.

As a result, as shown in fig. 7, the humanized anti-TSLP antibodies QP419423, QP420421, QP420422, QP420423 and the humanized chimeric antibody QP378379 all inhibited the downstream STAT5 signaling pathway caused by TSLP binding to its receptor.

Example 5: humanized anti-TSLP antibody QP420422 affinity maturation

1. Affinity matured antibody construction

Constructing a humanized phagemid vector: the humanized QP420422 was constructed into the phagemid vector in scFv format (VH- (GGGGS) 3-VL), respectively, as the wild-type sequence (i.e.the mutated sequence screened for affinity maturation). VH, (GGGGS) 3 linker, VL were spliced by overlap PCR (over-lap PCR) using NcoI and NotI cleavage sites to join phagemid vectors.

Constructing a phage display library: and (3) designing a mutation primer for each CDR by using the constructed wild scFv as a template, and introducing mutation in all CDR regions by adopting a degenerate primer to construct a mutation library. The PCR fragment was digested with NcoI and NotI, ligated into a phagemid vector, and finally transformed into E.coli TG1. Degenerate primers for each CDR create a separate library.

Library panning: after phage particles for panning are packaged from the library by phase, liquid phase panning is carried out by using biotinylated QP1005 (human TSLP recombinant protein) antigen and streptavidin magnetic beads, the concentration of the first round of panning is 5nM, and the antigen concentration of each round of panning is reduced by 10 times compared with the previous round. After three rounds of panning, 250 clones were picked for phage ELISA to detect binding activity, and positive clones were sequenced.

And (3) ELISA detection: after the redundant sequences are removed by performing alignment analysis on the sequenced clones, the non-redundant sequences are converted into full-length IG (hIgG 1, kappa) for mammalian cell 293E expression. Full-length IG after affinity purification was subjected to ELISA detection.

Finally, 3 mutant antibodies were obtained, in which the heavy chain CDR2 mutation using QD420 (SEQ ID NO: 5) as the parent was clone QD3611 (SEQ ID NO: 9) shown in Table 7, QD3616 (SEQ ID NO: 10), and the light chain CDR2 mutation using QD422 (SEQ ID NO: 7) as the parent was clone QD3625 (SEQ ID NO: 11) shown in Table 8, respectively.

Table 7: TSLP antibody affinity maturation molecule heavy chain CDR2 mutations

Light chain CDR2 mutation using QD422 as the parent clone QD3625 as shown in table 7:

table 8: TSLP antibody affinity maturation molecule light chain CDR2 mutations

Identification of TSLP antibody Activity (Binding-ELISA)

ELISA identification of antibody binding to human TSLP protein coating: protein QP1328 (FC fusion of human TSLP) was diluted to 1. Mu.g/ml with PBS, added to the ELISA plates at 60. Mu.l/well and incubated at 4 ℃ for 18-24h. And (3) sealing: the incubated ELISA plates were washed with a Biotek automatic plate washer, 2 times with PBS, and after washing, 3% BSA blocking solution was added to 200. Mu.l/well and blocked at room temperature for 1h. Incubation of the antibody: different concentrations of anti-TSLP antibody were incubated for 1h at room temperature, and the incubated ELISA plates were washed using a Biotek automated plate washer and 3 times with PBST. Incubation of enzyme-labeled antibodies: an enzyme-labeled antibody Goat Anti-Fab (HRP) needs to be diluted by 5000 times of a sealing solution, 60 mu l/hole, and incubated for 1h at normal temperature; TMB color development: washing the incubated ELISA plate with a Biotek automatic plate washing machine, washing with PBST for 5 times, adding substrate color development liquid TMB according to the dosage of 100 mul/hole, and developing for 10min at normal temperature in a dark place; and (4) terminating: after the color development is finished, stop solution 1M H2SO4 is added rapidly according to the dosage of 100 mu L/hole to stop the reaction. Reading: OD at a wavelength of 450nm was measured on a microplate reader. Results analysis, data entry and analysis were performed using Graphpad Prism software analysis.

The results are shown in FIG. 8, where different variants of the anti-TSLP antibody were able to bind human TSLP recombinant protein and outperformed the control antibody QP119120 (AMG 157).

3. Humanized TSLP antibody Activity identification (Blocking-ELISA)

The experimental steps are as follows: protein QP1171 (FC fusion protein of human TSLPR/IL7 Ra) was diluted to 2. Mu.g/ml with PBS, added to the ELISA plate at 60. Mu.l/well and incubated at 4 ℃ for 18-24h. And (3) sealing: the incubated ELISA plates were washed with a Biotek automatic plate washer, 2 times with PBS, and after washing, 3% BSA blocking solution was added to 200. Mu.l/well and blocked at room temperature for 1h. Incubation of antibody: different concentrations of chimeric human TSLP antibody and 0.02ug/ml Biotin-QP1006 (FC fusion protein of biotinylated human TSLP) were mixed according to 1: mix at a ratio of 1, add to the well-blocked ELISA plates at 60. Mu.l/well for 1h at room temperature, wash the incubated ELISA plates using a Biotek automatic plate washer, wash 3 times with PBST. Incubating enzyme-labeled Streptavidin (HRP) and diluting the Streptavidin by 5000 times by using a confining liquid, incubating the Streptavidin at 60 mu l/hole for 1h at normal temperature; TMB color development: washing the incubated ELISA plate with a Biotek automatic plate washing machine, washing with PBST for 5 times, adding substrate color development liquid TMB according to the dosage of 100 mul/hole, and developing for 10min at normal temperature in a dark place; and (4) terminating: after the color development is finished, adding 1 MH stop solution into the mixture according to the dosage of 100 mu L/hole ₂ SO ₄ The reaction was terminated. Reading: the OD value at a wavelength of 450nm was measured on a microplate reader. Results analysis, data entry and analysis were performed using Graphpad Prism software analysis. The results are shown in FIG. 9, where different variants of anti-TSLP antibodies were able to block binding of human TSLP to TSLPR/IL7 Ra.

Elisa identification of antibody binding to cynomolgus TSLP protein:

coating: protein QP313 (FC fusion of cynomolgus TSLP) was diluted to 1. Mu.g/ml in PBS and added to the ELISA plates at 60. Mu.l/well and incubated for 18-24h at 4 ℃. And (3) sealing: the incubated ELISA plates were washed with a Biotek automatic plate washer, 2 times with PBS, and after washing, 3% BSA blocking solution was added to 200. Mu.l/well and blocked at room temperature for 1h. Incubation of antibody: incubation of human TSLP chimeric antibody at different concentrations for 1h at room temperature, washing of incubated ELISA plates using a Biotek automatic plate washer, 3 times washing with PBST. Incubation of enzyme-labeled antibody: the enzyme-labeled antibody, goat Anti-Fab (HRP), was prepared by first blocking the mixture with a 1: diluting by 5000 times, performing incubation for 1h at normal temperature, wherein the concentration is 60 mu l/hole; TMB color development: washing the incubated ELISA plate with a Biotek automatic plate washing machine, washing with PBST for 5 times, adding substrate color development liquid TMB according to the dosage of 100 mul/hole, and developing for 10min at normal temperature in a dark place; and (4) terminating: after the color development is finished, stop solution 1M H2SO4 is added rapidly according to the dosage of 100 mu L/hole to stop the reaction. Reading: OD at a wavelength of 450nm was measured on a microplate reader. Results analysis, data entry and analysis were performed using Graphpad Prism software analysis. Results as shown in figure 10, each variant of the anti-TSLP antibody bound to cynomolgus monkey TSLP protein.

Biacore test of binding affinity and kinetics of anti-TSLP antibody to human TSLP recombinant protein

The affinity of anti-TSLP antibodies to human TSLP was determined by Biacore 8K, the experimental procedure was as follows:

the experimental steps are as follows: a certain amount of antibody to be detected is subjected to affinity capture by a Protein A chip, then human TSLP Protein (QP 1005) flows on the surface of the chip, and a reaction signal is detected in real time by using Biacore, so that an association and dissociation curve is obtained.

The experimental results are shown in table 9, and the binding affinity of anti-TSLP antibodies QP36110422, QP36160422 and QP04203625 to human TSLP protein is better than that of control antibody AMG157.

Table 9: binding affinity results of anti-TSLP antibodies to human TSLP

6. anti-TSLP antibody inhibition STAT5 signal pathway reporter gene experiment

The experimental steps are as follows: taking a stable transformed BaF3-TSLPR-IL-7Ra cell strain, washing the cell for 2 times by PBS, then paving 4E4/well onto an opaque reaction plate, namely 2E6/mL and 20 ul/hole, wherein the culture medium is a basic culture medium without IL-3, adding anti-TSLP antibodies with different concentrations (the concentration in the antibodies is 100ug/mL, and 4 times of gradient dilution), and incubating in an incubator for 20 min; adding TSLP recombinant protein (QP 1005) 20 ul/well with a final concentration of 2 ng/mL; after 6h of incubator culture, detection was performed by One-Glo Luciferase Assay System (promega, E6120) kit, 60 ul/well reagent was added, instrument Bio-TECK reading was taken, and the results were recorded.

As shown in fig. 11, the anti-TSLP antibodies QP04203625, QP36110422 and QP36160422 all inhibited downstream STAT5 signal pathway caused by TSLP and receptor binding thereof, and inhibited IC50 values were superior to the control antibody QP119120 (AMG 157).

Sequence listing

<110> recovery Biotechnology (Shanghai) Co., ltd

<120> anti-Thymic Stromal Lymphopoietin (TSLP) antibodies and uses thereof

<130> Q202115P

<160> 35

<170> SIPOSequenceListing 1.0

<210> 1

<211> 117

<212> PRT

<213> Artificial Sequence (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (Artificial Sequence)

<400> 12

Ser Tyr Trp Met His

1 5

<210> 13

<211> 17

<212> PRT

<213> Artificial Sequence (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 (Artificial Sequence)

<400> 14

Ser Leu Asp Gly Phe Phe Asp Tyr

1 5

<210> 15

<211> 11

<212> PRT

<213> Artificial Sequence (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 (Artificial Sequence)

<400> 16

Phe Ala Lys Thr Leu Ala Ala

1 5

<210> 17

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 17

Gln His His Tyr Asp Thr Pro Trp Thr

1 5

<210> 18

<211> 17

<212> PRT

<213> Artificial Sequence (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 (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 (Artificial Sequence)

<400> 20

Phe Ala Arg Arg Pro Ala Ala

1 5

<210> 21

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 21

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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 (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 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 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 Val Ala Tyr Arg Gln Glu Lys Asp Glu Asn Lys Trp 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 Ala Ala 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 (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 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 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 Val Ala Tyr Arg Gln Glu Lys Asp Glu Asn Lys Trp 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 Ala Ala 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 (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 (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 (11)

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:

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; or

HCDR1, HCDR2 and HCDR3 are respectively shown as SEQ ID NO. 12, SEQ ID NO. 19 and SEQ ID NO. 14, and LCDR1, LCDR2 and LCDR3 are respectively shown as SEQ ID NO. 15, SEQ ID NO. 16 and SEQ ID NO. 17; or

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; or

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.

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 NOs 1, 3, 4, 5, 9, or 10.

3. The antibody of claim 1, wherein the variable region of the light chain of the antibody has the amino acid sequence 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 shown as SEQ ID NO.1, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 2; or

The amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 5, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 7; or

The amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 5, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 11; or

The amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO. 9, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 7; or

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, an Fab fragment, an Fab 'fragment, an F (ab') 2 fragment, an Fv fragment, and an sc-Fv against a TSLP antigen.

6. The antibody of claim 1, wherein the antibody comprises a heavy chain constant region selected from the group consisting of an IgG1 subtype, an IgG2 subtype, an IgG3 subtype, or an IgG4 subtype, and conventional variants thereof, and the antibody comprises a light chain constant region selected from the group consisting of a kappa subtype or a lambda subtype, and conventional variants thereof.

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

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

10. The antibody of claim 9, wherein the TSLP-mediated disease comprises a cancer (tumor), an autoimmune disease, or an inflammatory disease.

11. The antibody of claim 9, wherein the TSLP-mediated disease comprises breast cancer, pancreatic cancer, gastric cancer, cervical cancer, colorectal cancer, lung cancer, melanoma, B-cell lymphoma, myeloma, asthma, idiopathic pulmonary fibrosis, atopic dermatitis, allergic conjunctivitis, allergic rhinitis, allergic rhinosinusitis, urticaria, endoxeon syndrome, eosinophilic esophagitis, food allergy, allergic diarrhea, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis, allergic fungal rhinosinusitis, chronic pruritus, systemic lupus erythematosus, rheumatoid arthritis, crohn'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 myasthenia syndrome, hypergranulocytic syndrome, eosinophilic granulomatosis with granulomatosis, inflammatory bowel disease, scleroderma, interstitial lung disease, B-or C-type hepatitis, fibrosis induced by radiation healing and wound induction.

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