Antigen-binding proteins that can bind to thymic interstitial lymphoid neogenetic factor.

Abstract

This invention provides an antagonist for human thymic stromal lymphoid neogenetic factor (TSLP), which is expected to be useful in treating inflammatory and fibrotic disorders. [Solution] This disclosure provides compositions and methods relating to antigen-binding proteins that bind to TSLP, including antibodies. In certain embodiments, this disclosure provides fully human anti-TSLP antibodies, humanized anti-TSLP antibodies and chimeric anti-TSLP antibodies, as well as derivatives of such antibodies. This disclosure further provides nucleic acids encoding such antibodies and antibody fragments and derivatives, as well as methods for producing and using such antibodies (including methods for treating and preventing TSLP-related inflammatory and fibrotic disorders).

Description

[Technical Field]

[0001] Cross-references to related applications This application claims the benefits of U.S. Provisional Patent Application No. 61 / 091,676, filed on 25 August 2008, and U.S. Provisional Patent Application No. 60 / 971,178, filed on 10 September 2007, both of which are incorporated herein by reference.

[0002] Field of Invention The field of this invention is human thymic stromal lymphoid neogenes (thymic stromal The present invention relates to a composition of an antigen-binding protein containing an antibody capable of binding to lymphopoietin, and to a related method. [Background technology]

[0003] Background of the Invention The prevalence of allergic diseases such as asthma, allergic rhinitis, atopic dermatitis, and food allergies appears to be increasing in recent years, particularly in developed countries, with a growing proportion of the affected population (Non-Patent Literature 1). Thymic-interstitial lymphoid neogenetic factor (TSLP) is an epithelial cell-derived cytokine produced in response to pro-inflammatory stimuli. TSLP has been found to promote allergic inflammatory responses primarily through its activity on dendritic and mast cells (Non-Patent Literature 2, Non-Patent Literature 3). Human TSLP expression has been reported to increase in the airways of asthma patients, correlating with disease severity (Non-Patent Literature 4). In addition, TSLP protein levels are detectable in concentrated bronchoalveolar lavage (BAL) fluid from asthma patients and other patients with allergic disorders. Elevated levels of TSLP protein and mRNA are also found in the lesional skin of patients with atopic dermatitis (AD). Therefore, TSLP antagonists are useful in treating inflammatory disorders.

[0004] Furthermore, TSLP has also been found to promote fibrosis, as reported in U.S. Patent Application No. 11 / 344,379. Fibrosis occurs when the fibrosis phase is unchecked during the tissue repair process, resulting in extensive tissue remodeling and the formation of permanent scar tissue (Non-Patent Literature 5). It is estimated that up to 45% of deaths in the United States may be attributable to fibroproliferative disorders that can affect many tissues and organ systems (Non-Patent Literature 5).

[0005] Currently, anti-inflammatory treatments are used to treat fibrotic disorders because fibrosis is common in many persistent inflammatory diseases such as idiopathic pulmonary fibrosis, progressive kidney disease, and cirrhosis. However, the mechanisms involved in controlling fibrosis appear to be quite different from those of inflammation, and anti-inflammatory therapy is not always effective in reducing or preventing fibrosis (Non-Patent Literature 5). Therefore, there remains a need to develop treatments to reduce and prevent fibrosis.

[0006] Therefore, antagonists to TSLP are expected to be useful in treating these inflammatory and fibrotic disorders. This disclosure provides such treatments and methods. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Kay, N Engl. J. Med. (2001)344:30-37 [Non-Patent Document 2] Soumelis et al., Nat Immun (2002) 3(7):673-680 [Non-Patent Document 3] Allakhverdi et al., J.Exp.Med.(2007)204(2):253~258 [Non-Patent Document 4] Ying et al., J. Immunol. (2005) 174:8183~8190

Non - Patent Document 5

Summary of the Invention

Means for Solving the Problems

[0008] Summary of the Invention In one aspect, the present disclosure provides an isolated antigen - binding protein, and this isolated antigen - binding protein is a. the following: i. A light - chain CDR3 sequence that differs from a CDR3 sequence selected from the group consisting of the light - chain CDR3 sequences of A1 - A27 by only additions, substitutions and / or deletions of up to 2 amino acids in total; ii. QQAX8SFPLT (SEQ ID NO: 251); a light - chain CDR3 sequence selected from, and b. the following: i. A heavy - chain CDR3 sequence that differs from a CDR3 sequence selected from the group consisting of the heavy - chain CDR3 sequences of A1 - A27 by only additions, substitutions and / or deletions of up to 3 amino acids in total; ii. GGGIX 12 VADYYX 13 YGMDV (SEQ ID NO: 255); iii. DX 21 GX 22 SGWPLFX 23 Y (SEQ ID NO: 259) including a heavy - chain CDR3 sequence selected from, where X8 is an N residue or a D residue; X 12 is a P residue or an A residue; X 13 is a Y residue or an F residue; X 21 is a G residue or a R residue; X 22 is a S residue or a T residue; X 23 is an A residue or a D residue, and the above antigen - binding protein specifically binds to TSLP.

[0009] In another aspect, the isolated antigen - binding protein of the present disclosure further has the following: a. the following: i. Light chain CDR1 sequences that differ from the A1-A27 light chain CDR1 sequences by only 3 amino acid additions, substitutions, and / or deletions; ii.RSSQSLX1YSDGX2TYLN(Sequence ID 246); iii.RASQX4X5SSWLA(Sequence ID 249); Selected light chain CDR1 sequence b. Below: i. Light chain CDR2 sequences that differ from the A1-A27 CDR2 sequences by only two amino acid additions, substitutions, and / or deletions; ii. KVSX3 (residues 1-4 of sequence number 247); iii.X6X7SSLQS(sequence number 250); or iv.QDX9KRPS(Sequence ID 252) A light chain CDR2 sequence selected from; and c. Below: i. Heavy chain CDR1 sequences that differ only by the addition, substitution, and / or deletion of 2 amino acids or less from the CDR1 sequences A1-A27; ii.X 10 YGMH (sequence number 253) and; iii.X 15 X 16 YMX 17 (Sequence No. 257) and; A heavy chain CDR1 sequence selected from, and d. Below: i. Heavy chain CDR2 sequences that differ only by the addition, substitution, and / or deletion of 3 amino acids or less from the CDR2 sequences A1-A27; ii.VIWX 11 DGSNKYYADSVKG (Sequence ID 254) and; iii.VISYDGSX 14 KYYADSVKG (Sequence ID 256) and; iv.WINPNSGGTNX 18 X 19 X 20 KFQG (Sequence ID 258) and; It comprises at least one of the heavy chain CDR2 sequences selected from, where X1 is a V residue or an I residue; X2 is an N residue or a D residue; X3 is a Y residue or an N residue; X4 is a G residue or an S residue; X5 is an L residue or an I residue; X6 is an N residue or a T residue; X7 is a T residue or an A residue; X9 is a K residue or an N residue; X 10 X is an S residue or an N residue; 11 X is a Y residue or an F residue; 14 is a Y residue or an N residue; X 15 X is a D residue or a G residue; 16 is a Y residue or a D residue; X 17 X is a Y residue or an H residue; 18 is a Y residue or an H residue; X 19 is a V residue or an A residue; X 20 This is a Q residue or an R residue, and the antigen-binding protein specifically binds to TSLP.

[0010] In another aspect of this disclosure, the isolated antigen-binding protein described in claim 1 is: a. The following: i. Light chain CDR1 sequence selected from A1 to A27; ii. Light chain CDR2 sequence selected from A1 to A27; iii. Light chain variable domain containing a light chain CDR3 sequence selected from A1 to A27; or b. The following: i. A heavy chain CDR1 sequence selected from A1 to A27; ii. A heavy chain CDR2 sequence selected from A1 to A27, and iii. A heavy chain variable domain containing a heavy chain CDR3 sequence selected from A1 to A27; or c. Light chain variable domain of (a) and heavy chain variable domain of (b) Includes any of the following.

[0011] In a further context, the isolated antigen-binding proteins described above are as follows: a. Below: i. Amino acids having at least 80% identical sequences to the light chain variable domain sequence selected from L1 to L27; ii. A sequence of amino acids encoded by a polynucleotide sequence that is at least 80% identical to the polynucleotide sequence encoding the L1-L27 light chain variable domain sequence; iii. A sequence of amino acids encoded by a polynucleotide sequence that hybridizes under moderately stringent conditions to a complementary chain of a polynucleotide consisting of L1-L27 light chain variable domain sequences; Selected light chain variable domain sequence b. Select from the following: heavy chain variable domain sequences i. A sequence of amino acids that is at least 80% identical to the heavy chain variable domain sequences of H1-H27; ii. A sequence of amino acids encoded by a polynucleotide sequence that is at least 80% identical to the polynucleotide sequence encoding the heavy chain variable domain sequences of H1-H27; iii. A sequence of amino acids encoded by a polynucleotide sequence that hybridizes under moderately stringent conditions to a complementary chain of a polynucleotide consisting of heavy chain variable domain sequences H1-H27; or c. Light chain variable domain of (a) and heavy chain variable domain of (b), It contains one of the above antigen-binding proteins, and the antigen-binding protein specifically binds to TSLP.

[0012] In a further context, the isolated antigen-binding protein of this disclosure comprises either a. a light chain variable domain sequence selected from L1 to L27; b. a heavy chain variable domain sequence selected from H1 to H27; or c. the light chain variable domain of (a) and the heavy chain variable domain of (b), wherein the antigen-binding protein specifically binds to TSLP.

[0013] In a further context, the isolated binding proteins are L1H1, L2H2, L3H3, L4H4, L5H5, L6H6, L7H7, L8H8, L9H9, L10H10, L11H11, L12H12, L13.1H13, L13.2H13, L14.1H14, L14.2H14, L15.1H15, L15.2H15, and L16.1H16. It includes light chain variable domain sequences and heavy chain variable domain sequences selected from L16.2H16, L17H17, L18.1H18, L18.2H18, L19.1H19, L19.2H19, L20.1H20, L20.2H20, L21H21, L22H22, L23H23, L24H24, L25H25, L26H26, and L27H27.

[0014] In a further aspect, the isolated antigen-binding proteins described above include binding proteins that bind to TSLP with a Kd substantially identical to that of a reference antibody selected from A2, A3, A4, and A5. In another aspect, the isolated antigen-binding proteins described above include binding proteins that inhibit TSLP activity with an IC50 identical to that of a reference antibody selected from A2, A3, A4, or A5 according to a primary cell OPG assay.

[0015] Furthermore, the isolated antigen-binding proteins described above cross-compete with the reference antibody for binding to TSLP. In another aspect, the isolated antigen-binding proteins bind to the same epitope as the reference antibody (e.g., A2, A4, A5, A6, A7, A10, A21, A23, or A26).

[0016] In one aspect, the isolated antigen-binding proteins described above are selected from human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, antigen-binding antibody fragments, single-chain antibodies, diabodies, triabodies, tetrabodies, Fab fragments, F(fa')x fragments, domain antibodies, IgD antibodies, IgE antibodies, and IgM antibodies, and IgG1 antibodies, and IgG2 antibodies, and IgG3 antibodies, and IgG4 antibodies, and IgG4 antibodies having at least one mutation in the hinge region that reduces the tendency to form intra-H chain disulfide bonds. In one aspect, the isolated antigen-binding proteins described above are human antibodies.

[0017] Also provided are isolated nucleic acid molecules comprising polynucleotide sequences encoding light chain variable domains, heavy chain variable domains, or both of the antigen-binding factors of the present disclosure. In one embodiment, the polynucleotide comprises light chain variable sequences L1-L27 and / or heavy chain variable sequences H1-H27, or both.

[0018] A vector comprising the polynucleotide of the present disclosure is also provided. In one embodiment, the vector is an expression vector. A host cell comprising the vector is also provided. A hybridoma capable of producing the antigen-binding protein of the present invention is also provided. A method for producing the antigen-binding protein is also provided, comprising the step of culturing the host cell under conditions that enable the host cell to express the antigen-binding protein.

[0019] A pharmaceutical composition comprising the antigen-binding protein of the present invention is also provided. In one embodiment, this pharmaceutical composition comprises a human antibody. A method for treating a TSLP-associated inflammatory condition in a subject requiring treatment of the TSLP-associated inflammatory condition is also provided, the method comprising the step of administering a therapeutically effective amount of the above composition to the subject. In one embodiment, this inflammation The conditions are allergic asthma, allergic rhinosinusitis, allergic conjunctivitis, or atopic dermatitis. A method for treating TSLP-associated fibrosis in subjects requiring treatment of TSLP-associated fibrosis is also provided, the method comprising the step of administering a therapeutically effective amount of the above composition to the subject. In one embodiment, the fibrosis is scleroderma, interstitial lung disease, idiopathic pulmonary fibrosis, fibrosis resulting from chronic hepatitis B or chronic hepatitis C, radiation-induced fibrosis, and fibrosis resulting from wound healing. [Brief explanation of the drawing]

[0020] [Figure 1A] The amino acid sequences of the light chain CDR1, CDR2, and CDR3 regions of A1 to A27 are provided. Furthermore, exemplary nucleotide sequences encoding each CDR are provided. [Figure 1B] The amino acid sequences of the light chain CDR1, CDR2, and CDR3 regions of A1 to A27 are provided. Furthermore, exemplary nucleotide sequences encoding each CDR are provided. [Figure 1C] The amino acid sequences of the light chain CDR1, CDR2, and CDR3 regions of A1 to A27 are provided. Furthermore, exemplary nucleotide sequences encoding each CDR are provided. [Figure 1D] The amino acid sequences of the light chain CDR1, CDR2, and CDR3 regions of A1 to A27 are provided. Furthermore, exemplary nucleotide sequences encoding each CDR are provided. [Figure 1E] The amino acid sequences of the light chain CDR1, CDR2, and CDR3 regions of A1 to A27 are provided. Furthermore, exemplary nucleotide sequences encoding each CDR are provided. [Figure 1F] The amino acid sequences of the light chain CDR1, CDR2, and CDR3 regions of A1 to A27 are provided. Furthermore, exemplary nucleotide sequences encoding each CDR are provided. [Figure 2A] The amino acid sequences of the heavy chain CDR1, CDR2, and CDR3 regions of A1 to A27 are provided. Furthermore, exemplary nucleotide sequences encoding each CDR are provided. [Figure 2B]The amino acid sequences of the heavy chain CDR1, CDR2, and CDR3 regions of A1 to A27 are provided. Furthermore, exemplary nucleotide sequences encoding each CDR are provided. [Figure 2C] The amino acid sequences of the heavy chain CDR1, CDR2, and CDR3 regions of A1 to A27 are provided. Furthermore, exemplary nucleotide sequences encoding each CDR are provided. [Figure 2D] The amino acid sequences of the heavy chain CDR1, CDR2, and CDR3 regions of A1 to A27 are provided. Furthermore, exemplary nucleotide sequences encoding each CDR are provided. [Figure 2E] The amino acid sequences of the heavy chain CDR1, CDR2, and CDR3 regions of A1 to A27 are provided. Furthermore, exemplary nucleotide sequences encoding each CDR are provided. [Figure 2F] The amino acid sequences of the heavy chain CDR1, CDR2, and CDR3 regions of A1 to A27 are provided. Furthermore, exemplary nucleotide sequences encoding each CDR are provided. [Modes for carrying out the invention]

[0021] Detailed description of the invention The present invention relates to antigen-binding factors (including antigen-binding proteins) that specifically bind to the cytokine human thymic stromal lymphoid neogenetic factor (TSLP), such as antagonistic TSLP antibodies, antibody fragments, and antibody derivatives, which are antigen-binding proteins that inhibit TSLP binding and signaling. These antigen-binding factors are useful in inhibiting or blocking TSLP from binding to its receptor and are useful in treating inflammatory diseases, fibrotic diseases, and other related conditions.

[0022] The present invention further provides compositions, kits, and methods relating to antigen-binding proteins that bind to TSLP. Also provided are nucleic acid molecules and their derivatives and fragments, including sequences of polynucleotides encoding all or part of a polypeptide that binds to TSLP, such as nucleic acids encoding all or part of an anti-TSLP antibody, antibody fragment, or antibody derivative. The present invention further provides vectors and plasmids containing such nucleic acids, as well as cells or cell lines containing such nucleic acids and / or vectors and plasmids. Methods provided include, for example, methods for producing, identifying, or isolating an antigen-binding protein (e.g., an anti-TSLP antibody) that binds to human TSLP, and methods for distributing antigen-binding proteins that bind to TSLP. The invention includes methods for determining whether or not to bind to TSLP, methods for preparing compositions (e.g., pharmaceutical compositions) containing antigen-binding proteins that bind to TSLP, methods for administering antigen-binding proteins that bind to TSLP to a subject (e.g., methods for treating conditions mediated by TSLP), and methods for modulating biological activity related to TSLP signaling in vitro or in vivo.

[0023] TSLP Thymic stromal lymphoid neogenesis factor (TSLP) refers to type I cytokines consisting of four α-helical bundles. These are members of the IL-2 family, but are most closely related to IL-7. Cytokines are small, regulatory proteins secreted in response to specific stimuli, acting on receptors on the membranes of target cells. Cytokines regulate various cellular responses. Cytokines are generally described in references such as *Cytokines*, edited by A. Mire-Sluis and R. Thorne, Academic Press, New York, (1998).

[0024] TSLP was originally cloned from a mouse thymic stromal cell line (Sims et al., J.Exp.Med 192(5), 671-680 (2000)) and was found to support the development of early B and T cells. Later, human TSLP was cloned and found to have 43% amino acid sequence identity with its mouse homolog (Quentmeier et al., Leukemia 15, 1286-1292 (2001) and U.S. Patent No. 6,555,520 (which is incorporated herein by reference)). The polynucleotide and amino acid sequences of human TSLP are presented in Sequence ID No. 1 and No. 2, respectively. TSLP has been found to bind with low affinity to receptor chains from the hematopoietin receptor family called TSLP receptor (TSLPR) (described in U.S. Patent Application No. 09 / 895,945 (Publication No. 2002 / 0068323)) (SEQ ID NOs. 3 and 4). The polynucleotide sequence encoding human TSLPR is presented as SEQ ID NO. 3 in this application, and its amino acid sequence is presented as SEQ ID NO. 4 in this application. The soluble domain of TSLPR is approximately amino acids 25-231 of SEQ ID NO. 4. TSLP binds with high affinity to the TSLPR heterodimer complex and the interleukin 7 receptor αIL-7Rα (Park et al., J.Exp.Med 192:5(2000), U.S. Patent Application No. 09 / 895,945, Publication No. US2002 / 0068323). The sequence of IL-7 receptor α is shown in Figure 2 of U.S. Patent No. 5,264,416 (which is incorporated herein by reference). The sequence of the soluble domain of IL-7 receptor α is amino acids 1-219 in Figure 2 of U.S. Patent No. 5,264,416.

[0025] As used herein, the term “TSLP polypeptide” refers to various forms of TSLP useful as an immunogen. These include TSLP expressed in a modified form in which the furin cleavage site is removed through amino acid sequence modification, as described in PCT Patent Application Publication WO03 / 032898. Modified TSLP retains activity, but the full sequence is more readily expressed in mammalian cells such as CHO cells. Examples of TSLP polypeptides include SEQ ID NO: 2, SEQ ID NO: 373, and SEQ ID NO: 375.

[0026] Furthermore, cynomolgus TSLP was identified, as shown in Example 1 below, and for example, as shown in Sequence ID No. 380.

[0027] TSLP is produced in human epithelial cells, including cutaneous epithelial cells, bronchial epithelial cells, tracheal epithelial cells, and airway epithelial cells, keratinocytes, stromal and mast cells, smooth muscle cells, and lung and cutaneous fibroblasts, as measured by quantitative mRNA analysis. melis et al., Nature Immunol. 3(7) 673-680 (2002). Both mouse and human TSLP are involved in promoting allergic inflammation.

[0028] [Table 1] TSLP activity TSLP activity involves the proliferation of BAF cells expressing human TSLPR (BAF / HTR), as described in PCT patent application publication WO03 / 032898. The BAF / HTR bioassay utilizes a mouse pro B lymphocyte cell line transfected with the human TSLP receptor. BAF / HTR cells are huTSLP-dependent in terms of growth and proliferate in response to active huTSLP added to the test sample. After the incubation period, cell proliferation is measured by adding Alamar Blue dye I or tritiated thymidine. Proliferation can also be measured using commercially available kits such as the CYQUANT cell proliferation assay kit (Invitrogen).

[0029] Further assays for huTSLP activity include assays that measure the induction of T cell growth from human bone marrow by TSLP, such as those described in U.S. Patent No. 6,555,520. Another TSLP activity is the activity that activates STAT5, as described in relation to Levin et al., J.Immunol. 162:677-683 (1999) and PCT patent application WO03 / 032898.

[0030] Further assays can be found in U.S. Patent Publication No. 2006 / 0039910 (sequential number 11 / 2 This includes the production of TSLP-induced CCL17 / TARC from primary human monocytes and dendritic cells, as described in 05,909).

[0031] Cell-based assays useful for measuring TSLP activity are described in the following examples. These include the BAF cell proliferation assay described above, and the primary cell assay described below for measuring TSLP-induced osteoprotegerin (OPG) production from primary human dendritic cells, and the cynomolgus monkey peripheral blood mononuclear cell assay, also described below.

[0032] TSLP activity also includes in vivo activity. These can be measured in mouse models (e.g., as described in Zhou et al., Nat Immunol 6(10)1047-1053 (2005) and Yoo et al., J Exp Med. 202(4)541-549 (2005)). For example, anti-mouse TSLP antibodies have been shown to reduce BALF cell integrity and BALF levels of IL-5 and IL-13 in the Ova-asthma model (Zhou et al.).

[0033] definition Polynucleotide and polypeptide sequences are indicated using standard one- or three-letter abbreviations. Unless otherwise specified, polypeptide sequences have their amino terminus on the left and their carboxyl terminus on the right, and single-stranded nucleic acid sequences, and the top strand of double-stranded nucleic acid sequences, have their 5' terminus on the left and their 3' terminus on the right. Furthermore, specific polypeptide or polynucleotide sequences may be described by explaining how they differ from a reference sequence.

[0034] Polynucleotide and polypeptide sequences of specific light chain variable domains and heavy chain variable domains (e.g., L1 ("light chain variable domain 1"), H1 ("heavy chain variable domain 1")). Antibodies containing light and heavy chains are indicated by combining the names of the light chain variable domain and the heavy chain variable domain. For example, "L4H7" indicates an antibody containing the L4 light chain variable domain and the H7 heavy chain variable domain.

[0035] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention have meanings generally understood by those skilled in the art. Furthermore, unless otherwise required by the context, singular terms include plural terms, and plural terms include singular terms. Generally, the scientific terms and techniques related to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein chemistry, nucleic acid chemistry, and hybridization described herein are well known and commonly used in the art. Unless otherwise specified, the methods and techniques of the present invention are generally well known in the art and are performed in accordance with conventional methods described in various general and more specific references cited and discussed throughout this specification. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual See Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990) (these are incorporated herein by reference). Enzyme reactions and purification techniques are carried out according to the manufacturer's specifications, as is generally done in the art, or as described herein. The terminology and laboratory procedures and techniques used herein in relation to analytical chemistry, synthetic organic chemistry, and medicinal chemistry and pharmaceutical chemistry are well known and applicable. These are commonly used in the technical field. Standard techniques may be used for chemical synthesis, chemical analysis, pharmaceutical preparations, formulations, and for delivery and patient treatment.

[0036] The following terms, unless otherwise specified, are understood to have the following meanings: The term “isolated molecule” (if the molecule is, for example, a polypeptide, polynucleotide, or antibody) is, based on its origin or the source of its derivative, a molecule that (1) does not have naturally associated components that are present in its natural state, (2) substantially does not contain other molecules from the same species, (3) is expressed by cells of a different species, or (4) does not exist in nature. Thus, a molecule that is chemically synthesized or expressed in a cell system different from the cells in which it occurs in nature is “isolated” from its naturally associated components. A molecule may also be made substantially free of naturally associated components by isolation using purification techniques well known in the art. The purity or homogeneity of a molecule can be assayed by many methods well known in the art. For example, the purity of a polypeptide sample can be assayed using polyacrylamide gel electrophoresis and staining of a gel to visualize polypeptides using techniques well known in the art. For specific purposes, higher resolution may be provided by using HPLC or other means well known in the art for purification.

[0037] The terms “TSLP inhibitor” and “TSLP antagonist” are used interchangeably. Each is a molecule that detectably inhibits TSLP signaling. This inhibition caused by the TSLP inhibitor does not need to be complete, as long as it is detectable using the assay. For example, the cell-based assay described in Example 4 below demonstrates a useful assay for measuring inhibition of TSLP signaling.

[0038] The terms "peptide," "polypeptide," and "protein" refer to molecules containing two or more amino acid residues linked together by peptide bonds, respectively. These terms encompass, for example, natural and artificial proteins, polypeptide analogs of protein fragments and protein sequences (e.g., mutaines, variants, and fusion proteins), and proteins modified post-translation or otherwise covalently or non-covalently. Peptides, polypeptides, or proteins may exist as monomers or polymers.

[0039] As used herein, the term “polypeptide fragment” means a polypeptide having deletions at the amino terminus and / or carboxyl terminus compared to the corresponding full-length protein. For example, a fragment may have a length of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 50, 70, 80, 90, 100, 150, or 200 amino acids. For example, a fragment may also have a length of at most 1,000, 750, 500, 250, 200, 175, 150, 125, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 14, 13, 12, 11, or 10 amino acids. A fragment may further contain one or more additional amino acids at either or both of its ends (e.g., a sequence of amino acids derived from a different naturally occurring protein (e.g., Fc or leucine zipper domain) or an artificial amino acid sequence (e.g., an artificial linker sequence)).

[0040] The polypeptides of the present invention include polypeptides modified in any way and for any reason to (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter the binding affinity for forming protein complexes, (4) alter the binding affinity, and (5) impart or alter other physicochemical or functional properties. Analogs include the mutaine of the polypeptide. For example, single or multiple amino acid substitutions (e.g., conserved amino acid substitutions) may be made in naturally occurring sequences (e.g., parts of the polypeptide outside domains that form intermolecular contacts). A "conserved amino acid substitution" does not substantially alter the structural features of the parent sequence (e.g., the substituted amino acid is present in the parent sequence). It should not tend to disrupt the rix, nor should it tend to disrupt other types of secondary structures that characterize the parent sequence or are required for its functionality. Examples of secondary and tertiary structures of polypeptides recognized in the art are described in Proteins, Structures and Molecular Principles (Creighton, ed., WH Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, NY (1991)); and Thornton et al., Nature 354:105 (1991) (these are incorporated herein by reference, respectively).

[0041] A "modified" polypeptide includes an amino acid sequence in which one or more amino acid residues are inserted into, deleted from, and / or substituted into another polypeptide sequence, in comparison to that amino acid sequence. Modifieds of the present invention include fusion proteins. Modified antibodies described herein also include those derived from processing. Such modifieds include, for example, those having one, two, three, four, five, six, seven, eight, nine, or more additional amino acids at the N-terminus of the light or heavy chain as a result of inefficient signal sequence cleavage. Such modifieds also include those having one or more amino acids deleted from the N-terminus or C-terminus of the light or heavy chain.

[0042] A "derivative" of a polypeptide is a polypeptide (e.g., an antibody) that has been chemically modified, for example, by conjugation to another chemical moiety (e.g., polyethylene glycol, albumin (e.g., human serum albumin)), phosphorylation, and glycosylation. Unless otherwise specified, the term "antibody" includes antibodies, which consist of two full-length heavy chains and two full-length light chains, as well as their derivatives, variants, fragments, and mutaines. Examples of these are listed below.

[0043] An "antigen-binding protein" according to this disclosure is a protein capable of binding to an antigen, and optionally a scaffold or framework portion that allows the antigen-binding portion to adopt a conformation that facilitates the binding of the antigen-binding protein to the antigen. In one embodiment, the antigen-binding protein of the present invention comprises at least one CDR. Examples of antigen-binding proteins include antibodies, antibody fragments (e.g., the antigen-binding portion of an antibody), antibody derivatives, and antibody analogs. This antigen-binding protein may include, for example, an alternative protein scaffold or artificial scaffold having a grafted CDR or CDR derivative. Such scaffolds include, but are not limited to, antibody-derived scaffolds containing introduced mutations that stabilize the three-dimensional structure of the antigen-binding protein, and, for example, complete synthetic scaffolds containing biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, Vol. 53, No. 1: 121-129; Roque et al., 2004, Biotechnol. Prog. 20: 639-654. Furthermore, peptide antibody mimes ("PAMs") and scaffold-based antibody mimes utilizing fibronectin components as scaffolds may be used.

[0044] Antigen-binding proteins can have structures similar to, for example, naturally occurring immunoglobulins. "Immunoglobulins" are tetrameric molecules. In naturally occurring immunoglobulins, each tetramer consists of two identical pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa). The amino-terminus of each chain contains a variable region of approximately 100-110 or more amino acids, which plays a major role in antigen recognition. The carboxyl-terminus of each chain defines a constant region that plays a major role in effector activity. (Human) Light chains are classified into κ and λ light chains. Heavy chains are classified as μ, δ, γ, α, or ε, which define the antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by "J" regions of approximately 12 or more amino acids, and the heavy chain also contains "D" regions of approximately 10 or more amino acids. For general information, see Fundamental Immunology Ch.7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989)) (which, for all purposes, is incorporated herein by reference in its entirety). The variable regions of each light / heavy chain pair form antibody binding sites, resulting in an intact immunoglobulin having two binding sites.

[0045] Naturally occurring immunoglobulin chains exhibit a similar general structure of a relatively conserved framework region (FR) linked by three hypervariable regions (also called complementarity-determining regions or CDRs). From the N-terminus to the C-terminus, both the light and heavy chains contain domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The amino acid assignments to each domain are as follows: Kabat et al., Sequences of Proteins. The definition follows that of Immunological Interest, 5th edition, US Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242, 1991. Intact antibodies include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies with full-length heavy and light chains.

[0046] Unless otherwise specified, “antibody” refers to an intact immunoglobulin or its antigen-binding moiety that competes with an intact antibody for specific binding. Antigen-binding moieties can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of an intact antibody. Antigen-binding moieties include Fab, Fab', F(ab')2, Fd, Fv, and domain antibodies (dAb), as well as complementarity-determining region (CDR) fragments, single-chain antibodies (scFv), diabodies, triabodies, tetrabodies, and polypeptides containing at least a portion of an immunoglobulin sufficient to confer specific antigen binding to a polypeptide.

[0047] Fab fragment is V L , V H , C L and C H The F(ab')2 fragment is a monovalent fragment having one domain; the F(ab')2 fragment is a divalent fragment having two Fab fragments linked by disulfide bridges in the hinge region; the Fd fragment is V H Domain and C H It has one domain; the Fv fragment is the V of a single arm of the antibody. L Domain and V H It has a domain; and the dAb fragment is V H Domain, V L Domain, or V H Or V L It has an antigen-binding fragment of the domain (U.S. Patent Nos. 6,846,634, 6,696,245, U.S. Patent Publication Nos. 05 / 0202512, 04 / 0202995, 04 / 0038291, 04 / 0009507, 03 / 0039958, Ward et al., Nature 341:544-546, 1989).

[0048] Single-chain antibodies (scFv) are V L and V HThe region is linked by a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain (where this linker is long enough to allow this protein chain to fold over itself), and is an antibody that forms a monovalent antigen-binding site (see, e.g., Bird et al., 1988, Science 242:423-26, and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-83). The diabody is a bivalent antibody containing two polypeptide chains, where each polypeptide chain is linked by a linker. H Domain and V L The linker contains a domain that is too short to pair with two domains on the same chain, and therefore allows each domain to pair with a complementary domain on another polypeptide chain (e.g., Holliger et al.). See 1993, Proc. Natl. Acad. Sci. USA 90:6444-48, and Poljak et al., 1994, Structure 2:1121-23. When the two polypeptide chains of a diabody are identical, the diabody resulting from their pairing has two identical antigen-binding sites. Polypeptide chains with different sequences may be used to create diabodies with two different antigen-binding sites. Similarly, tribodies and tetrabodies are antibodies containing three and four polypeptide chains, respectively, that form three and four antigen-binding sites, which may be identical or different.

[0049] The complementarity-determining regions (CDRs) and framework regions (FRs) of a given antibody can be identified using the systematic method described by Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, US Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242, 1991. One or more CDRs can be incorporated into a molecule either covalently or noncovalently, making that molecule an antigen-binding protein. The antigen-binding protein can incorporate the CDR as part of a longer polypeptide chain, covalently ligate the CDR to another polypeptide chain, or incorporate the CDR noncovalently. These CDRs enable this antigen-binding protein to specifically bind to a particular antigen of interest.

[0050] Antigen-binding proteins may have one or more binding sites. If more than one binding site is present, the binding sites may be identical or distinct. For example, naturally occurring human immunoglobulins typically have two identical binding sites, while "bispecific" or "bifunctional" antibodies have two distinct binding sites.

[0051] The term "human antibody" includes all antibodies having one or more variable and constant regions derived from a human immunoglobulin sequence. In one embodiment, all variable and constant domains are obtained from a human immunoglobulin sequence (full human antibody). These antibodies can be prepared by various methods (including, for example, immunization using the antigen of interest in mice genetically modified to express antibodies derived from genes encoding human heavy and / or light chains, as described below).

[0052] Humanized antibodies have a sequence different from that of antibodies obtained from non-human species due to the substitution, deletion, and / or addition of one or more amino acids, and as a result, when administered to a human subject, these humanized antibodies are less likely to elicit an immune response and / or elicit a less severe immune response compared to antibodies from non-human species. In one embodiment, specific amino acids in the framework domains and constant domains of the heavy and / or light chains of a non-human antibody are mutated to produce a humanized antibody. In another embodiment, a constant domain derived from a human antibody is fused to a variable domain of a non-human species. In yet another embodiment, one or more amino acid residues in one or more CDR sequences of a non-human antibody are modified to reduce the potential immunogenicity of the non-human antibody when administered to a human subject, where the modified amino acid residues are either not important for the antibody's immune-specific binding to its antigen, or the changes made to the amino acid sequence are conservative changes, and as a result, the binding of the humanized antibody to the antigen is not significantly inferior to the binding of the non-human antibody to the antigen. Examples of methods for producing humanized antibodies can be found in U.S. Patent Nos. 6,054,297, 5,886,152, and 5,877,293.

[0053] The term "chimeric antibody" refers to an antibody that contains one or more regions derived from one antibody and one or more regions derived from one or more other antibodies. In one embodiment, one or more of the CDRs are obtained from a human anti-TSLP antibody. In another embodiment, of the CDRs All of these are obtained from human anti-TSLP antibodies. In another embodiment, CDRs derived from more than one human anti-TSLP antibody are mixed and harmonized within the chimeric antibody. For example, the chimeric antibody may include CDR1 derived from the light chain of a first human anti-TSLP antibody, CDR2 and CDR3 derived from the light chain of a second human anti-TSLP antibody, and CDR derived from the heavy chain of a third anti-TSLP antibody. Furthermore, the framework region may be obtained from one of the same anti-TSLP antibodies, one or more different antibodies (e.g., human antibodies), or a humanized antibody. In an example of a chimeric antibody, the heavy chain and / or part of the light chain are derived from a particular species or are identical to, homologous to, or derived from an antibody belonging to a particular antibody class or subclass, while the rest of the chain is derived from a different species or is identical to, homologous to, or derived from an antibody belonging to a different antibody class or subclass. This also includes fragments of such antibodies that exhibit the desired biological activity (i.e., the ability to specifically bind to the human TSLP receptor).

[0054] Antibody fragments or analogs can be readily prepared by those skilled in the art in accordance with the teachings herein and using techniques well known in the art. The preferred amino and carboxyl termini of the fragments or analogs are located near the boundaries of the functional domains. Structural and functional domains can be identified by comparing nucleotide and / or amino acid sequence data with publicly available or proprietary sequence databases. Computerized comparison methods can be used to identify sequence motifs or predicted protein conformation domains present in other proteins with known structures and / or functions. Methods for identifying protein sequences that fold into known three-dimensional structures are known; see, for example, Bowie et al., 1991, Science 253:164.

[0055] A "CDR-grafted antibody" is an antibody that contains a framework of one or more CDRs derived from an antibody of a specific species or isotype, and another antibody of the same or a different species or isotype.

[0056] A "polyspecific antibody" is an antibody that recognizes more than one epitope on one or more antigens. A subclass of this type of antibody is a "bispecific antibody" that recognizes two different epitopes on the same or different antigens.

[0057] Antigen-binding proteins containing antibodies are, it is, 10 -7 When binding to an antigen such as TSLP with high binding affinity, as determined by a Kd (or the corresponding Kb, as defined below) value less than or equal to M, it is said to "specifically bind" to that antigen.

[0058] An "antigen-binding domain," "antigen-binding region," or "antigen-binding site" is a part of an antigen-binding protein that contains amino acid residues (or other parts) that interact with the antigen and contribute to the specificity and affinity of the antigen-binding protein for that antigen. With respect to an antibody that specifically binds to an antigen, this includes at least one part of its CDR domain.

[0059] The "percent identity" of two polynucleotide sequences or two polypeptide sequences is determined by comparing those sequences using the GAP computer program (part of the GCG Wisconsin Package, version 10.3 (Accelrys, San Diego, CA)) with its default parameters.

[0060] The terms "polynucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably throughout, and they refer to DNA or RNA produced using DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), and nucleotide analogs (e.g., peptide nucleic acids and naturally occurring nucleotide analogs). This includes analogs and hybrids thereof. The nucleic acid molecule may be single-stranded or double-stranded. In one embodiment, the nucleic acid molecule of the present invention includes a continuous open reading frame encoding the antibody of the present invention, or a fragment, derivative, mutain, or variant thereof.

[0061] Two single-stranded polynucleotides can be aligned in an antiparallel orientation, and as a result, all nucleotides in one polynucleotide face their complementary nucleotide in the other polynucleotide without introducing gaps and without any nucleotides that do not pair at the 5' or 3' ends of either sequence, then those two single-stranded polynucleotides are "complementary" to each other. Two polynucleotides can hybridize to each other under moderately stringent conditions, then this polynucleotide is "complementary" to another polynucleotide. Thus, a polynucleotide can be complementary to another polynucleotide that is not its complementary strand.

[0062] A "vector" is a nucleic acid that can be used to introduce another nucleic acid, to which it is ligated, into a cell. One type of vector is a "plasmid," which is a linear or circular double-stranded DNA molecule to which an additional nucleic acid segment can be ligated. Another type of vector is a viral vector (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses), to which an additional DNA segment can be introduced into the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors containing bacterial origins of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the host cell's genome upon introduction into the host cell and thereby replicate together with the host genome. An "expression vector" is a type of vector that can induce the expression of a selected polynucleotide.

[0063] A nucleotide sequence is "operably ligated" to a regulatory sequence if the regulatory sequence affects the expression of that nucleotide sequence (e.g., the level, timing, or location of expression). A "regulatory sequence" is a nucleic acid that affects the expression of the nucleic acid to which it is operably ligated (e.g., the level, timing, or location of expression). A regulatory sequence may exert its influence on the regulated nucleic acid, for example, directly or through the action of one or more other molecules (e.g., polypeptides that bind to the regulatory sequence and / or the nucleic acid). Examples of regulatory sequences include promoters, enhancers, and other expression regulators (e.g., polyadenylation signals). Further examples of regulatory sequences are described, for example, in Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA, and Baron et al., 1995, Nucleic Acids Res. 23:3605~06.

[0064] A “host cell” is a cell that can be used to express nucleic acids (e.g., the nucleic acids of the present invention). The host cell may be a prokaryote (e.g., E. coli), or it may be a eukaryote, such as a single-celled eukaryote (e.g., yeast or other fungi), a plant cell (e.g., tobacco or tomato plant cell), an animal cell (e.g., human cell, monkey cell, hamster cell, rat cell, mouse cell or insect cell), or a hybridoma. Exemplary host cells include the Chinese hamster ovary (CHO) cell line, or the DHFR-deficient CHO cell line DXB-11 (see Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216-20), CHO cell lines that grow in serum-free medium (see Rasmussen et al., 1998, Cytotechnology 28:31), CS-9 cells (a derivative of DXB-11 CHO cells), and AM-1 / D cells (described in U.S. Patent No. 6,210,924), as well as their derivatives. Other CHO cell lines include CHO-K1 (ATCC# CCL-61), EM9 (ATCC# CRL-1861), and UV20 (ATCC# CRL-1862). Examples of other host cells include the monkey kidney cell line COS-7 (ATCC#). Examples include CRL 1651 (see Gluzman et al., 1981, Cell 23:175), L cells, C127 cells, 3T3 cells (ATCC CCL 163), HeLa cells, BHK (ATCC CRL 10) cell line, CV1 / EBNA cell line obtained from African green monkey kidney cell line CV1 (ATCC CCL 70) (see McMahan et al., 1991, EMBO J.10:2821), human embryonic kidney cells such as 293, 293 EBNA or MSR 293, human epithelial A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell lines obtained from in vitro cultures of primary tissue, primary explants, HL-60, U937, HaK or Jurkat cells. Typically, a host cell is a cultured cell that can be transformed or transfected with a nucleic acid encoding a polypeptide (which can then be expressed within that host cell). The phrase “recombinant host cell” can be used to mean a host cell that has been transformed or transfected with the nucleic acid to be expressed. A host cell can also be a cell that contains a nucleic acid but does not express that nucleic acid at the desired level unless a regulatory sequence is introduced into that host cell so as to be operably linked to that nucleic acid. It is understood that the term host cell refers not only to a specific target cell but also to the offspring or potential offspring of such a cell. For example, since certain modifications may occur in subsequent generations due to mutation or environmental influences, such offspring may not actually be identical to the parent cell, but are still included within the scope of the term as used herein.

[0065] Antigen-binding protein In one aspect, the disclosure provides antigen-binding proteins such as antibodies, antibody fragments, antibody derivatives, antibody mutaines, and antibody modifiers that bind to human TSLP. Antigen-binding proteins according to the disclosure include antigen-binding proteins that bind to human TSLP and thereby reduce TSLP activity. For example, an antigen-binding protein may interfere with the binding of TSLP to its receptor and thus reduce TSLP activity.

[0066] In one embodiment, the present invention provides an antigen-binding protein comprising one or more CDR sequences that differ from the CDR sequences shown in Figures 1A-1F or 2A-2F by only 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, 1 or fewer, or 0 or fewer amino acid residues.

[0067] In another embodiment, at least one of the CDR3 sequences of the antigen-binding protein is the sequence shown in Figures 1A-1F or Figures 2A-2F. In another embodiment, the light chain CDR3 sequence of the antigen-binding protein is the A1-A27 light chain sequence, and the heavy chain CDR3 sequence of the antigen-binding protein is the A1-A27 heavy chain CDR3 sequence.

[0068] In another embodiment, the antigen-binding proteins described above further comprise 1, 2, 3, 4, or 5 CDR sequences, each independently differing from the A1-A27 CDR sequences by only 5, 4, 3, 2, 1, or 0 single amino acid additions, substitutions, and / or deletions. The light chain CDR sequences of exemplary antigen-binding proteins A1-A27 and the heavy chain CDR sequences of exemplary binding proteins A1-A27 are shown in Figures 1A-1F and 2A-2F, respectively. The polynucleotide sequences encoding the amino acid sequences of the CDRs are also shown. Furthermore, the consensus sequences of the CDR sequences are provided below.

[0069]

number

[0070]

number

[0071]

number

[0072]

number

[0073]

number

[0074] Table 2 below provides the nucleic acid (DNA) sequences encoding the variable heavy domain (H#) and variable light domain (L#) for exemplary TSLP antigen-binding proteins A1–A27, as well as the amino acid sequences of the variable heavy domain and variable light domain, respectively. CDR1, 2, and 3 for each variable domain are continuous from the beginning to the end of each sequence. Framework (Fr) regions are underlined. Frameworks 1, 2, 3, and 4 for each variable domain are continuous from the beginning to the end of each sequence. (For example, in each sequence, the first underlined part of the sequence is Fr1, the second is Fr2, the third is Fr3, and the last is Fr4.)

[0075] [Table 2-1]

[0076] [Table 2-2]

[0077] [Table 2-3]

[0078] Table 2-4

[0079] Table 2-5

[0080] Table 2-6

[0081] Table 2-7

[0082] Table 2-8

[0083] Table 2-9

[0084] Table 2-10

[0085] Table 2-11

[0086] Table 2-12

[0087] Table 2-13

[0088] [Table 2-14]

[0089] [Table 2-15]

[0090] [Table 2-16] Certain embodiments of the antigen-binding protein of the present invention may comprise one or more amino acid sequences identical to one or more amino acid sequences of the CDR, and further comprise one or more FRs shown earlier. In one embodiment, the antigen-binding protein comprises the light chain CDR1 sequence shown earlier. In another embodiment, the antigen-binding protein comprises the light chain CDR2 sequence shown earlier. In another embodiment, the antigen-binding protein comprises the light chain CDR3 sequence shown earlier. In another embodiment, the antigen-binding protein comprises the heavy chain CDR1 sequence shown earlier. In another embodiment, the antigen-binding protein comprises the heavy chain CDR2 sequence shown earlier. In another embodiment, the antigen-binding protein comprises the heavy chain CDR3 sequence shown earlier. In another embodiment, the antigen-binding protein comprises Furthermore, it includes the previously shown light chain FR1 sequence. In another embodiment, this antigen-binding protein further includes the previously shown light chain FR2 sequence. In another embodiment, this antigen-binding protein further includes the previously shown light chain FR3 sequence. In another embodiment, this antigen-binding protein further includes the previously shown light chain FR4 sequence. In another embodiment, this antigen-binding protein further includes the previously shown heavy chain FR1 sequence. In another embodiment, this antigen-binding protein further includes the previously shown heavy chain FR2 sequence. In another embodiment, this antigen-binding protein further includes the previously shown heavy chain FR3 sequence. In another embodiment, this antigen-binding protein further includes the previously shown heavy chain FR4 sequence.

[0091] In one embodiment, the disclosure provides an antigen-binding protein comprising a light chain variable domain comprising an amino acid sequence that differs by only 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 residues from the sequence of a light chain variable domain selected from the group consisting of L1 to L27, where each such sequence difference is independently either a deletion, insertion, or substitution of one amino acid residue. In another embodiment, the light chain variable domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% identical to the sequence of a light chain variable domain selected from the group consisting of L1 to L27. In another embodiment, the light chain variable domain includes a sequence of amino acids encoded by a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% identical to the nucleotide sequence encoding the light chain variable domain selected from the group consisting of L1 to L27. In another embodiment, the light chain variable domain includes a sequence of amino acids encoded by a polynucleotide that hybridizes under moderately stringent conditions to a complementary chain of a polynucleotide encoding the light chain variable domain selected from the group consisting of L1 to L27. In another embodiment, the light chain variable domain includes a sequence of amino acids encoded by a polynucleotide that hybridizes under highly stringent conditions to a complementary chain of light chain polynucleotides L1 to L27.

[0092] In another embodiment, the present invention provides an antigen-binding protein comprising a heavy chain variable domain comprising an amino acid sequence that differs by only 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 residues from the sequence of a heavy chain variable domain selected from the group consisting of H1 to H27, where each such sequence difference is independently either a deletion, insertion, or substitution of one amino acid residue. In another embodiment, the heavy chain variable domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% identical to the sequence of a heavy chain variable domain selected from the group consisting of H1 to H27. In another embodiment, the heavy chain variable domain includes a sequence of amino acids encoded by a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% identical to the nucleotide sequence encoding the heavy chain variable domain selected from the group consisting of H1 to H27. In another embodiment, the heavy chain variable domain includes a sequence of amino acids encoded by a polynucleotide that hybridizes under moderately stringent conditions to a complementary chain of polynucleotides encoding the heavy chain variable domain selected from the group consisting of H1 to H27. In another embodiment, the heavy chain variable domain includes a sequence of amino acids encoded by a polynucleotide that hybridizes under highly stringent conditions to a complementary chain of polynucleotides encoding the heavy chain variable domain selected from the group consisting of H1 to H27.

[0093] In some embodiments provided in Table 2 above, two light chains are coupled to a single heavy chain and are identified, for example, as L-12.1, L-12.2, etc. These alternative light chains each form a pair with a single heavy chain. In these embodiments, the combination of light chains and heavy chains Combinations may be assayed as described below, and combinations of light and heavy chains that provide higher TSLP neutralizing activity may be selected.

[0094] Further embodiments include antigen-binding proteins comprising combinations of L1H1, L2H2, L3H3, L4H4, L5H5, L6H6, L7H7, L8H8, L9H9, L10H10, L11H11, L12H12, L13H13, L14H14, L15H15, L16H16, L17H17, L18H18, L19H19, L20H20, L21H21, L22H22, L23H23, L24H24, L25H25, L26H26 and L27H27.

[0095] The antigen-binding proteins of the present invention (e.g., antibodies, antibody fragments, and antibody derivatives) may further include any constant region known in the art. The light chain constant region may be, for example, a κ-type or λ-type light chain constant region (e.g., a human κ-type or λ-type light chain constant region). The heavy chain constant region may be, for example, an α-type, δ-type, ε-type, γ-type, or μ-type heavy chain constant region (e.g., a human α-type, δ-type, ε-type, γ-type, or μ-type heavy chain constant region). In one embodiment, the light chain or heavy chain constant region is a naturally occurring constant region fragment, derivative, variant, or mutaine.

[0096] In one embodiment, the antigen-binding protein described above includes IgG such as IgG1, IgG2, IgG3, or IgG4.

[0097] Techniques for obtaining antibodies of a different subclass or isotype from the target antibody (i.e., subclass switching) are known. For example, an IgG antibody can be obtained from an IgM antibody, and conversely, an IgM antibody can be obtained from an IgG antibody. Such techniques enable the preparation of novel antibodies that possess the antigen-binding properties of a given antibody (parent antibody) but also exhibit biological properties associated with a different antibody isotype or subclass than that of the parent antibody. Recombinant DNA techniques can be used. Cloned DNA encoding a specific antibody polypeptide (e.g., DNA encoding the constant domain of the desired isotype of antibody) can be used in such procedures. See also Lantto et al., 2002, Methods Mol. Biol. 178:303-16.

[0098] In one embodiment, the antigen-binding protein of the present invention comprises an IgG1 heavy chain constant domain or a fragment of an IgG1 heavy chain domain. In another embodiment, the antigen-binding protein of the present invention further comprises a light chain κ constant domain or a light chain λ constant domain or fragments thereof. The light chain constant regions and the polynucleotides encoding them are provided in Table 3 below. In yet another embodiment, the antigen-binding protein of the present invention further comprises a heavy chain constant domain or fragment thereof, such as the IgG2 heavy chain constant region shown in Table 3 below.

[0099] The nucleic acids (DNA) encoding the heavy chain constant domain and the light chain constant domain, as well as the amino acid sequences of the heavy chain domain and the light chain domain, are provided below. The λ variable domain may be fused to the λ constant domain, and the κ variable domain may be fused to the κ constant domain.

[0100] [Table 3] The antigen-binding proteins of the present invention include, for example, combinations of variable domains having a preferred isotype (e.g., IgA, IgG1, IgG2, IgG3, IgG4, IgM, IgE, and IgD), such as L1H1, L2H2, L3H3, L4H4, L5H5, L6H6, L7H7, L8H8, L9H9, L10H10, L11H11, L12H12, L13.1H13, L13.2H13, L14.1H14, and L14. Examples include those containing 2H14, L15.1H15, L15.2H15, L16.1H16, L16.2H16, L17H17, L18.1H18, L18.2H18, L19.1H19, L19.2H19, L20.1H20, L20.2H20, L21H21, L22H22, L23H23, L24H24, L25H25, L26H26, and L27H27, as well as their Fab or F(ab')2 fragments. Furthermore, if IgG4 is desired, it may also be desirable to introduce point mutations within the hinge region to mitigate the tendency to form intra-H chain disulfide bonds that can result in heterogeneity of IgG4 antibodies, as described in Bloom et al., 1997, Protein Science 6:407 (informatively incorporated herein).

[0101] Antibodies and antibody fragments As used herein, the term “antibody” means an intact antibody or its antigen-binding fragment, as set forth in the Definitions section herein. An antibody may include a complete antibody molecule (including polyclonal, monoclonal, chimeric, humanized, or human versions having full-length heavy and / or light chains) or an antigen-binding fragment thereof. Examples of antibody fragments include F(ab')2 fragments, Fab fragments, Fab' fragments, Fv fragments, Fc fragments, and Fd fragments, which may be incorporated into single-domain antibodies, monovalent antibodies, single-chain antibodies, maxibodies, minibodies, intrabody antibodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, 2005, Nature Biotechnology, 23, 9, 1126–1136). Antibody polypeptides containing fibronectin polypeptide monobodies are also disclosed in U.S. Patent No. 6,703,199. Other antibody polypeptides are disclosed in U.S. Patent Application Publication No. 2005 / 0238646 (which are single-chain polypeptides). Monovalent antibody fragments are disclosed in U.S. Patent Application Publication No. 20050227324.

[0102] Antigen-binding fragments derived from antibodies can be obtained, for example, by proteolytic hydrolysis of the antibody (e.g., pepsin or papain digestion of the entire antibody according to conventional methods). For example, an antibody fragment may be produced by enzymatic cleavage of an antibody using pepsin to provide a 5S fragment designated F(ab')2. This fragment may be further cleaved using a thiol reducing agent to yield a 3.5S monovalent Fab' fragment. If necessary, the cleavage reaction may be carried out using a blocking group for the sulfhydryl group resulting from the cleavage of the disulfide bond. As an alternative, enzymatic cleavage using papain directly yields two monovalent Fab fragments and one Fc fragment. These methods are described, for example, in Goldenberg, U.S. Patent No. 4,331,647; Nisonoff et al., Arch. Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959; Edelman et al., Methods. This is described in *in Enzymology* 1:422 (Academic Press 1967), as well as in Andrews, SM and Titus, JA, *Current Protocols in Immunology* (Coligan JE et al., eds.), John Wiley & Sons, New York (2003), pp. 2.8.1-2.8.10 and 2.10A.1-2.10A.5. Other methods for cleaving antibodies, such as separating the heavy chain to form a monovalent light-heavy chain fragment (Fd), further cleaving the fragment, or other enzymatic, chemical, or genetic techniques, may also be used, insofar as the fragment binds to an antigen recognized by the intact antibody.

[0103] Antibody fragments may also be any synthetic protein or a genetically engineered protein. Examples of antibody fragments include isolated fragments consisting of a light chain variable region, "Fv" fragments consisting of a heavy chain and a light chain variable region, and recombinant single-chain polypeptide molecules (scFv proteins) in which the light chain variable region and the heavy chain variable region are linked by a peptide linker.

[0104] Another form of antibody fragment is a peptide containing one or more complementarity-determining regions (CDRs) of the antibody. CDRs (also called "minimum recognition units" or "hypervariable regions") can be obtained by constructing a polynucleotide encoding the desired CDR. Such polynucleotides are prepared, for example, by using polymerase chain reactions that synthesize the variable regions using mRNA from antibody-producing cells as a template (e.g., Larrick et al., Methods: A Companion to Methods in Enzyme ology 2:106, 1991;Courtenay-Luck, “Genetic See also “Manipulation of Monoclonal Antibodies,” in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), p. 166 (Cambridge University Press 1995); and Ward et al., “Genetic Manipulation and Expression of Antibodies,” in Monoclonal Antibodies: Principles and Applications, Birch et al. (eds.), p. 137 (Wiley-Liss, Inc. 1995).

[0105] Therefore, in one embodiment, the binding factor comprises at least one CDR as described herein. This binding factor may comprise at least two, three, four, five, or six CDRs as described herein. This binding factor may further comprise at least one variable region domain of an antibody as described herein. This variable region domain may be of any size or amino acid composition and generally comprises at least one CDR sequence involved in binding to TSLP (e.g., heavy chain CDR1, CDR2, CDR3 and / or light chain CDR as specifically described herein), and this CDR sequence is adjacent to or in-frame with one or more framework sequences. Generally, the variable (V) region domain is an immunoglobulin heavy (V) region domain. H ) Chain variable domain and / or light (V L ) can be any suitable sequence (arrangement) of the chain variable domain. For example, the V region domain can be a monomer and, as described below, at least 1 × 10⁻¹⁶ -7 V can independently bind to human TSLP with affinity of M or less. H or V L It could be a domain. Alternatively, this V-region domain could be a dimer, and V H -V H , V H -V L or V L -V L It may contain a dimer. This V-region dimer may contain at least one V that can be bonded noncovalently. H Chain and at least one V L Including chains (hereafter, F) V (For example). If desired, these chains may be covalently coupled either directly via a disulfide bond between two variable domains or via a linker (e.g., a peptide linker), resulting in a single chain Fv(scF). V ) can form.

[0106] The variable region domains described above may be any naturally occurring variable domains or their manipulated versions. By "manipulated version," we mean a variable region domain created using recombinant DNA manipulation techniques. Such manipulated versions include, for example, those created from the variable region of a particular antibody by insertion, deletion, or modification of the amino acid sequence of that antibody. A specific example is a manipulated variable region domain comprising at least one CDR and optionally one or more framework amino acids derived from a first antibody, as well as the remainder of the variable region domain derived from a second antibody.

[0107] The variable region domain described above can be covalently attached to at least one other antibody domain or fragment at its C-terminal amino acid. Therefore, for example, the VH domain present in the variable region domain can be linked to an immunoglobulin CH1 domain or fragment. Similarly, V L The domain is C K It can be linked to a domain or a fragment thereof. In this method, for example, an antibody has an antigen-binding domain that is linked to the V H Domain and V L Domain (this V H Domain and V L The domains are the CH1 domain and the C domain at their C-terminus, respectively. K The Fab fragment may contain a domain covalently linked to the CH1 domain. This CH1 domain may be extended with further amino acids, for example, to provide a hinge region or part of a hinge region domain found in the Fab' fragment, or to provide further domains such as antibody CH2 and CH3 domains.

[0108] antigen-binding protein derivatives The nucleotide sequences shown in Figures 1A-1F, Figures 2A-2F, and Table 2 above can be modified, for example, by random mutagenesis or site-directed mutagenesis (e.g., oligonucleotide-induced site-specific mutagenesis), to produce modified polynucleotides that include the substitution, deletion, or insertion of one or more specific nucleotides compared to the unmutated polynucleotides. Examples of techniques for producing such modifications are described in Walder et al., 1986, Gene 42:133; Bauer et al., 1985, Gene 37:73; Craik, BioTechniques, January 1985, 12-19; ​​Smith et al., 1981, Genetic Engineering: Principles and Methods, Plenum Press; and U.S. Patents 4,518,584 and 4,737,462. These and other methods may be used, for example, to produce derivatives of TSLP antigen-binding proteins that have desired properties (e.g., increased affinity, avidity or specificity to TSLP, increased activity or stability in vivo or in vitro, or reduced in vivo side effects compared to underivativeized antigen-binding proteins).

[0109] Other derivatives of anti-TSLP antigen-binding proteins containing antibodies within the scope of the present invention include covalent or aggregated conjugates of an anti-TSLP antibody or fragment thereof with other proteins or polypeptides, such as those resulting from the expression of a recombinant fusion protein containing a heterologous polypeptide fused to the N-terminus or C-terminus of an anti-TSLP antibody polypeptide. For example, the conjugated peptide may be a heterologous signal (or leader) polypeptide (e.g., a yeast α-factor leader) or an epitope tag. The antigen-binding protein-containing fusion protein may contain a peptide (e.g., polyHis) added to facilitate the purification or identification of the antigen-binding protein. The antigen-binding protein may also be linked to a FLAG peptide, as described in Hopp et al., Bio / Technology 6:1204, 1988 and U.S. Patent No. 5,011,912. The FLAG peptide is highly antigenic and provides an epitope that is reversibly bound by a specific monoclonal antibody (mAb), enabling rapid assay and easy purification of the expressed recombinant protein. Reagents useful for preparing fusion proteins in which FLAG peptides are fused to a given polypeptide are commercially available (Sigma, St. Louis, MO).

[0110] Oligomers containing one or more antigen-binding proteins can be used as TSLP antagonists. Oligomers may be in the form of covalently or non-covalently linked dimers, trimers, or higher-order oligomers. Oligomers containing two or more antigen-binding proteins are intended for use, one example being homodimers. Other oligomers include heterodimers, homotrimers, heterotrimers, homotetramers, and heterotetramers.

[0111] One embodiment relates to an oligomer comprising multiple antigen-binding proteins linked via covalent or non-covalent interactions between peptide moieties fused to antigen-binding proteins. Such peptides may be peptide linkers (spacers) or peptides having properties that promote oligomerization. Certain polypeptides derived from leucine zippers and antibodies are among the peptides that can promote the oligomerization of antigen-binding proteins attached to peptides, as described in more detail below.

[0112] In certain embodiments, the above oligomer comprises 2 to 4 antigen-binding proteins capable of binding to TSLP. The antigen-binding proteins of the oligomer may be in any form, such as any of the above forms (e.g., modified or fragmented).

[0113] In one embodiment, the oligomer is prepared using a polypeptide derived from immunoglobulin. The preparation of fusion proteins containing specific heterologous polypeptides fused to various parts of an antibody-derived polypeptide (including the Fc domain) is described, for example, by Ashkenazi et al., 1991, PNAS USA 88:10535; Byrn et al., 1990, Nature 344:677; and Hollenbaugh et al., 1992 “Construction of Immunoglobulin Fusion Proteins”, Current Protocols in Immunology, Appendix 4, pp. 10.19.1-10.19.11.

[0114] One embodiment of the present invention relates to a dimer containing two fusion proteins, which is produced by fusing a fragment of an anti-TSLP antibody to the Fc region of the antibody. This dimer can be produced, for example, by inserting a gene fusion product encoding the fusion protein into a suitable expression vector, expressing this gene fusion product in host cells transformed with a recombinant expression vector, and assembling the expressed fusion protein to closely resemble an antibody molecule, in which case an interchain disulfide bond is formed between the Fc portions to obtain a dimer.

[0115] As used herein, the term “Fc polypeptide” includes the native and mutaine forms of polypeptides derived from the Fc region of an antibody. It also includes truncated forms of such polypeptides that include a hinge region that facilitates dimerization. Fusion proteins (and oligomers formed therefrom) containing the Fc moiety offer the advantage of easier purification by affinity chromatography, superior to that of protein A or protein G columns.

[0116] One suitable Fc polypeptide, described in PCT Patent Application WO93 / 10151 (incorporated herein by reference), is a single-chain polypeptide extending from the N-terminal hinge region of the Fc region of a human IgG1 antibody to the native C-terminus. Another useful Fc polypeptide is Fc mutein, described in U.S. Patent No. 5,457,035 and Baum et al., 1994, EMBO J.13:3992-4001. The amino acid sequence of this mutein is identical to that of the native Fc sequence presented in WO93 / 10151, except that amino acid 19 is changed from Leu to Ala, amino acid 20 is changed from Leu to Glu, and amino acid 22 is changed from Gly to Ala. This mutein exhibits reduced affinity for the Fc receptor.

[0117] In other embodiments, the variable portions of the heavy and / or light chains of the anti-TSLP antibody may be replaced by the variable portions of the antibody heavy and / or light chains.

[0118] Alternatively, the above oligomer is a fusion protein containing multiple antigen-binding proteins, with or without a peptide linker (spacer peptide). Suitable peptide linkers include those described in U.S. Patents 4,751,180 and 4,935,233.

[0119] Another method for preparing oligomeric antigen-binding proteins involves the use of leucine zippers. Leucine zipper domains are peptides that promote the oligomerization of the protein in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., 1988, Science 240:1759) and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and their derivatives that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble oligomeric proteins are described in PCT patent application WO94 / 10308, and a leucine zipper derived from lung surfactant protein D (SPD) is described in Hoppe et al., 1994, FEBS Letters. This is described in 344:191 (which is incorporated herein by reference). The use of a modified leucine zipper that enables stable trimerization of heterologous proteins fused to it is described in Fanslow et al., 1994, Semin.Immunol.6:267-78. In one approach, a recombinant fusion protein containing an anti-TSLP antibody fragment or derivative fused to a leucine zipper peptide is expressed in a suitable host cell, and the anti-TSLP antibody fragment or derivative of the soluble oligomer formed is recovered from the culture supernatant.

[0120] As described herein, the antibody comprises at least one CDR. For example, one or more CDRs may be incorporated into a known antibody framework region (e.g., IgG1, IgG2) or conjugated to a suitable vehicle that increases its half-life. Suitable vehicles include, but are not limited to, Fc, polyethylene glycol (PEG), albumin, and transferrin. These and other suitable vehicles are known in the art. Such conjugated CDR peptides may be in monomeric, dimeric, tetrameric, or other forms. In one embodiment, one or more water-soluble polymers are conjugated to one or more specific positions (e.g., the amino terminus) of the conjugating factor.

[0121] In certain preferred embodiments, the antibody comprises one or more water-soluble polymer attachments, including but not limited to polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol. See, for example, U.S. Patents 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, and 4,179,337. In certain embodiments, the derivative binding factors include one or more monomethoxy-polyethylene glycol, dextran, cellulose, or other carbohydrate-based polymers, poly(N-vinylpyrrolidone)-polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyol (e.g., glycerol), and polyvinyl alcohol, as well as mixtures of such polymers. In certain embodiments, one or more water-soluble polymers are randomly attached to one or more side chains. In certain embodiments, PEG may act to improve the therapeutic ability of a binding factor (e.g., an antibody). Specific such methods are discussed, for example, in U.S. Patent No. 6,133,426 (which is incorporated herein by reference for any purpose).

[0122] It is recognized that the antibodies of the present invention may include substitution, deletion, or addition of at least one amino acid, provided that the antibody retains its binding specificity. Therefore, modifications to the antibody structure are included within the scope of the present invention. These may include amino acid substitutions, which may be conserved or non-conservative, that do not disrupt the antibody's human TSLP binding ability. Conservative amino acid substitutions may include amino acid residues that do not exist naturally and are typically incorporated by chemical peptide synthesis rather than by synthesis in a biological system. These include peptidomimetic and other reversed or inverted forms of the amino acid moiety. Conservative amino acid substitutions may also include substitutions of native amino acid residues at standard residues, resulting in little to no effect on the polarity or charge of the amino acid residue at that position.

[0123] Non-conservative substitutions may involve replacing a member of one class of amino acids or amino acid mime with a member from another class having different physical properties (e.g., size, polarity, hydrophobicity, charge). Such substituted residues may be introduced into regions of a human antibody homologous to a non-human antibody, or into non-homologous regions of that molecule.

[0124] Furthermore, those skilled in the art can generate test variants containing a single amino acid substitution at each desired amino acid residue. These variants can then be screened using activity assays known to those skilled in the art. Such variants can be used to gather information about suitable variants. For example, if it is found that a change to a particular amino acid residue disrupts, undesirably reduces, or results in inadequate activity, variants with such changes can be avoided. In other words, based on the information gathered from such conventional experiments, those skilled in the art can easily determine which amino acids should be avoided, either alone or in combination with other mutations.

[0125] Those skilled in the art can determine suitable variants of the polypeptides described herein using well-known techniques. In certain embodiments, those skilled in the art can identify suitable regions of the molecule that can be modified without disrupting activity by targeting regions that are not considered important to activity. In certain embodiments, residues and portions of molecules that are conserved among similar polypeptides can be identified. In certain embodiments, even regions that may be important to biological activity or structure can be subjected to conserved amino acid substitution without disrupting biological activity and without adversely affecting the polypeptide structure.

[0126] Furthermore, those skilled in the art may re-examine structure-function studies that have identified residues in similar polypeptides that are important for activity or structure. In light of such comparisons, the importance of amino acid residues in a protein can be predicted, corresponding to amino acid residues that are important for activity or structure in similar proteins. Those skilled in the art may then select amino acid substitutions that are chemically similar to these predicted important amino acid residues.

[0127] Those skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to the structure of a similar polypeptide. In light of such information, those skilled in the art can predict the alignment of amino acid residues of an antibody with respect to its three-dimensional structure. In certain embodiments, those skilled in the art may choose not to make dramatic changes to the amino acid residues predicted to be on the protein surface, because such residues may be involved in important interactions with other molecules.

[0128] Many scientific publications have focused on predicting secondary structures. See Moult J., Curr. Op. in Biotech., 7(4):422-427 (1996); Chou et al., Biochemistry, 13(2):222-245 (1974); Chou et al., Biochemistry, 113(2):211-222 (1974); Chou et al., Adv. Enzymol. Relat. Areas Mol. Biol., 47:45-148 (1978); Chou et al., Ann. Rev. Biochem., 47:251-276 and Chou et al., Biophys. J., 26:367-384 (1979). Furthermore, computer programs are now available to assist in predicting secondary structures. One method of predicting secondary structures is based on homology modeling. For example, two polypeptides or proteins with more than 30% sequence identity or more than 40% similarity often have similar structural topologies. The recent growth of protein databases (PDBs) has provided increased predictability of secondary structures, including the number of possible folds within a polypeptide or protein structure. See Holm et al., Nucl. Acid. Res., 27(1):244-247 (1999). It has been suggested that a limited number of folds exist within a given polypeptide or protein, and that once a critical number of structures are elucidated, structural prediction becomes dramatically faster (Brenner et al., Curr. Op. Struct. Biol., 7(3):369-376 (1997)).

[0129] Further methods for predicting secondary structure include "threading" (Jones, D., Curr. Opin. Struct. Biol., 7(3):377-87 (1997); Sippl et al., Structure, 4(1):15-19 (1996)), "profile analysis" (Bowie et al., Science, 253:164-170 (1991); Gribskov et al., Meth. Enzym., 183:146-159 (1990); Gribskov et al., Proc. Nat. Acad. Sci., 84(13):4355-4358 (1987)), and "evolutionary linkage" (Holm, see above (1999) and Brenner, see above (1997)).

[0130] Those skilled in the art will understand that some proteins (e.g., antibodies) can undergo various post-translational modifications. The type and extent of these modifications often depend on the host cell line and culture conditions used to express the protein. Such modifications include changes in glycosylation, oxidation of methionine, diketopiperidine formation, aspartate isomerization, and asparagine adiposition. A frequently occurring modification is the loss of basic residues at the carboxyl terminus (e.g., lysine or arginine) due to the action of carboxypeptidases (as described in Harris, RJ, Journal of Chromatography 705:129-134, 1995).

[0131] In certain embodiments, antibody variants include glycosylation variants in which the number and / or type of glycosylation sites are altered compared to the amino acid sequence of the parent polypeptide. In certain embodiments, the variant contains more or fewer N-linked glycosylation sites than the native protein. Alternatively, substitutions that eliminate this sequence remove existing N-linked carbohydrate chains. Rearrangements of N-linked carbohydrate chains are also provided in which one or more N-linked glycosylation sites (typically naturally occurring) are eliminated and one or more novel N-linked sites are created. Further preferred antibody variants include cysteine ​​variants in which one or more cysteine ​​residues are deleted or substituted with another amino acid (e.g., serine) compared to their parent amino acid sequence. Cysteine ​​variants may be useful, for example, when the antibody must be refolded into a biologically active conformation after isolation of an insoluble inclusion body. Generally, cysteine ​​variants have fewer cysteine ​​residues than natural proteins, and typically have an even number of cysteine ​​residues to minimize interactions arising from unpaired cysteine.

[0132] The desired amino acid substitutions (whether conserved or non-conservative) can be determined by those skilled in the art when such substitutions are desired. In certain embodiments, amino acid substitutions may be used to identify key residues of antibodies against human TSLP or to increase or decrease the affinity of antibodies against human TSLP as described herein.

[0133] In particular embodiments, preferred amino acid substitutions are those that (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for protein complex formation, (4) alter binding affinity, and / or (4) confer or modify other physicochemical or functional properties to such polypeptides. In particular embodiments, one or more amino acid substitutions (conservative amino acid substitutions in particular embodiments) may be made in naturally occurring sequences (in particular embodiments, parts of the polypeptide outside the domains that form intermolecular contacts). In particular embodiments, conservative amino acid substitutions typically do not substantially alter the structural features of the parent sequence (for example, the substituted amino acid should not tend to disrupt helices present in the parent sequence, nor should it tend to disrupt other types of secondary structures that characterize the parent sequence). Examples of secondary and tertiary structures of polypeptides recognized in the art include Proteins, This is described in Structures and Molecular Principles (Creighton, ed., WH Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, NY (1991)); and Thornton et al., Nature 354:105 (1991) (these are incorporated herein by reference, respectively).

[0134] In certain embodiments, the antibodies of the present invention may be chemically bound to polymers, lipids, or other parts.

[0135] Furthermore, the antigen-binding protein described above may include at least one of the CDRs described herein, incorporated into a biocompatible framework structure. In one example, this biocompatible framework structure may include a polypeptide or a portion thereof sufficient to form a conformally stable structural support, framework, or scaffold, which may present a sequence of one or more amino acids (e.g., a CDR, variable region, etc.) that binds to an antigen in a localized surface region. Such structures may be naturally occurring polypeptides or polypeptide "folds" (structural motifs), or may have one or more modifications, such as the addition, deletion, or substitution of amino acids, compared to naturally occurring polypeptides or folds. These scaffolds may be derived from polypeptides of any species (or more than one species), such as humans, other mammals, other vertebrates, invertebrates, plants, bacteria, or viruses.

[0136] Typically, biocompatible framework structures are based on protein scaffolds or backbones other than immunoglobulin domains. For example, those based on fibronectin, ankyrin, lipocalin, neocarzinostatin, cytochrome b, CP1 zinc finger, PST1, coiled coil, LACI-D1, Z domain, and tendamistat domain may be used (see, e.g., Nygren and Uhlen, 1997, Current Opinion in Structural Biology, 7, 463-469).

[0137] Furthermore, in another embodiment, those skilled in the art will recognize that the antigen-binding protein may comprise one or more heavy chain CDR1, CDR2, CDR3 and / or light chain CDR1, CDR2, and CDR3 having one or more amino acid substitutions, provided that the antibody retains binding specificity for unsubstituted CDRs. The non-CDR portion of the antibody may be a non-protein molecule, and this binding factor cross-blocks the binding of the antibodies disclosed herein to human TSLP and / or inhibits TSLP activity. The non-CDR portion of the antibody may be a non-protein molecule, where the antibody exhibits a binding pattern to human TSLP protein similar to that shown by at least one of antibodies A1-A27 in a competitive binding assay and / or neutralizes the activity of TSLP. The non-CDR portion of this antibody may consist of amino acids, where this antibody is a recombinant binding protein or synthetic peptide, and this recombinant binding protein cross-blocks the binding of the antibodies disclosed herein to human TSLP and / or neutralizes TSLP in vivo or in vitro. The non-CDR portion of this antibody may consist of amino acids, where this antibody is a recombinant antibody, and this recombinant antibody exhibits a binding pattern to human TSLP polypeptide similar to that shown by at least one of antibodies A1-A27 in a competitive binding assay and / or neutralizes the activity of TSLP.

[0138] Methods for creating antigen-binding proteins (especially antibodies).

[0139] As described above, antigen-binding proteins, such as antibodies containing one or more heavy chain CDR1, CDR2, and CDR3, and / or light chain CDR1, CDR2, and CDR3, can be obtained by expression from host cells containing DNA encoding these sequences. The DNA encoding each CDR sequence can be determined based on the amino acid sequence of the CDR and can be synthesized together with any desired antibody variable region framework and constant region DNA sequence using appropriate oligonucleotide synthesis techniques, site-directed mutagenesis, and polymerase chain reaction (PCR) techniques. DNA encoding variable region frameworks and constant regions are widely available to those skilled in the art from gene sequence databases such as GenBank®.

[0140] Additional embodiments include chimeric antibodies (e.g., humanized versions of non-human (e.g., mouse) monoclonal antibodies). Such humanized antibodies can be prepared by known techniques and, when administered to humans, offer the advantage of reduced immunogenicity. In one embodiment, the humanized monoclonal antibody comprises the variable domain (or all or part of its antigen-binding site) of a mouse antibody and a constant domain derived from a human antibody. Alternatively, the humanized antibody fragment may comprise the antigen-binding site of a mouse monoclonal antibody and a variable domain fragment (lacking the antigen-binding site) derived from a human antibody. Procedures for the production of chimeric and further manipulated monoclonal antibodies are described in Riechmann et al., 1988, Nature 332:323, and Liu et al., 1987, Proc. Nat. Acad. Sci. The procedure includes those described in USA 84:3439, Larrick et al., 1989, Bio / Technology 7:934, and Winter et al., 1993, TIPS 14:139. In one embodiment, the chimeric antibody is an antibody grafted with a CDR. Techniques for humanizing antibodies are discussed, for example, in U.S. Patents 5,869,619, 5,225,539, 5,821,337, 5,859,205, and 6,881,557, Padlan et al., 1995, FASEB J. 9:133-39, and Tamura et al., 2000, J. Immunol. 164:1432-41. Additional techniques for producing such humanized antibodies are described in Zhang, W. et al., Molecular Immunology. 42(12):1445~1451, 2005; Hwang W. et al., Methods. 36(1):35~42, 2005; Dall'Acqua WF et al., Methods 36(1):43~60, 2005; and Clark, M., Immunology Today. This is described in 21(8):397~402, 2000).

[0141] Procedures have been developed for generating human antibodies or partially human antibodies in non-human animals. For example, mice were created in which one or more endogenous immunoglobulin genes were inactivated by various methods. Human immunoglobulin genes were introduced into the mice to replace the inactivated mouse genes. The antibodies produced in the animals incorporate human immunoglobulin polypeptide chains encoded by the human genetic material introduced into the animals. In one embodiment, a non-human animal (e.g., a transgenic mouse) is immunized with a TSLP protein, resulting in the production of antibodies directed against various TSLP polypeptides in the animal. Examples of suitable immunogens are provided in the following examples.

[0142] Examples of technologies for the production and use of transgenic animals for the production of human antibodies or partially human antibodies include U.S. Patents No. 5,814,318, No. 5,569,825, and No. 5,545,806, Davis et al., 2003, Production of human antibodies from transgenic mice in Lo, ed. Antibody Engineering: Methods and Protocols, Humana Press, NJ: 191-200, K ellermannら、2002,Curr Opin Biotechnol. 13:593~97.Russell.2000,Infect Immun. 68:1820~26、Cockら、2000,Eur J Immun. Rev. 30:534~40, Davis, 1999, Cancer Metastasis. 18:421~25、Green,1999,J Immunol Methods. 231:11~23、Jakobovits,1998,Advanced Drug Delivery Reviews 31:33~42、Greenら、1998,J Exp Med. 188:483~95;Jacobovits A,1998,Exp. Open. Invest. Drugs. 7:607~14, Tsuda, 1997, Genomics. 42:413~21.Mendez.1997,Night Genet. 15:146~56;Jacobovits,1994,Curr Biol. 4:761~63、Arbonesら、1994,Immunity. 1:247~60, Green, 1994, Nat Genet. 7:13~21, Jacobovits, 1993, Nature. 362:255~58;Jakobovitsら,1993,Proc Natl Acad Sci USA.90:2551~55; Huszar.“Immunoglobulin gene rearrangement in B-cell deficient mice generated by targeted deletion of the JH locus.” International Immunology 5 (1993): 647~656, Choi et al., 1993, Nature Genetics 4: 117~23, Fishwild et al., 1996, Nature Biotechnology 14: 845~51, Harding et al., 1995, Annals of the New York Academy of Sciences, Lonberg et al., 1994, Nature 368:856~59, Lonberg, 1994, Transgenic Approaches to Human Monoclonal Antibodies in Handbook of Experimental Pharmacology 113: 49~101, Lonberg et al., 1995, Internal Review of Immunology 13:65~93, Neuberger, 1996, Nature Biotechnology 14:826, Taylor et al., 1992, Nucleic Acids Research 20: This is described in 6287-95, Taylor et al., 1994, International Immunology 6: 579-91, Tomizuka et al., 1997, Nature Genetics 16: 133-43, Tomizuka et al., 2000, Proceedings of the National Academy of Sciences USA 97: 722-27, Tuaillon et al., 1993, Proceedings of the National Academy of Sciences USA 90: 3720-24, and Tuaillon et al., 1994, Journal of Immunology 152: 2912-20.

[0143] In another aspect, the present invention provides a monoclonal antibody that binds to human TSLP. The monoclonal antibody can be produced after the completion of an immunization schedule using any technique known in the art (e.g., by immortalizing spleen cells taken from a transgenic animal). Spleen cells can be immortalized using any technique known in the art (e.g., by fusing them with myeloma cells to generate hybridoma cells). Myeloma cells for use in the hybridoma generation fusion procedure preferably have an enzyme deficiency that prevents the myeloma cells from growing in a specific selective medium that does not produce antibodies, supports high fusion efficiency, and promotes the growth of only the desired fusion cells (hybridoms). An example of a suitable cell line for use in mouse fusion is Sp-20, P3- Cell lines include X63 / Ag8, P3-X63-Ag8.653, NS1 / 1.Ag 4 1, Sp210-Ag14, FO, NSO / U, MPC-11, MPC11-X45-GTG 1.7, and S194 / 5XX0 Bul; examples of cell lines used in rat fusion include R210.RCY3, Y3-Ag 1.2.3, IR983F, and 4B210. Other cell lines useful for cell fusion are U-266, GM1500-GRG2, LICR-LON-HMy2, and UC729-6.

[0144] In one embodiment, a hybridoma cell line is generated by immunizing an animal (e.g., a transgenic animal having a human immunoglobulin sequence) with a TSLP immunogen; collecting spleen cells from the immunized animal; fusing the collected spleen cells with a myeloma cell line to generate hybridoma cells; establishing a hybridoma cell line from the hybridoma cells; and identifying a hybridoma cell line that produces antibodies conjugating TSLP polypeptides. Such hybridoma cell lines and the TSLP monoclonal antibodies produced by them are encompassed by the present invention.

[0145] Monoclonal antibodies secreted by hybridoma cell lines can be purified using any technique known in the art. Hybridomas or mAbs can be further screened to identify mAbs with specific properties (e.g., inhibiting TSLP activity such as osteoprotegerin (OPG) production from primary human dendritic cells). Examples of such assays are provided in the following examples.

[0146] Molecular evolution of the complementarity-determining region (CDR) at the center of the antibody binding site has been used to isolate antibodies with increased affinity, as described, for example, by Schier et al., J. Mol. Biol. 263:551. Therefore, such techniques are useful in preparing antibodies against human TSLP.

[0147] Antigen-binding proteins that target human TSLP can be used, for example, in an assay to detect the presence of TSLP, either in vitro or in vivo.

[0148] Human antibodies, partially human antibodies, or humanized antibodies are suitable for many applications, particularly those involving the administration of such antibodies to human subjects, but other types of antigen-binding proteins are suitable for specific applications. The non-human antibodies of the present invention may be derived from any antibody-producing animal, such as mice, rats, rabbits, goats, donkeys, or non-human primates (monkeys (e.g., cynomolgus or rhesus monkeys) or apes (e.g., chimpanzees)). The non-human antibodies of the present invention may be used, for example, in in vitro and cell culture-based applications, or in any other application where an immune response to the antibodies of the present invention is absent, insignificant, preventable, unimportant, or desired. In one embodiment, the non-human antibodies of the present invention are administered to a non-human subject. In another embodiment, the non-human antibodies do not induce an immune response in a non-human subject. In another embodiment, the non-human antibodies are derived from the same species as the non-human subject (e.g., the mouse antibodies of the present invention are administered to mice). Antibodies from a particular species can be produced, for example, by immunizing an animal of a species possessing the desired immunogen, by using an artificial system for producing antibodies of that species (e.g., a bacterial or phage display-based system for producing antibodies of a specific species), by converting an antibody from one species to an antibody from another species (e.g., by replacing the constant region of the antibody with a constant region from another species), or by replacing one or more amino acid residues of the antibody to make it more closely resemble the sequence of an antibody from another species. In one embodiment, the antibody is a chimeric antibody containing amino acid sequences derived from antibodies from two or more different species.

[0149] Antigen-binding proteins can be prepared by any of a number of conventional techniques. For example, antigen-binding proteins can be purified from cells that naturally express antigen-binding proteins (for example, antibodies can be purified from hybridomas that produce antibodies), or they can be generated in recombinant expression systems using any technique known in the art. For example, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New York (1980); and Antibodies: A Laboratory Manual, Harlow and Land (eds.), Cold Spring Harbor Laboratory See Press, Cold Spring Harbor, NY, (1988).

[0150] Any expression system known in the art may be used to produce the recombinant peptides of the present invention. Generally, host cells are transformed with a recombinant expression vector containing DNA encoding the desired polypeptide. Among the host cells that may be used are prokaryotes, yeast, or higher eukaryotic cells. Prokaryotes include Gram-negative or Gram-positive organisms, e.g., E. coli or Bacillus. Higher eukaryotic cells include insect cells and established cell lines of mammalian origin. An example of a suitable mammalian host cell line is the COS-7 strain of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., 1981, Cell 23:175), L cells, 293 cells, C127 cells, 3T3 cells (ATCC CCL 163), this includes Chinese hamster ovary (CHO) cells, HeLa cells, BHK (ATCC CRL 10) cell lines, and CVI / EBNA cell lines obtained from the African green monkey kidney cell line CVI (ATCC CCL 70) as described by McMahan et al., 1991, EMBO J. 10: 2821. Suitable cloning and expression vectors for use in bacterial, fungal, yeast, and mammalian cell hosts are described by Pouwels et al., (Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985).

[0151] Transformed cells may be cultured under conditions that promote polypeptide expression, and the polypeptide may be recovered by conventional protein purification procedures. One such purification procedure is described in the following examples. The polypeptides intended for use herein include substantially homogeneous recombinant mammalian anti-TSLP antibody polypeptides that are substantially free of contaminating endogenous substances.

[0152] Antigen-binding proteins can be prepared and screened for desired properties by any of a number of known techniques. Some of these techniques involve isolating a nucleic acid encoding the polypeptide chain (or a portion thereof) of the antigen-binding protein of interest (e.g., a TSLP antibody) and manipulating that nucleic acid via recombinant DNA techniques. The nucleic acid can be fused with another nucleic acid of interest, or modified to add, delete, or substitute one or more amino acid residues (e.g., by mutagenesis or other conventional techniques).

[0153] Single-chain antibodies can be formed by linking heavy chain and light chain variable domain (Fv region) fragments via amino acid bridges (short peptide linkers) to produce a single polypeptide chain. Such single-chain Fvs (scFvs) consist of two variable domain polypeptides (V L and V H The polypeptides were prepared by fusing DNA encoding a peptide linker between DNA encoding the peptide linker. The resulting polypeptides can fold over themselves to form antigen-binding monomers, or, depending on the length of the flexible linker between the two variable domains, they can form multimers (e.g., dimers, trimers, or It can form tetramers (Kortt et al., 1997, Prot. Eng. 10:423; Kortt et al., 2001, Biomol. Eng. 18:95~108). Different V L and V HBy binding the contained polypeptide, multimer scFvs that bind to different epitopes can be formed (Kriangkum et al., 2001, Biomol. Eng. 18:31-40). The technology developed for the production of single-chain antibodies is U.S. Patent No. 4,946,778; Bird, 1988, Science 242:423; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879; Ward et al., 1989, Nature This includes techniques described in 334:544, de Graaf et al., 2002, Methods Mol Biol. 178:379~87. Single-chain antibodies obtained from antibodies provided herein include, but are not limited to, scFvs containing the variable domain combinations L1H1, L2H2, L3H3, L4H4, L5H5, L6H6, L7H7, L8H8, L9H9, L10H10, L11H11, L12H12, L13H13, L14H14, L15H15, L16H16, L17H17, L18H18, L19H19, L20H20, L21H21, L22H22, L23H23, L24H24, L25H25, L26H26, and L27H27 as encompassed by the present invention.

[0154] Once synthesized, the DNA encoding the antibody or fragment of the present invention can be amplified and expressed by any of the various well-known methods for nucleic acid excision, ligation, transformation, and transfection using any number of known expression vectors. Therefore, in certain embodiments, expression of the antibody fragment may be preferred in a prokaryotic host such as Escherichia coli (see, e.g., Pluckthun et al., 1989 Methods Enzymol. 178:497-515). In certain other embodiments, expression of the antibody or fragment may be preferred in eukaryotic host cells including yeast (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris), animal cells (including mammalian cells), or plant cells. Suitable examples of animal cells include, but are not limited to, myeloma (e.g., mouse NSO strain), COS, CHO, or hybridoma cells. Examples of plant cells include tobacco, maize, soybean, and rice cells. One or more replicable expression vectors containing DNA encoding the variable and / or constant regions of an antibody can be prepared and used to transform a suitable cell line, such as a non-producing myeloma cell line like the mouse NSO strain, or a bacterium like E. coli, into which antibody production will occur. To obtain effective transcription and translation, the DNA sequence in each vector includes a promoter and leader sequence operably ligated to appropriate regulatory sequences, particularly the variable domain sequence. Specific methods for generating antibodies in this manner are generally well-known and routinely used. For example, basic molecular biology procedures are described by Maniatis et al. (Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, New York, 1989; see also Maniatis et al., 3rd ed., Cold Spring Harbor Laboratory, New York, (2001)).DNA sequencing can be performed as described by Sanger et al. (PNAS 74:5463, (1977)) and the Amersham International plc sequencing handbook, and site-directed mutagenesis can be performed using methods known in the field (Kramer et al., Nucleic Acids Res. 12:9441, (1984); Kunkel Proc. Natl. Acad. Sci. USA). This can be carried out according to 82:488~92 (1985); Kunkel et al., Methods in Enzymol. 154:367~82 (1987); the Anglian Biotechnology Ltd. handbook). Furthermore, numerous publications describe appropriate techniques for preparing antibodies by manipulating DNA, creating expression vectors, and transforming and culturing appropriate cells (Mountain A and Adair, JR in Biotechnology and Genetic Engineering Reviews (ed. Tombs, MP, 10, Chapter 1, 1992, Intercept, Andover, UK); “Current Protocols in Molecular Biology”, 1999, FM Ausubel (ed.), Wiley Interscience, New York).

[0155] If it is desired to improve the affinity of the antibody according to the present invention containing one or more of the above-mentioned CDRs, CDR maintenance (Yang et al., J. Mol. Biol., 254, 392~403, 1995), chain shuffling (Marks et al., Bio / Technology, 10, 779~783, 1992), use of E. coli mutants (Low et al., J. Mol. Biol., 250, 350~368, 1996), DNA shuffling (Patten et al., Curr. Opin. Biotechnol., 8, 724~733, 1997), phage display (Thompson et al., J. Mol. Biol., 256, 7~88, 1996), and PCR (Crameri et al., Nature, 391, 288~291) may be performed. This can be obtained by many affinity maturation protocols, including (1998). All of these methods of affinity maturation are discussed by Vaughan et al. (Nature Biotechnology, 16, 535-539, 1998).

[0156] Other antibodies according to the present invention may be obtained by conventional immunization and cell fusion procedures described herein and known in the art. Monoclonal antibodies of the present invention may be produced using a variety of known techniques. Generally, monoclonal antibodies that bind to a specific antigen may be obtained by methods known to those skilled in the art (e.g., Kohler et al., Nature). 256:495, 1975; Coligan et al., (eds.), Current Protocols in Immunology, 1:2.5.12.6.7 (John Wiley & Sons 1991), U.S. Patent Nos. 32,011, 4,902,614, 4,543,439 and 4,411,993, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, Kennett, McKearn, and Bechtol (eds.) (1980), and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press (1988), Picksley et al., “Production of monoclonal antibody against proteins expressed in E. coli,” See *in DNA Cloning 2: Expression Systems*, 2nd Edition, Glover et al., (eds.), page 93 (Oxford University Press 1995). Antibody fragments can be obtained from them using any suitable standard technique (e.g., proteolytic digestion), or optionally by proteolytic digestion followed by mild reduction and alkylation of disulfide bonds (e.g., using papain or pepsin). Alternatively, such fragments can also be generated by recombinant gene manipulation techniques as described herein.

[0157] Monoclonal antibodies are obtained by methods known in the art and methods described herein, by immunogens containing the human TSLP of SEQ ID NO: 2, other TSLP polypeptide sequences as described herein, or fragments thereof, in animals (e.g., rats, hamsters, rabbits, etc.). Alternatively, it may be obtained by injection into mice (e.g., including transgenic or knockout mice) as known in the art. The presence of specific antibody production may be monitored by taking serum samples after the initial injection and / or booster injection and detecting the presence of antibodies or fragments thereof that bind to human TSLP using any one of the several immunodetection methods known in the art and described herein. From animals producing the desired antibody, lymphoid cells, the most common cells derived from the spleen or lymph nodes, are collected to obtain B lymphocytes. The B lymphocytes are then fused with a fusion partner of drug-sensitive myeloma cells, preferably homologous to an immunized animal and optionally possessing other desired characteristics (e.g., cells unable to express the endogenous Ig gene product, e.g., P3X63-Ag 8.653 (ATCC No. CRL 1580); NSO, SP20) to generate a hybridoma, an immortal eukaryotic cell line.

[0158] Lymphoid (e.g., spleen) cells and myeloma cells can be conjugated within minutes using a membrane fusion promoter (e.g., polyethylene glycol or a nonionic surfactant) and seeded at low density on a selective medium that supports the proliferation of hybridoma cells but not non-fused myeloma cells. A preferred selective medium is HAT (hypoxanthine, aminopterin, thymidine). After a sufficient time (usually about 1-2 weeks), colonies of cells are observed. Single colonies are isolated, and antibodies produced by these cells can be tested for binding activity against human TSLP using any of the various immunoassays known in the art and described herein. The hybridomas are cloned (e.g., by limiting dilution cloning or by soft agar plaque isolation), and clones that produce antibodies specific to human TSLP are selected and cultured. Monoclonal antibodies from the hybridoma cultures can be isolated from the supernatant of the hybridoma cultures.

[0159] An alternative method for generating mouse monoclonal antibodies is to inject hybridoma cells into the peritoneal cavity of mice of the same genotype (e.g., mice treated to promote the formation of ascites fluid containing monoclonal antibodies (e.g., pristane-sensitized mice)). Monoclonal antibodies can be isolated and purified by a variety of well-established techniques. Such isolation techniques include affinity chromatography, size exclusion chromatography, and ion exchange chromatography using protein A (e.g., Coligan pp. 2.7.1-2.7.12 and 2.9.1-2.9.3; see Baines et al., “Purification of Immunoglobulin G (IgG),” Methods in Molecular Biology, Vol. 10, pp. 79-104 (The Humana Press, Inc. 1992)). Monoclonal antibodies can be purified by affinity chromatography using appropriate ligands selected based on the specific properties of the antibody (e.g., heavy or light chain isotype, binding characteristics, etc.). Examples of suitable ligands immobilized on a solid support include protein A, protein G, anti-constant region (light chain or heavy chain) antibodies, anti-idiotype antibodies, and TSLP, or fragments or variants thereof.

[0160] The antibodies of the present invention are also complete human monoclonal antibodies. Complete human monoclonal antibodies can be produced by any number of techniques, such as those described above. Such methods further include, but are not limited to, Epstein-Barr virus (EBV) transformation of human peripheral blood cells (e.g., including B lymphocytes), in vitro immunization of human B cells, fusion of spleen cells from immunized transgenic mice carrying inserted human immunoglobulin genes, isolation from human immunoglobulin V region phage libraries, or other procedures known in the art and disclosed herein. For example, complete human monoclonal antibodies can be obtained from engineered transgenic mice that produce specific human antibodies in response to antigenic attack. A method for obtaining complete human antibodies from transgenic mice is, for example, Green et al., Nature Genet. 7:13, 1994; Lonberg et al., Nature 368:856, 1994; Taylor et al., Int. Immun. 6:579, 1994; U.S. Patent No. 5,877,397; Bruggemann et al., 1997; Curr. Opin. Biotechnol. 8:455~58; Jakobovits et al., 1995 This technique is described in Ann. NY Acad. Sci. 764:525~35. In this technique, elements of human heavy and light chain loci are introduced into mouse lines obtained from embryonic stem cell lines having targeted disruption of endogenous heavy and light chain loci (see also Bruggemann et al., Curr. Opin. Biotechnol. 8:455~58 (1997)). For example, the human immunoglobulin transgene may be a minigene construct that undergoes B cell-specific DNA rearrangement and hypermutation in mouse lymphoid tissue, or a transloci on a yeast artificial chromosome. Complete human monoclonal antibodies can be obtained by immunizing transgenic mice, which can then produce human antibodies specific to human TSLP. Lymphocytes from immunized transgenic mice can be used to generate human antibody-secreting hybridomas by the methods described herein. Polyclonal serum containing complete human antibodies can also be obtained from the blood of immunized animals.

[0161] One exemplary method for generating human antibodies of the present invention involves immortalizing human peripheral blood cells by EBV transformation, as described, for example, in U.S. Patent No. 4,464,456. Such immortalized B cell lines (or lymphoblastoid cell lines) that produce monoclonal antibodies specifically binding to human TSLP can be identified by immunodetection methods (e.g., ELISA) as provided herein and then isolated by standard cloning techniques. The stability of lymphoblastoid cell lines producing anti-TSLP antibodies can be improved by fusing the transformed cell lines with mouse myeloma according to methods known in the art (see, for example, Glasky et al., Hybridoma 8:377-89 (1989)) to generate mouse-human hybrid cell lines. Yet another method for generating human monoclonal antibodies is in vitro immunization, which involves first immunizing human spleen B cells with human TSLP and then fusing the first immunized B cells with a heterohybrid fusion partner. For example, see Boerner et al., 1991 J. Immunol. 147:86-95.

[0162] In certain embodiments, B cells producing anti-human TSLP antibodies are selected, and their light and heavy chain variable regions are cloned from these B cells by techniques known in the art (International Publication No. 92 / 02551; U.S. Patent No. 5,627,052; Babcook et al., Proc. Natl. Acad. Sci. USA 93:7843~48 (1996)) and molecular biology techniques described herein. B cells derived from immunized animals can be isolated from spleen, lymph node, or peripheral blood samples by selecting cells that produce antibodies that specifically bind to TSLP. B cells can also be isolated from humans (e.g., peripheral blood samples). Methods for detecting single B cells that produce antibodies with desired specificity (e.g., by plaque formation, fluorescence-activated cell sorting, detection of specific antibodies after in vitro stimulation, etc.) are well known in the art. A method for selecting B cells that produce a specific antibody includes, for example, preparing a single suspension of B cells in soft agar containing human TSLP. The binding of specific antibodies produced by B cells to antigens leads to the formation of complexes that can be observed as immunoprecipitates. After B cells producing the desired antibody are selected, the specific antibody gene can be cloned by isolating and amplifying the DNA or mRNA by methods known in the art and described herein.

[0163] A further method for obtaining the antibody of the present invention is by phage display. For example, Winter et al., 1994 Annu. Rev. Immunol. 12:433. See ~55; Burton et al., 1994 Adv. Immunol. 57:191~280. Combinatorial libraries of human or mouse immunoglobulin variable region genes can be created in phage vectors, which can be screened to select Ig fragments (Fab, Fv, sFv, or multimers thereof) that specifically bind to TSLP or its variants or fragments. For example, see U.S. Patent No. 5,223,409; Huse et al., 1989 Science 246:1275~81; Sastry et al., Proc. Natl. Acad. Sci. USA 86:5728~32 (1989); Alting-Mees et al., Strategies in Molecular Biology 3:1~9 (1990); Kang et al., 1991 Proc. Natl. Acad. Sci. USA 88:4363~66; Hoogenboom et al., 1992 J. Molec. Biol. 227:381~388; Schlebusch et al., 1997 Hybridoma 16:47~52 and the references cited therein. For example, a library containing numerous polynucleotide sequences encoding Ig variable region fragments can be inserted into the genome of a filamentous bacteriophage, such as M13 or a variant thereof, in frame with a sequence encoding a phage shell protein. The fusion protein may be a fusion of the shell protein with the light chain variable region domain and / or the heavy chain variable region domain. According to specific embodiments, immunoglobulin Fab fragments can also be presented on phage particles (see, for example, U.S. Patent No. 5,698,426).

[0164] Heavy chain or light chain immunoglobulin cDNA expression libraries are, for example, λlmmunoZap TM (H) and λImmunoZap TM(L) vector (Stratagene, La Jolla, California) can be used to prepare λ phages. Briefly, mRNA is isolated from a B cell population and used in λImmunoZap(H) and λImmunoZap(L) vectors to generate heavy and light chain immunoglobulin cDNA expression libraries. These vectors may be screened individually or co-expressed to form Fab fragments or antibodies (see Huse et al., above; also see Sastry et al., above). Positive plaques are then converted to non-lytic plasmids, enabling high levels of expression of monoclonal antibody fragments from E. coli.

[0165] In one embodiment, the variable region of the gene that expresses the desired monoclonal antibody in the hybridoma is amplified using nucleotide primers. These primers can be synthesized by those skilled in the art or purchased from commercially available sources (e.g., in addition to the above, V Ha , V Hb , V Hc , V Hd , C H1 , V L and C L See Stratagene (La Jolla, California), which sells primers for mouse and human variable regions, including primers for the region itself. These primers can be used to amplify heavy chain or light chain variable regions, and then they are used in ImmunoZAP, respectively. TM H or ImmunoZAP TM It can be inserted into vectors such as L(Stratagene). These vectors can then be introduced for expression in E. coli, yeast, or mammal-based systems. H and V L Large quantities of single-chain proteins, including domain fusions, can be generated using these methods (see Bird et al., Science 242:423-426, 1988).

[0166] Once cells producing antibodies according to the present invention are obtained using either the immunization or other techniques described above, specific antibody genes can be cloned by isolating and amplifying DNA or mRNA from the cells in accordance with standard methods as described herein. The antibodies produced from the cells are sequenced, and the identified CDRs and the DNA codes for those CDRs can be manipulated as previously described to produce other antibodies according to the present invention.

[0167] The antigen-binding proteins of the present invention preferably modulate TSLP activity in one of the cell-based assays and / or in vivo assays described herein, and / or cross-block the binding of one of the antibodies described herein, and / or are cross-blocked by one of the antibodies described herein. Particularly useful are antigen-binding proteins that cross-compete with one of the exemplary antibodies described herein (i.e., cross-block the binding of one of the exemplary antibodies described herein, and are cross-blocked by one of the exemplary antibodies to bind to TSLP). Thus, such binders can be identified using the assays described herein.

[0168] In certain embodiments, antibodies are generated by first identifying antibodies that bind to TSLP and / or neutralize in a cell-based assay described herein and / or cross-block antibodies described herein and / or whose binding to TSLP is cross-blocked by one of the antibodies described herein. The CDR regions derived from these antibodies are then inserted into a suitable biocompatible framework to generate an antigen-binding protein. The non-CDR portion of the conjugate may consist of amino acids or non-protein molecules. The assays described herein allow for the characterization of the conjugate. Preferably, the conjugate of the present invention is an antibody as defined herein.

[0169] The antigen-binding proteins of the present invention include proteins that bind to the same epitope, such as the exemplary antibodies described herein. As discussed in Example 9, the epitope may be structural or functional. A structural epitope can be thought of as a patch of target covered by the antibody. A functional epitope is a part of a structural epitope and contains residues that directly contribute to the affinity of interactions (e.g., hydrogen bonding, ionic interactions). One method for determining the epitope of an antibody is to scan for mutations in the target molecule and measure the effect of those mutations on binding. By giving a three-dimensional structure to the antibody-binding region, mutations in the epitope can decrease or increase the binding affinity of the antibody to the mutated target.

[0170] Antigen-binding proteins can be defined by their epitopes. As seen in Table 6, all antibodies can bind to TSLP, but they are affected differently by mutations in specific residues in TSLP, indicating that their respective epitopes do not completely overlap. Preferred antigen-binding proteins include proteins that share at least some of the structural epitopes of the reference antibodies described herein.

[0171] For example, a preferred antigen-binding protein is one that shares at least some of the same structural epitopes as A2. This is demonstrated by an increase in binding affinity compared to wild-type TSLP when TSLP has mutations in K67E, K97E, K98E, R100E, K101E, or K103E. This can also be demonstrated by a decrease in binding affinity compared to wild-type TSLP when TSLP has mutations in K21E, T25R, S28R, S64R, or K73E. While the antigen-binding protein and A2 are similarly affected by some mutations and not by others, the greater the identity between the antigen-binding protein and A2 in terms of the effect of mutations in specific residues of TSLP, the greater the structural epitope shared between the antigen-binding protein and the reference antibody.

[0172] Another preferred antigen-binding protein is one that shares at least part of the same structural epitope as A4. This includes TSLP, K97E, K98E, R100E, and K If TSLP has the 101E or K103E mutation, this is demonstrated by an increase in binding affinity compared to wild-type TSLP. This can also be demonstrated by a decrease in binding affinity compared to wild-type TSLP if TSLP has the K10E, A14R, K21E, D22R, K73E, K75E, or A76R mutation.

[0173] Another preferred antigen-binding protein is one that shares at least a portion of the same structural epitope as A5. This can be demonstrated by a decrease in binding affinity compared to wild-type TSLP when TSLP has mutations in K12E, D22R, S40R, R122E, N124E, R125E, or K129E.

[0174] Another preferred antigen-binding protein is one that shares at least a portion of the same structural epitope as A6. This can be demonstrated by a decrease in binding affinity compared to wild-type TSLP when TSLP has mutations in S40R, S42R, H46R, R122E, or K129E.

[0175] Another preferred antigen-binding protein is one that shares at least a portion of the same structural epitope as A7. This is demonstrated by the increased binding affinity of TSLP compared to wild-type TSLP when it has the K101E mutation. This can also be demonstrated by the decreased binding affinity of TSLP compared to wild-type TSLP when it has the D2R, T4R, D7R, S42R, H46R, T49R, E50R, Q112R, R122E, R125E, or K129E mutations.

[0176] Another preferred antigen-binding protein is one that shares at least a portion of the same structural epitope as A10. This is demonstrated by increased binding affinity compared to wild-type TSLP when TSLP has mutations in K97E, K98E, R100E, K101E, or K103E. This can also be demonstrated by decreased binding affinity compared to wild-type TSLP when TSLP has mutations in N5R, S17R, T18R, K21E, D22R, T25R, T33R, H46R, A63R, S64R, A66R, E68R, K73E, K75E, A76R, A92R, T93R, Q94R, or A95R.

[0177] Another preferred antigen-binding protein is one that shares at least a portion of the same structural epitope as A21. This is demonstrated by increased binding affinity compared to wild-type TSLP when TSLP has mutations in K97E, K98E, R100E, K101E, or K103E. This can also be demonstrated by decreased binding affinity compared to wild-type TSLP when TSLP has mutations in K21E, K21R, D22R, T25R, T33R, S64R, K73E, K75E, E111R, or S114R.

[0178] Another preferred antigen-binding protein is one that shares at least a portion of the same structural epitope as A23. This is demonstrated by increased binding affinity compared to wild-type TSLP when TSLP has mutations in K67E, K97E, K98E, R100E, K101E, or K103E. This can also be demonstrated by decreased binding affinity compared to wild-type TSLP when TSLP has mutations in E9R, K10E, K12E, A13R, S17R, S20R, K21E, K21R, K73E, K75E, N124E, or R125E.

[0179] Another preferred antigen-binding protein is one that shares at least a portion of the same structural epitope as A26. This is demonstrated by the increased binding affinity when TSLP has mutations in K97E, K98E, R100E, K101E, or K103E compared to wild-type TSLP. This is also demonstrated when TSLP has mutations in A14R, K21E, D22R, A63R, S64R, K67E, K73E, A76R, A92R, or A95R. If present, this can be demonstrated by a decrease in binding affinity compared to wild-type TSLP.

[0180] Comparing binding-affecting mutations between antibodies suggests that certain residues of TSLP tend to be part of an antibody's ability to bind TSLP and inhibit TSLP activity. Such residues include K21, D22, K73, and K129. Therefore, preferred antigen-binding proteins include proteins with a higher affinity for wild-type TSLP than those with a K21E mutation, proteins with a higher affinity for wild-type TSLP than those with a D21R mutation, proteins with a higher affinity for wild-type TSLP than those with a K73E mutation, and proteins with a higher affinity for wild-type TSLP than those with a K129E mutation.

[0181] Furthermore, many of the exemplary antigen-binding proteins described herein share the property that their affinity for TSLP increases when the basic patch of amino acids at positions 97-103 is replaced with an acidic amino acid.

[0182] nucleic acid In one aspect, the invention provides an isolated nucleic acid molecule. The nucleic acid encodes, for example, a polynucleotide encoding all or a portion of an antigen-binding protein (e.g., one or both chains of an antibody of the invention, or a fragment, derivative, mutein, or variant thereof), a polynucleotide sufficient for use as a hybridization probe, a PCR primer or sequencing primer for identifying, assaying, mutating, or amplifying a polynucleotide encoding a polypeptide, an antisense nucleic acid for inhibiting expression of a polynucleotide, and complementary sequences of the foregoing. The nucleic acid can be of any length. They can be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1,000, 1,500, 3,000, 5,000 or more nucleotides in length, and / or can include one or more additional sequences (e.g., regulatory sequences), and / or can be part of a larger nucleic acid (e.g., a vector). The nucleic acid can be single-stranded or double-stranded and can include RNA and / or DNA nucleotides, and variants thereof (e.g., peptide nucleic acids).

[0183] Nucleic acids encoding antibody polypeptides (e.g., heavy or light chains, variable domains only, or full length) can be isolated from B cells of mice immunized with a TSLP antigen. The nucleic acids can be isolated by conventional methods such as polymerase chain reaction (PCR).

[0184] Nucleic acid sequences encoding the variable regions of the heavy and light chain variable regions are shown above. Those skilled in the art will appreciate that due to the degeneracy of the genetic code, each polypeptide sequence disclosed herein is encoded by a large number of other nucleic acid sequences. The invention provides each degenerate nucleotide sequence encoding each antigen-binding protein of the invention.

[0185] The present invention further provides nucleic acids that hybridize to other nucleic acids (e.g., nucleic acids containing any of the nucleotide sequences A1-A27) under specific hybridization conditions. Methods for hybridizing nucleic acids are well known in the art. See, for example, Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989) 6.3.1-6.3.6. Moderately stringent hybridization conditions, as defined herein, are 5× sodium chloride / sodium citrate (SCC), 0.5% SDS, 1.0 mM Pre-washing solution containing EDTA (pH 8.0), approximately 50% formamide hybridize. The hybridization conditions used are a saturation buffer, 6×SSC, and a hybridization temperature of 55°C (or other similar hybridization solutions, e.g., a hybridization solution containing about 50% formamide at a hybridization temperature of 42°C), and a washing condition of 60°C in 0.5×SSC, 0.1% SDS. One stringent hybridization condition is hybridization in 6×SSC at 45°C, followed by one or more washes at 68°C in 0.1×SSC, 0.2% SDS. Furthermore, those skilled in the art can manipulate the hybridization and / or washing conditions to increase or decrease the stringency of the hybridization, resulting in nucleic acids containing nucleotide sequences that are at least 65, 70, 75, 80, 85, 90, 95, 98, or 99% identical to one another, generally remaining hybridized to one another. Basic parameters influencing the selection of hybridization conditions and guidance for devising appropriate conditions are established, for example, by Sambrook, Fritsch, and Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, chapters 9 and 11; and Current Protocols in Molecular Biology, 1995, Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4), and can be readily determined by those skilled in the art, for example, based on DNA length and / or base composition.

[0186] Changes can be introduced into the nucleic acid by mutation, resulting in a change in the amino acid sequence of the polypeptide (e.g., antigen-binding protein) it encodes. The mutation can be introduced using any technique known in the art. In one embodiment, one or more specific amino acid residues are changed, for example, using a site-directed mutagenesis protocol. In another embodiment, one or more randomly selected residues are changed, for example, using a random mutagenesis protocol. However made, the mutant polypeptide can be expressed and screened for desired properties.

[0187] The mutation can be introduced into the nucleic acid without significantly changing the biological activity of the polypeptide encoded by the nucleic acid. For example, nucleotide substitutions can be made that result in amino acid substitutions at non-essential amino acid residues. In one embodiment, the nucleotide sequence given herein for A1 - A27, or a desired fragment, variant, or derivative thereof, is mutated such that one or more deletions or substitutions of the amino acid residues shown herein for A1 - A27 result in two or more residues having different sequences. In another embodiment, mutagenesis inserts one amino acid residue adjacent to one or more of the amino acid residues shown herein for A1 - A27 such that two or more residues have different sequences. Alternatively, one or more mutations that selectively change the biological activity (e.g., binding to TSLP) of the polypeptide encoded by the nucleic acid can be introduced into the nucleic acid. For example, the mutation can change the biological activity quantitatively or qualitatively. Examples of quantitative changes include an increase, decrease, or elimination of the activity. Examples of qualitative changes include changing the antigen specificity of an antigen-binding protein.

[0188] In another aspect, the present invention provides nucleic acid molecules suitable for use as primers or hybridization probes for detecting the nucleic acid sequence of the present invention. The nucleic acid molecules of the present invention may include only a portion of the nucleic acid sequence encoding the full-length polypeptide of the present invention, for example, a fragment that can be used as a probe or primer, or a fragment that encodes the active portion of the polypeptide of the present invention (for example, a TSLP binding portion).

[0189] The nucleic acid sequence-based probe of the present invention is the nucleic acid or a similar nucleic acid (for example, the present invention). It can be used to detect transcripts encoding the polypeptide. The probe may include a labeling group (e.g., a radioisotope), a fluorescent compound, an enzyme, or an enzyme cofactor). Such a probe can be used to identify cells expressing the polypeptide.

[0190] In another aspect, the present invention provides vectors comprising nucleic acids encoding the polypeptide or a portion thereof. Examples of vectors include, but are not limited to, plasmids, viral vectors, non-episomal mammalian vectors, and expression vectors (e.g., recombinant expression vectors).

[0191] The recombinant expression vector of the present invention may contain the nucleic acid of the present invention in a form suitable for expression of the nucleic acid in a host cell. The recombinant expression vector includes one or more regulatory sequences selected based on the host cell used for expression, the regulatory sequences being operably ligated to the nucleic acid sequence to be expressed. The regulatory sequences include regulatory sequences that lead to constitutive expression of a nucleotide sequence in many types of host cells (e.g., the SV40 early gene enhancer, the Roussarcoma virus promoter, and the cytomegalovirus promoter), regulatory sequences that lead to expression of the nucleotide sequence only in specific host cells (e.g., tissue-specific regulatory sequences (see Voss et al., 1986, Trends Biochem. Sci. 11:287, Maniatis et al., 1987, Science 236:1237, the whole of which is incorporated herein by reference)), and regulatory sequences that lead to induced expression of a nucleotide sequence in response to a specific treatment or condition (e.g., the metallothionein promoter in mammalian cells and the tet response and / or streptomycin response promoter in both prokaryotes and eukaryotes (see ibid.)). Those skilled in the art will understand that the design of an expression vector may depend on factors such as the selection of host cells to be transformed and the level of expression of the desired protein. The expression vector of the present invention can be introduced into host cells to produce a protein or peptide, which may include a fusion protein or peptide encoded by a nucleic acid as described herein.

[0192] In another aspect, the present invention provides host cells into which the recombinant expression vector of the present invention has been introduced. The host cells may be any eukaryotic cell (e.g., E. coli) or a mammalian cell (e.g., yeast, insect, or CHO cell). The vector DNA may be introduced into prokaryotic or eukaryotic cells via common transformation or transfection techniques. With regard to stable transfection of mammalian cells, it is known that, depending on the expression vector and transfection technique used, only a small number of cells can incorporate the foreign DNA into their genome. To identify and select these integrants, genes encoding selection markers (e.g., for antibiotic resistance) are commonly introduced into host cells along with the gene of interest. Preferred selection markers include genes that confer resistance to drugs (e.g., G418, hygromycin, and methotrexate). Cells stably transfected with the introduced nucleic acid may be identified by drug selection, in addition to other methods (e.g., cells incorporating the selection marker gene survive, while other cells die).

[0193] Indications TSLP is involved in promoting a variety of inflammatory disorders, particularly allergic inflammatory disorders. As used herein, the term "allergic inflammation" refers to the expression of immunoglobulin E (IgE)-related immune responses (Manual of Allergy and Immunology, Chapter 2, Alvin M. Sanico, Bruce S. Bochner, and Sarbjit S. Saini, Adelman et al., ed., Lippincott, Williams, Wilkins, Philadelphia, PA, (2002)). Allergic inflammation involves the release of type 2 helper T cells (T) into the affected tissue. HIt is generally characterized by the invasion of 2 cells (Kay, see above). Allergic inflammation includes inflammatory skin conditions such as atopic dermatitis, as well as allergic sinusitis, inflammatory lung diseases such as asthma, and allergic conjunctivitis (Manual of Allergy and (Immunology, as described above). As used herein, the term “TSLP-associated allergic inflammation” refers to an allergic inflammatory condition in which TSLP is upregulated or otherwise demonstrated to be involved.

[0194] Allergic asthma is a chronic inflammatory disease of the airways characterized by eosinophilia of the airways, high levels of serum IgE, and mast cell activation involved in increased airway responsiveness, epithelial damage, and excessive mucus secretion (Wills-Karp, M, Ann. Rev.). Immunol. 17:255~281 (1999), Manual of Allergy and Immunology (see above). Studies have shown that varying degrees of chronic inflammation are present in the airways of all asthmatic individuals, even during asymptomatic periods. In susceptible individuals, this inflammation causes recurrent symptoms of wheezing, shortness of breath, chest tightness, and cough (Manual of Allergy and Immunology, see above).

[0195] Atopic dermatitis is a chronic pruritic skin disease characterized by skin disorders, including elevated serum total IgE, eosinophilia, and increased histamine release from basophils and mast cells. Individuals suffering from atopic dermatitis exhibit a pronounced T H 2. The response is observed, and the onset of atopic dermatitis disorder is accompanied by the release of high levels of IL-4, IL-5, and IL-13. HIt is thought to be mediated by the initial cutaneous infiltration of 2 lymphocytes (Leung, J. Allergy Clin Immunol 105:860~76 (2000)). The relationship between TSLP and other inflammatory cytokines is described in U.S. Patent Application No. 11 / 205,904, Publication No. 2006 / 0039910, which is incorporated herein by reference.

[0196] Human TSLP expression, as detected by in-situ hybridization, has been reported to increase in asthmatic airways in correlation with disease severity (Ying et al., J. Immunology 174:8183~8190 (2005)). Assays of TSLP mRNA levels in lung samples from asthma patients showed increased TSLP expression compared to controls. In addition, TSLP protein levels are detectable in concentrated bronchoalveolar lavage (BAL) fluid from asthma patients, lung transplant patients, and cystic fibrosis patients. It has recently been found that TSLP is released in responses to microorganisms and trauma, as well as inflammation, and activates mast cells (Allakhverdi et al., J Exp. Med 20492: 253~258 (2007)).

[0197] Human TSLP protein has been shown to be associated with disease in bronchial mucus and BAL fluid in patients with moderate / severe asthma and COPD (Ying et al., J Immunol 181(4):2790~8(2008)).

[0198] Overexpression of TSLP in the lungs of transgenic mice induces asthma-like airway inflammation (Zhou et al., Nat. Immunol 10:1047~1053 (2005)). In addition, it has been reported that TSLPR-deficient mice do not experience asthma progression in an OVA-asthma model, indicating that TSLP is required for asthma progression in airway inflammation models (Zhou et al., see above, Carpino et al., Mol. Cell Biol. 24:2584~2592 (2004)).

[0199] In addition to asthma, elevated levels of TSLP protein and mRNA were found in damaged skin and inflamed tonsil epithelial cells of patients with atopic dermatitis (AD). (Soumelis et al., Nature Immunol: 3 (7): 673~680 (2002)). Overexpression of TSLP in the skin of transgenic mice produces an AD-like phenotype (Yoo et al., J Exp Med 202: 541~549 (2005)).

[0200] Therefore, TSLP antagonists, particularly the TSLP antigen-binding proteins and antibodies of this application, are useful as therapeutic treatments for allergic inflammation, especially asthma and atopic dermatitis.

[0201] In addition, TSLP antagonists, particularly the TSLP antigen-binding proteins and antibodies of this disclosure, are also useful for treating fibrotic disorders. TSLP has been shown to be involved in promoting fibrotic disorders, as described in application number 11 / 344,379. TSLP has been found to induce fibroblast accumulation and collagen deposition in animals. For example, intradermal injection of mouse TSLP into mice resulted in fibrosis in the subcutaneous tissue of mice, characterized by fibroblast proliferation and collagen deposition. Antagonizing TSLP activity prevented or reduced fibroblast proliferation and collagen deposition in the tissue.

[0202] As used herein, the terms “fibrous proliferative disorder” or “fibrotic disorder or condition” refer to conditions related to fibrosis in one or more tissues. As used herein, the term “fibrosis” refers to the formation of fibrous tissue as a reparative or reactive process rather than as a normal component of an organ or tissue. Fibrosis is characterized by the accumulation of fibroblasts and collagen deposition beyond normal levels in any particular tissue. As used herein, the term “fibrosis” is used synonymously with “fibroblast accumulation and collagen deposition.” Fibroblasts are connective tissue cells distributed throughout the connective tissue of the body. Fibroblasts secrete a flexible extracellular matrix containing type I and / or type III collagen. In response to tissue injury, nearby fibroblasts migrate to the wound, proliferate, and produce large amounts of collagenous matrix. Collagen is a glycine and proline-rich fibrous protein and is a major component of the extracellular matrix and connective tissue, cartilage, and bone. Collagen molecules are triple-stranded helical structures called α-chains, which are wrapped around each other in a rope-like helix. Collagen exists in several forms or types, the most common of which, type I, is found in skin, tendons, and bones; and type III is found in skin, blood vessels, and internal organs.

[0203] Fibrotic disorders include, but are not limited to, systemic and focal scleroderma, keloids and hyperplastic scars, atherosclerosis, restenosis, pulmonary inflammation and fibrosis, idiopathic pulmonary fibrosis, cirrhosis, fibrosis as a result of chronic hepatitis B or C infection, kidney disease, heart disease resulting from scar tissue, and eye diseases such as macular degeneration and vitreal retinopathy. Further fibrotic disorders include fibrosis caused by chemotherapy agents, radiation-induced fibrosis, and injuries and burns.

[0204] Scleroderma is a fibrotic disorder characterized by skin thickening and hardening resulting from overproduction of new collagen by fibroblasts in the skin and other organs. Systemic scleroderma can affect multiple organs. Systemic sclerosis is characterized by hyalinization and thickened collagenous fibrous tissue, particularly in the hands and face, with skin thickening and adhesion to underlying tissues. The disease can also be characterized by dysphagia due to loss of peristalsis and submucosal fibrosis of the esophagus, difficulty breathing due to pulmonary fibrosis, myocardial fibrosis, and vascular changes in the kidneys (Stedman’s Medical Dictionary, 26 th Edition, Williams & Wilkins, 1995). Pulmonary fibrosis affects 30 - 70% of scleroderma patients and often results in restrictive lung disease (Atamas et al., Cyto kine and Growth Factor Rev 14: 537 - 550 (2003)). Idiopathic pulmonary fibrosis is a chronic, progressive and usually fatal lung disorder and is thought to be the result of a chronic inflammatory process (Kelly et al., Curr Pharma Design 9: 39 - 49 (2003)).

[0205] Therefore, TSLP antagonists, particularly the TSLP antigen - binding proteins and antibodies of the present application, are useful as therapeutic treatments for fibrotic diseases including, but not limited to, scleroderma, interstitial lung disease, idiopathic pulmonary fibrosis, fibrosis resulting from chronic hepatitis B or chronic hepatitis C, radiation - induced fibrosis, and fibrosis resulting from wound healing.

[0206] While the above indications are preferred, other diseases, disorders, or conditions may be treated or prevented by the administration of antigen conjugates to the subject. Such diseases, disorders, and conditions include inflammation, autoimmune diseases, inflammation of cartilage, fibrotic diseases and / or osteolysis, arthritis, rheumatoid arthritis, juvenile arthritis, juvenile rheumatoid arthritis, oligoarticular juvenile rheumatoid arthritis, polyarticular juvenile rheumatoid arthritis, systemic onset juvenile rheumatoid arthritis, juvenile ankylosing spondylitis, juvenile arthritis based on juvenile bowel disease, juvenile reactive arthritis, juvenile Reter syndrome, SEA syndrome (seronegative, enthesopathy, arthropathy syndrome), juvenile dermatomyositis, juvenile psoriatic arthritis, juvenile scleroderma, juvenile lupus erythematous erythematous, juvenile vasculitis, oligoarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic onset (onset) Rheumatoid arthritis, ankylosing spondylitis, arthritis due to intestinal disease, reactive arthritis, Reter syndrome, SEA syndrome (seronegative, enthesopathy, arthropathy syndrome), dermatomyositis, psoriatic arthritis, scleroderma, lupus erythematosus, vasculitis, myolitis, polymyositis, dermatomyositis, osteoarthritis, polyarteritis, Wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, scleroderma, sclerosis, primary biliary tract This includes, but is not limited to, sclerosing cholangitis, Sjögren's syndrome, psoriasis, psoriasis vulgaris, guttate psoriasis, inverse psoriasis, pustular psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis, atherosclerosis, lupus, Still's disease, systemic lupus erythematosus (SLE), myasthenia gravis, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, celiac disease, multiple sclerosis (MS), asthma, COPD, Guillain-Barré disease, type 1 diabetes, Graves' disease, Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, GVHD, etc. In certain embodiments, a pharmaceutical composition containing a therapeutically effective amount of TSLP antigen-binding protein is provided.

[0207] The term “treatment” includes, but is not limited to, the alleviation or prevention of at least one symptom or other aspect of a disorder, or a reduction in the severity of a disease. Antigen-binding proteins do not need to produce a complete cure or eradicate all symptoms or manifestations of a disease in order to constitute a promising therapeutic agent. As recognized in the relevant field, a drug used as a therapeutic agent may reduce the severity of a given disease state, but does not need to eliminate all symptoms of the disease in order to be considered a useful therapeutic agent. Similarly, a prophylactically administered treatment does not need to be completely effective in preventing the onset of a state in order to constitute a promising drug. It is sufficient to simply reduce the impact of the disease (e.g., by reducing the number or severity of its symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect), or by reducing the likelihood that the disease will develop or worsen in a subject. One embodiment of the present invention relates to a method comprising administering an antigen-binding protein to a patient in a quantity and for a duration sufficient to induce a sustained improvement above baseline in an index reflecting the severity of a particular disorder.

[0208] Pharmaceutical composition In some embodiments, the present invention provides a therapeutically effective amount of one or more antigen-binding proteins of the present invention to a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and The present invention provides a pharmaceutical composition comprising a and / or an adjuvant. In addition, the present invention provides a method of treating a patient by administering such a pharmaceutical composition. The term “patient” includes human and animal subjects.

[0209] A pharmaceutical composition comprising one or more antigen-binding proteins may be used to reduce TSLP activity. A pharmaceutical composition comprising one or more antigen-binding proteins may be used in the treatment of consequences, symptoms, and / or pathologies associated with TSLP activity. A pharmaceutical composition comprising one or more antigen-binding proteins may be used in a method to inhibit the binding of TSLP to TSLPR and / or signal transduction, comprising providing the antigen-binding proteins of the present invention to TSLP.

[0210] In certain embodiments, the acceptable formulation substances are preferably nontoxic to the recipient at the dose and concentration used. In certain embodiments, the pharmaceutical composition may include formulation substances for modifying, maintaining, or preserving, for example, pH, osmolality by weight, viscosity, clarity, color, isotonicity, aroma, sterility, stability, rate of degradation or release, or absorption or permeation of the composition.In such embodiments, suitable formulations include amino acids (e.g., glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (e.g., ascorbic acid, sodium sulfite, or sodium bisulfite); buffering agents (e.g., borates, bicarbonates, Tris-HCl, citrates, phosphates, or other organic acids); and bulking agents. Agents (e.g., mannitol or glycine); chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); complexing agents (e.g., caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (e.g., glucose, sucrose, mannose or dextrin); proteins (e.g., serum albumin, gelatin or immunoglobulin); colorants, flavorings and diluents; emulsifiers; hydrophilic polymers (e.g., polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (e.g., sodium); preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben) This includes, but is not limited to, chlorhexidine, sorbic acid, or hydrogen peroxide; solvents (e.g., glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (e.g., mannitol or sorbitol); suspending agents; surfactants or wetting agents (e.g., Pluronic acid, PEG, sorbitan esters, polysorbates (e.g., polysorbate 20, polysorbate, Triton, tromethamine, lecithin, cholesterol, tyroxapal); stability enhancers (e.g., sucrose or sorbitol); tonicity enhancers (e.g., alkali metal halides, preferably sodium chloride or potassium chloride, mannitol, sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants. See REMINGTON'S PHARMACEUTICAL SCIENCES, 18'' Edition, (AR Genrmo, ed.), 1990, Mack Publishing Company.

[0211] In certain embodiments, the optimal pharmaceutical composition is determined by those skilled in the art, depending, for example, on the intended route of administration, the method of delivery, and the desired dose. See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES above. In certain embodiments, such a composition may affect the physical state, stability, in vivo release rate, and in vivo removal rate of the antigen-binding protein of the present invention. In certain embodiments, the first vehicle or carrier in the pharmaceutical composition may be substantially aqueous or non-aqueous. For example, a suitable vehicle or carrier may be water for infusion, physiological saline solution, or artificial cerebrospinal fluid, which may be supplemented with other substances common in compositions for parenteral administration. Neutral buffered physiological saline or physiological saline mixed with serum albumin is a more exemplary vehicle. In certain embodiments, The pharmaceutical composition comprises Tris buffer at approximately pH 7.0–8.5, or acetate buffer at approximately pH 4.0–5.5, and may further comprise sorbitol or a suitable substitution thereof. In certain embodiments of the present invention, the TSLP antigen-binding protein composition may be prepared for storage in the form of a lyophilized mass or aqueous solution by mixing a selected composition having an optimal formulation agent (REMINGTON'S PHARMACEUTICAL SCIENCES, as described above) at a desired degree of purity. Furthermore, in certain embodiments, the TSLP antigen-binding protein product may be formulated as a lyophilized product using a suitable excipient such as sucrose.

[0212] The pharmaceutical compositions of the present invention may be selected for parenteral delivery. Alternatively, the compositions may be selected for inhalation or for delivery through the gastrointestinal tract (e.g., orally). The formulation components are preferably present at concentrations acceptable at the site of administration. In certain embodiments, buffers are used to maintain the composition within a pH range of physiological pH or slightly lower, generally from about 5 to about 8. These include about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, and about 8.0.

[0213] When parenteral administration is intended, the therapeutic composition for use in the present invention may be provided in the form of a parenterally acceptable aqueous solution containing the desired TSLP antigen-binding protein in a pharmaceutically acceptable vehicle that does not contain pyrogens. A particularly suitable vehicle for parenteral infusion is sterile distilled water, in which the TSLP antigen-binding protein is formulated to be sterile, isotonic, and properly preserved. In certain embodiments, the preparation may contain a formulation of the desired molecule together with a drug (e.g., injectable microspheres, bio-erodible particles, high molecular weight compounds (e.g., polylactic acid or polyglycolic acid), beads, or liposomes) that can provide controlled or sustained release of the product that can be delivered via accumulation injection. In certain embodiments, hyaluronic acid may also be used, having the effect of increasing the duration in circulation. In certain embodiments, an implantable drug delivery device may be used to introduce the desired antigen-binding protein.

[0214] The pharmaceutical compositions of the present invention may be formulated for inhalation. In these embodiments, the TSLP antigen-binding protein may be conveniently formulated as a dry, inhalable powder. In certain embodiments, the TSLP antigen-binding protein inhalation solution may also be formulated with a propellant for aerosol delivery. In certain embodiments, the solution may be atomized. Methods of administration to the lungs and thus formulations are further described in International Patent Application No. PCTUS94 / 001875, which is incorporated by reference and describes the delivery of chemically modified proteins to the lungs.

[0215] The formulation is also intended to be administered orally. TSLP antigen-binding proteins administered in this manner may be formulated with or without carriers conventionally used in formulations of solid dosage forms such as tablets and capsules. In certain embodiments, capsules are designed to release the active portion of the formulation in the gastrointestinal tract, while bioavailability is maximized and pre-systemic degradation is minimized. Additional agents may be included to enhance the absorption of TSLP antigen-binding proteins. Diluents, fragrances, low-melting-point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrants, and binders may also be used.

[0216] The pharmaceutical composition of the present invention is preferably provided to contain an effective amount of one or more TSLP antigen-binding proteins in a mixture with a non-toxic excipient suitable for the manufacture of tablets. The solution can be prepared in unit dose form by dissolving the tablet in sterile water or another suitable vehicle.

[0217] Suitable excipients include, but are not limited to, inert diluents (e.g., calcium carbonate, sodium carbonate or sodium bicarbonate, lactose, or calcium phosphate); binders (e.g., starch, gelatin, or acacia); or lubricants (e.g., magnesium stearate, stearic acid, or talc).

[0218] Additional pharmaceutical compositions are obvious to those skilled in the art and include formulations containing TSLP antigen-binding proteins in sustained or controlled delivery formulations. Methods for formulating a variety of other sustained or controlled delivery methods (e.g., liposome carriers, bio-feedable microparticles or porous beads and accumulation injections) are also known to those skilled in the art. See, for example, International Patent Application PCT / US93 / 00829, incorporated by reference, which describes the controlled release of porous polymer microparticles for the delivery of pharmaceutical compositions.

[0219] The sustained-release preparation may contain a semipermeable polymer material in the form of a molded product (e.g., a film or microcapsules). The sustained-release preparation may include polyesters, hydrogels, polylactides (disclosed in U.S. Patent No. 3,773,919 and European Patent Application Publication No. EP058481, respectively, as incorporated herein by reference), copolymers of L-glutamic acid and gammaethyl-L-glutamate (Sidman et al., 1983, Biopolymers 2:547-556), poly(2-hydroxyethyl-inethacrylate) (Langer et al., 1981, J. Biomed. Mater. Res. 15:167-277 and Langer, 1982, Chem. Tech. 12:98-105), ethylene vinyl acetate (Langer et al., 1981, as above), or poly-D(-)-3-hydroxybutyric acid (European Patent Application Publication No. EP 133,988).

[0220] The sustained-release composition may also include liposomes, which may be prepared by any of several methods known in the art. See, for example, Eppstein et al., 1985, Proc. Natl Acad. ScL USA 82.3688~3692; and, as incorporated by reference, European Patent Application Publications EP 036,676, EP 088,046 and EP 143,949.

[0221] Pharmaceutical compositions used for in vivo administration are generally provided as sterile preparations. Sterilization can be achieved by filtration through a sterile filter membrane. If the composition is lyophilized, sterilization using this method can be performed either before or after lyophilization and reconstitution. Compositions for parenteral administration can be stored in lyophilized form or in solution. Parenteral compositions are generally placed in containers with a sterile access port (e.g., bags of intravenous solutions or vials with stoppers that can be pierced by a subcutaneous needle).

[0222] An aspect of the present invention comprises a self-buffered TSLP antigen-binding protein formulation, which may be used as a pharmaceutical composition as described in the international patent application (International Publication No. 06138181A2 (PCT / US2006 / 022599)). International Publication No. 06138181A2 (PCT / US2006 / 022599) is incorporated herein by reference in its entirety. One embodiment provides a self-buffered TSLP antigen-binding protein formulation comprising a TSLP antigen-binding protein with a total salt concentration less than 150 mM.

[0223] The therapeutically effective dose used of a pharmaceutical composition containing TSLP antigen-binding protein is, for example, However, this depends on the treatment situation and purpose. Those skilled in the art will understand that the appropriate dose level for treatment varies in part depending on the molecule being delivered, the indication for which the TSLP antigen-binding protein is used, the route of administration, and the size (body weight, body surface or organ size) and / or the patient's condition (age and general health).

[0224] In certain embodiments, clinicians may determine the dose and alter the route of administration to obtain the optimal therapeutic effect. Typical doses can vary from approximately 0.1 μg / kg to approximately 30 mg / kg or more, depending on the factors mentioned above. In certain embodiments, the dose may vary from 0.1 μg / kg to approximately 30 mg / kg, optionally from 1 μg / kg to approximately 30 mg / kg, or from 10 μg / kg to approximately 5 mg / kg.

[0225] The frequency of administration depends on the pharmacokinetic parameters of the specific TSLP antigen-binding protein in the formulation used. Generally, clinicians administer the composition until a dose is reached that achieves the desired effect. The composition may therefore be administered as a single dose, or as two or more doses during a period (which may or may not contain the same amount of the desired molecule), or as continuous infusions via an implantable device or catheter. Further improvements to the appropriate dose are routinely made by those skilled in the art and are within the scope of practice.

[0226] The appropriate dose can be determined through the use of appropriate dose-response data. In certain embodiments, the antigen-binding protein of the present invention may be administered to a patient over an extended period. Long-term administration of the antigen-binding protein of the present invention minimizes adverse immune or allergic responses commonly associated with antigen-binding proteins that are not entirely human (e.g., antibodies produced against human antigens in non-human animals, e.g., incomplete human antibodies or non-human antibodies produced in non-human species).

[0227] The route of administration of the pharmaceutical composition follows known methods (e.g., by oral, intravenous, intraperitoneal, intracerebral (intraparum), intraventricular, intramuscular, intraocular, intraarterial, intraportal, or intrafocal routes; by continuous-release systems or implantable devices). In certain embodiments, the composition may be administered by large-volume instantaneous doses, by intravenous infusion, or by implantable devices.

[0228] The composition may also be administered topically via the embedding of a membrane, sponge, or other suitable material in which the desired molecule is absorbed or encapsulated. In certain embodiments in which an implantation device is used, the device may be embedded in a suitable tissue or organ, and the delivery of the desired molecule may be via diffusion, time-release bolus, or continuous administration.

[0229] Combination therapy In further embodiments, the antigen-binding protein is administered in combination with other agents useful for treating the condition the patient is suffering from. Examples of such agents include both protein-like and non-protein-like agents. When multiple treatments are administered concurrently, the doses may be adjusted on a case-by-case basis, as is recognized in the relevant art. "Concurrent administration" and combination therapy also include the administration of a treatment in which the antigen-binding protein is administered at least once during a course of treatment, which involves administering at least one other therapeutic agent to the patient, but is not limited to concurrent administration. [Examples]

[0230] The present invention is described, and the following examples are provided for illustrative purposes only and are not limiting.

[0231] Example 1: Preparation of antigen Several forms of recombinant TSLP were used as immunogens. Human TSLP was expressed in both E. coli and mammalian cells. The human TSLP produced by E. coli was a full-length protein without a tag. A TSLP protein with a furin cleavage site deletion, generated by deleting nucleotides 382-396 (AGAAAAAGGAAAGTC, SEQ ID NO: 370) corresponding to amino acids 128-132 (RKRKV, SEQ ID NO: 371), was generated in COS PKB cells. This protein contains a C-terminal poly-HIS-Flag tag (nucleotide sequence = ATGTTCCCTTTTGCCTTACTATATGTTCTGTCAGTTTCTTTCAGGAAAATCTTCATCTTACAACTTGTAGGGCTGGTGTTAACTTACGACTTCACTAACTGTGACTTTGAGAAGATTAAAGCAGCC TATCTCAGTACTATTTCTAAAGACCTGATTACATATATGAGTGGGACCAAAAGTACCGAGTTCAACAACACCGTCTCTTGTAGCAATCGGCCACATTGCCTTACTGAAATCCAGAGCCTAACCTTC AATCCCACCGCCGGCTGCGCGTCGCTCGCCAAAGAAATGTTCGCCATGAAAACTAAGGCTGCCTTAGCTATCTGGTGCCCAGGCTATTCGGAAACTCAGATAAATGCTACTCAGGCAATGAAGAAG AGGACAACCAATAAATGTCTGGAACAAGTGTCACAATTACAAGGATTGTGGCGTCGCTTCAATCGACCTTTACTGAAACAACAGCATCACCATCACCATCACGACTACAAAGACGATGACGACAAA (Sequence code 372); Protein sequence = MFPFALLYVLSVSFRKIFILQLVGLVLTYDFTNCDFEKIKAAYLSTISKDLITYMSGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEMFAMKTKAALAIWCPGYSETQINATQAMKKRTTNKCLEQVSQLQGLWRRFNRPLLKQQHHHHHHDYKDDDDK (Sequence ID 373).

[0232] In another campaign, full-length TSLP protein tagged with a C-terminal poly-HIS-Flag was generated in COS PKB cells (nucleotide sequence = ATGTTCCCTTTTGCCTTACTATATGTTCTGTCAGTTTCTTTCAGGAAAATCTTCATCTTACAACTTGTAGGGCTGGTGTTAACTTACGACTTCACTAACTGTGACTTTGAGAAGATTAAAGCAGCCTAT CTCAGTACTATTTCTAAAGACCTGATTACATATATGAGTGGGACCAAAAGTACCGAGTTCAACAACACCGTCTCTTGTAGCAATCGGCCACATTGCCTTACTGAAATCCAGAGCCTAACCTTCAATCCCA CCGCCGGCTGCGCGTCGCTCGCCAAAGAAATGTTCGCCATGAAAACTAAGGCTGCCTTAGCTATCTGGTGCCCAGGCTATTCGGAAACTCAGATAAATGCTACTCAGGCAATGAAGAAGAGGAGAAAAAGGAAAGTCACAACCAATAAATGTCTGGAACAAGTGTCACAATTACAAGGATTGTGGCGTCGCTTCAATCGACCTTTACTGAAACAACAGCATCACCATCACCATCACGACTACAAAGACGATGACGACAAA (Sequence ID 374); Protein sequence = MFPFALLYVLSVSFRKIFILQLVGLVLTYDFTNCDFEKIKAAYLSTISKDLITYMSGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEMFAMKTKAALAIWCPGYSETQINATQAMKKRRKRKVTTNKCLEQVSQLQGLWRRFNRPLLKQQHHHHHHDYKDDDDK (Sequence ID 375). Note that amino acid sequences 1-28 (MFPFALLYVLSVSFRKIFILQLVGLVLT, SEQ ID NO: 376) are signal peptides cleaved from the mature products of both of these proteins.

[0233] In addition, cynomolgus monkey TSLP was cloned and fused to the same C-terminal poly-HIS-Flag, nucleotides 358-372 (AGAAAAAGGAAAGTC) corresponding to the furin cleavage site (amino acids 120-124 (RKRKV, SEQ ID NO: 371) were used. , SEQ ID NO: 370) was deleted (DNA =ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACCGGTTACGACTTCACTAACTGTGACTTTCAGAAGATTGAAGCAGACTATCTCCGTACTATTTCTAAAGACCTGATTACATATATGAGTGGGACTAAAAGTACCGACTTCAACAACACCGTCTCCTGTAGCAATCGGCCACACTGCCTTACTGAAATCCAGAGCCTAACCTTCAATCCCACCCCC CGCTGCGCGTCGCTCGCCAAGGAAATGTTCGCCAGGAAAACTAAGGCTACCCTCGCTCTCTGGTGCCCAGGCTATTCGGAAACTCAGATAAATGCTACTCAGGCAATGAAGAAGAGGACAACCAATAAATGTCTGGAACAAGTGTCACAATTACTAGGATTGTGGCGTCGCTTCATTCGAACTTTACTGAAACAACAGCACCACCACCACCACCATGACTATAAAGACGATGACGACAAAT (Sequence ID 377); Protein = METDTLLLWVLLLWVPGSTGYDFTNCDFQKIEADYLRTISKDLITYMSGTKSTDFNNTVSCSNRPHCLTEIQSLTFNPTPRCASLAKEMFARKTKATLALWCPGYSETQINATQAMKKRTTNKCLEQVSQLLGLWRRFIRTLLKQQHHHHHHDYKDDDDK (Sequence No. 378)) or as full-length / natural product (nucleotide sequence = ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACCGGTTACGACTTCACTAACTGTGACTTTCAGAAGATTGAAGCAGACTATCTCCGTACTATTTCTAAAGACCTGATTACATATATGAGTGGGACTAAAAGTACCGACTTCAACAACACCGTCTCCTGTAGCAATCGGCCACACTGCCTTACTGAAATCCAGAGCCTAACCTTCAAT CCCACCCCCCGCTGCGCGTCGCTCGCCAAGGAAATGTTCGCCAGGAAAACTAAGGCTACCCTCGCTCTCTGGTGCCCAGGCTATTCGGAAACTCAGATAAATGCTACTCAGGCAATGAAGAAGAGGAGAAAA AGGAAAGTCACAACCAATAAATGTCTGGAACAAGTGTCACAATTACTAGGATTGTGGCGTCGCTTCATTCGAACTTTACTGAAACAACAGCACCACCACCACCACCATGACTATAAAAGACGATGACGACAAA (SEQ ID NO: 379); The protein was subcloned and expressed in COS PKB cells using one of the following proteins: METDTLLLWVLLLWVPGSTGYDFTNCDFQKIEADYLRTISKDLITYMSGTKSTDFNNTVSCSNRPHCLTEIQSLTFNPTPRCASLAKEMFARKTKATLALWCPGYSETQINATQAMKKRRKRKVTTNKCLEQVSQLLGLWRRFIRTLLKQQHHHHHHDYKDDDDK (SEQ ID NO: 380).Note that the amino acid sequence 1-20 (METDTLLLWVLLLWVPGSTG, SEQ ID NO: 381) is the signal peptide cleaved from the mature products of both of these cynomolgus monkey proteins.

[0234] Example 2 Mouse Anti-Human TSLP Antibody hTSLP-Fc was used for immunization of Balb / c mice (Jackson Laboratories, Bar Harbor, Maine). After several immunizations, lymphocytes were released from the spleen and fused with NS1 (ATCC) of mouse myeloma cells by chemical fusion using 50% PEG / DMSO (Sigma). The fused cells were seeded in a 96-well plate at a density of 2×10 cells / well in 200 μl of DMEM HAT (0.1 mM hypoxanthine, 0.16 mM thymidine, 4 mM aminopterin, Sigma) medium supplemented with 10% FBS, 5% Origen Cloning Factor (BioVe TM )、1× penicillin-streptomycin-glutamine, sodium pyruvate (Invitrogen). The medium was replaced with DMEM HT (0.1 mM hypoxanthine, 0.16 mM thymidine) medium supplemented with 10% FBS, 5% Origen Cloning Factor (BioVeris 4 )、1× penicillin-streptomycin-glutamine, sodium pyruvate (Invitrogen) 7 days after fusion. The conditioned medium was collected 2 days after the medium replacement and proceeded to the first screening. TM )、1× penicillin-streptomycin-glutamine, sodium pyruvate (Invitrogen).

[0235] Example 3: Generation of a Complete Human Antibody A complete human monoclonal antibody specific for TSLP can be obtained, for example, from U.S. 2005 / 0118643, U.S. Patent Nos. 6114598, 6162963, 7049426, 7064244, Green et al., Nature Genetics The compounds were produced using the XenoMouse® technology in accordance with the methods described in 7:13~21 (1994), Medez et al., Nature Genetics 15:146~156 (1997), Green and Jakobovitis J. Ex. Med. 188:483~495 (1998) (all of which are incorporated herein by reference), and as described below.

[0236] Two campaigns were conducted. In Campaign 1, XenoMouse® IgG2 and IgG4 cohorts were used. 50% of the mice received human TSLP produced by E. coli, and 50% received human TSLP produced by mammals (as described above). Serum titers were monitored by ELISA (as described below), and the mice with the best titers were fused to generate hybridomas using the following method.

[0237] Selected mice were killed, and the exuded lymph nodes were collected from each cohort. The lymph node cells were enriched with B cells, and these B cells were fused with myeloma cells to create hybridomas. The fused hybridoma strains were then seeded in hybridoma medium and cultured at 37°C for 10–14 days. The supernatant of these hybridomas was screened for IgG antibodies that bind to TSLP by ELISA as described below.

[0238] A second campaign was launched in which IgG2 XenoMouse® cells from two cohorts were immunized with mammalian-produced human TSLP, and one cohort was further immunized with cynomolgus monkey TSLP. After several immunizations, lymphocytes derived from lymph nodes were fused and cultured as described above. Following culturing, the supernatant of the hybridomas was screened for binding to TSLP by ELISA as described below.

[0239] Polyclonal supernatants from both campaigns were selected for further subcloning based on the assays described below. Hybridomas containing antibodies that are potentially inhibitors of TSLP activity were identified, and their cross-reactivity with cynomolgus monkey TSLP was further determined. The results are shown in Example 5 below. Supernatants from promising hybridomas were selected based on their ability in the primary DC assay described later. These hybridomas were cloned into single cells and spread for further testing. The antibodies were then purified as described below.

[0240] Antibodies were purified from hybridoma conditioned medium using Mab Select (GE Healthcare) resin. 100 μl of a 1:2 slurry of Mab Select resin equilibrated in PBS was added to conditioned medium (CM) between 7 and 10 ml. The test tube was placed in a rotator overnight at 4-8°C. The test tube was centrifuged at 1,000 × g for 5 minutes and then removed. The supernatant of the binding fraction was transferred. The resin was washed with 5 ml of PBS, and then centrifuged as described above, and the supernatant was transferred. The resin was then transferred to a 2 ml SPIN-X 0.45 μm test tube. The resin was washed twice more with 0.5 ml of PBS and then centrifuged. Mab was eluted by incubation at room temperature for 10 minutes with 0.2 ml of 0.1 M acetic acid, stirring occasionally. The test tube was centrifuged, and 30 μl of 1 M Tris buffer at pH 8.0 was added to the eluate. The purified Mab was stored at 4-8°C.

[0241] Example 4: Antibody assay A. ELISA to detect the presence of anti-TSLP antibodies ELISA was performed by coating a 96-well plate bound to Costar 3368 medium with recombinant wtHuTSLP or pHisFlag at 2 μg / ml in 1 × PBS / 0.05% azide at a rate of 50 μl / well and incubating overnight at 4°C. The plate was washed and blocked with 250 μl of 1 × PBS / 1% milk (assay diluent) and incubated at room temperature for at least 30 minutes.

[0242] Approximately 50 μl / well of hybridoma supernatant, positive control mouse antibody M385, or negative control was added, and the plates were incubated at room temperature for 2 hours. The plates were washed, and the secondary antibody, goat anti-human IgG Fc HPR (Pierce), or alternatively goat anti-mouse IgG HPR (Jackson Labs), was applied at 400 ng / ml in the assay diluent. The plates were incubated at room temperature for 1 hour, washed, and the OD was read at 450 nm.

[0243] Screening of anti-TSLP hybridoma supernatant was performed using one of the following functional assays.

[0244] 1. 96-well plates were coated with soluble huIL-7Ra-huTSLPR-Fc protein having an 8-amino acid linker (SGGAPMLS, SEQ ID NO: 382) between the receptor and human Fc, and incubated overnight at 4°C.

[0245] 2. The plate was washed and then blocked at room temperature for 1 hour using PBS + 1% BSA + 5% sucrose.

[0246] 3. The plates were incubated with biotinylated huTSLPHFdel (HF stands for polyHis Flag, where TSLP is a furin cleavage site deletion)(del). The plates were then incubated at room temperature for 2 hours with (+ / -) hybridoma supernatant or mouse anti-human TSLP (M385) as a positive control.

[0247] 4. SA-HRP detection (streptavidin-horseradish peroxidase). SA strongly binds to the biotinylated huTSLPHFdel, and HRP catalyzes the oxidation of the pigment TMB by hydrogen peroxide (turning it blue).

[0248] B. Cell-based assays 1) Inhibition of TSLP-induced proliferation of stable BAF cell lines expressing the human TSLPR-IL7R complex, using hybridoma supernatant or purified antibody, was determined by the following protocol.

[0249] 1. BAF in growth medium (RPMI 1640 + 10% FBS + 1% L-glutamine + 0.1% Pen / Strep + 0.1% 2-ME): Hu TSLPR stable cell lines were washed, and used TSLP was removed in maintenance medium (the same as the growth medium, but with the addition of 10 ng / mL of huTSLPHFwt).

[0250] 2. HuTSLPwtpHF (+ / -) or cynomolgus monkey TSLPwtpHF (+ / -) were incubated in wells with hybridoma supernatant / purified antibody / or mouse anti-human TSLP (M385) at room temperature for 30 minutes.

[0251] 3. 5 × 10 4 Cells were added to the wells and incubated for 3 days.

[0252] 4. The cells were pulsed overnight with tritiated thymidine (1 uCi / well). Cell proliferation or inhibition of the BAF cells was evaluated by the amount of tritiated thymidine uptake by the cells (CPM).

[0253] 2) Primary cell assay. Inhibition of TSLP-induced osteoprotegerin (OPG) production (described in U.S. Patent No. 6,284,728) from primary human dendritic cells by hybridoma or purified antibody was determined using the following protocol.

[0254] 1. Peripheral blood CD11c+ myeloid dendritic cells were enriched from normal in-house donor leukocyte packs using a CD1c (BDCA-1) dendritic cell isolation kit (Miltenyi Biotec).

[0255] 2. huTSLPwtpHF (+ / -) or cynomolgus monkey TSLPwtpHF was incubated with supernatant, purified antibody, or mouse anti-human TSLP at room temperature for 30 minutes.

[0256] 3.1 × 10⁵ cells / well were added and incubated for 48 hours. The supernatant was collected and assayed for human OPG production by ELISA, and inhibition of OPG production by hybridoma supernatant or purified antibody was determined. OPG ELISA was performed using the R&D systems DuoSet® development kit. Anti-TSLP antibody inhibited OPG production from cells in a dose-dependent manner.

[0257] 3) Cynomolgus monkey peripheral blood mononuclear cell assay. Inhibition of CynoTSLP-induced CCL22 / MDC production by hybridoma supernatant or purified antibody was determined using the following protocol.

[0258] 1. Peripheral blood unicellular cells (PBMCs) were obtained from peripheral blood acquired from cynomolgus monkeys (SNBL) by covering an isolymph with a 1:1 blood:PBS mixture.

[0259] 2. Cynomolgus monkey TSLPwtpHF (+ / -) supernatant / purified antibody or soluble huIL-7Ra-huTSLPR-Fc was incubated at room temperature for 30 minutes.

[0260] 3. 4×10 5Cells were added to the wells and incubated for 5 days. The supernatant was collected and assayed for CCL22 / MDC production in cynomolgus monkeys by ELISA.

[0261] Example 5: K D decision The surface plasmon resonance experiments described in this patent application were performed at 25°C using a Biacore 3000 instrument (Biacore International AB, Uppsala, Sweden) equipped with a CM4 sensor chip. Anti-Fcγ-specific capture antibodies were covalently immobilized on two flow cells on the CM4 chip using standard amine coupling chemistry with HBS-EP as the running buffer. Specifically, each flow cell was activated with a 1:1 (v / v) mixture of 0.1 M NHS and 0.4 M EDC. AffiniPure goat anti-human IgG, Fcγ fragment-specific antibody (Jackson ImmunoResearch Inc. West Grove, PA) was immobilized at a target level of 3,000 RU on two flow cells with 30 ug / ml in 10 mM sodium acetate, pH 5.0. The remaining reactive surface was deactivated by injection of 1 M ethanolamine. The running buffer was then switched to HBS-EP + 0.1 mg / ml BSA for all remaining steps.

[0262] The following antibodies were tested. A5 IgG2 is a purified monoclonal antibody, A2 IgG1 and IgG2 are recombinant purified antibodies, and A3 IgG4 and A4 IgG4 are the supernatants of the clones. The antibodies were appropriately diluted in the running buffer, such that a 2-minute injection at 10 μl / min on the test flow cell resulted in approximately 110 - 175 response units of the antibody captured on the surface of the test flow cell. No antibody was captured on the surface of the control flow cell. Subsequently, various concentrations of human, cynomolgus monkey, or murine TSLP were flowed over two flow cells together with a buffer blank. The concentration ranges of human and cynomolgus monkey TSLP were 0.44 - 100 nM, while the concentration range of murine TSLP was 8.2 - 6000 nM. A flow rate of 50 μl / min was used, and after a 2-minute binding step, a 10 - 30-minute dissociation step followed. After each cycle, the surface was regenerated with a 30-second injection of 10 mM glycine pH 1.5. Next, a new antibody was captured on the test flow cell and the next cycle was prepared.

[0263] The data were double referenced by subtracting the control surface response to remove changes in the bulk refractive index and by subtracting the average buffer blank response to remove systematic artifacts from the experimental flow cell. The TSLP data were processed and overall were fit to a 1:1 interaction model with local Rmax using BIA evaluation Software v 4.1. (Biacore International AB, Uppsala, Sweden). Association (k a ) and dissociation (k d ) rate constants were determined and used to calculate the dissociation equilibrium constant (K D ). The dissociation rate constants and dissociation equilibrium constants are summarized in the table found in Example 6.

[0264] Example 6: In Vitro Activity of Antibodies ⏎ The following antibodies were characterized for kd and KD using the Biacore assay described above. Primary dendritic cell assays were used to determine IC50 (pM). Data for A5 were generated using purified clonal antibodies, data for A2 were generated using recombinant purified antibodies, and data for A3 and A4 were generated using clonal supernatants. All TSLP variants were generated from mammalian cells.

[0265] [Table 4-1] Example 7: Recombinant expression and purification of antibodies Development of stable cell lines that express antibodies Duplicate oligonucleotides were synthesized to correspond to the primary sequences of the light and heavy chain variable domains of both the sense and antisense strands. This oligonucleotide pool was used in standard PCR. The product from this first reaction was used as a template for a second PCR amplification. The amplified variable heavy chain and variable light chain fragments were subcloned into intermediate vectors and sequenced to identify error-free products. The variable heavy chain fragment was cloned into a transient expression vector containing a signal peptide and the human IgG2 constant region. The variable light chain fragment was cloned into a transient expression vector containing a signal peptide and the human lambda constant region. The complete heavy chain gene was transferred into vector pDC324. The complete light chain gene was transferred into expression vector pDC323.

[0266] The CS-9 host cells used for transfection with anti-TSLP expression plasmids were CHO cell lines obtained from DXB-11 cells through adaptation to serum-free medium (Rasmussen et al., Cytotechnology 28:31~42, 1998). Anti-TSLP cell lines were prepared by transfecting CS-9 host cells with the expression plasmids pDC323-anti-TSLP-lambda and pDC324-anti-TSLP-IgG2 using standard electroporation or lipofection procedures. After transfection of the host cell line with the expression plasmids, the cells were grown in selective medium for 2-3 weeks to allow plasmid selection and cell recovery. In some cases, the medium was supplemented with 3% dialyzed fetal bovine serum (ds or dFBS). When serum was used, it was removed from the medium after the selection period. Cells were grown in selective medium until a viability of greater than 85% was achieved. This pool of transfected cells was then cultured in culture medium.

[0267] Cell line cloning A cell bank of selected clones was created using the following procedure. The cloning step ensures that a clonal population and cell bank are generated that will enable reproducible execution in commercial production. The amplified pool of antibody-expressing cells was seeded at limiting dilutions in 96-well plates, and candidate clones were evaluated for their growth and productivity capabilities in small-scale studies.

[0268] Example 8: Cross-competition of antibodies A common method for defining an epitope is through competitive testing. Antibodies that compete with each other may bind to the same site on the target. This example describes a method for determining competition for binding to TSLP and the results of this method when applied to many of the antibodies described herein.

[0269] Binning experiments can be performed in many ways, and the method used can have a certain effect on the assay results. What these methods have in common is that, typically, TSLP is bound by one reference antibody and probed by the other. If this reference antibody interferes with the binding of this probe antibody, then these antibodies are said to be in the same bin. The order in which these antibodies are used is important. When antibody A is used as a reference antibody and it blocks the binding of antibody B, the reverse is not always true: antibody B used as a reference antibody does not necessarily block antibody A. There are many factors at play here: antibody binding can cause conformational changes within the target, which can interfere with the binding of the second antibody, or overlapping epitopes that do not completely occlude each other may still have a high-affinity interaction with the target sufficient to allow binding. Antibodies with much higher affinity may have a higher ability to bump into blocking antibodies without interfering. Generally, if competition is observed in either order, the antibodies are said to go into the same bin, and if both antibodies can block each other, their epitopes may overlap more completely.

[0270] For this embodiment, a modification of the Multiplexed Binning method described by Jia et al. (J. Immunological Methods, 288(2004)91-98) was used. Because the presence of furin cleavage sites in TSLP can lead to heterogeneity in TSLP protein preparations (preps), TSLP in which arginine in the furin cleavage site was mutated to alanine was used. See U.S. Patent No. 7,288,633. Streptavidin-coated Luminex beads (Luminex, #L100-L1XX-01, where XX specifies the bead code) of each bead code were incubated in 100 µl of 6 pg / bead biotinylated monovalent mouse anti-human IgG capture antibody (BD Pharmingen, #555785) at room temperature in the dark for 1 hour, and then washed three times with PBSA (phosphate-buffered saline (PBS) + 1% bovine serum albumin (BSA)). Each bead code was incubated separately for 1 hour with 100 µl of 1:10 dilution of anti-TSLP antibody (Coating Antibody), and then washed. These beads were pooled and then distributed into a 96-well filter plate (Millipore, #MSBVN1250). 100 µl of 2 µg / ml parent TSLP was added to half of the wells, buffer was added to the other half, incubated for 1 hour, and then washed. 100 µl of 1:10 dilution of anti-TSLP antibody (Detection Ab) was added to one well containing TSLP and one well without TSLP, incubated for 1 hour, and then washed. Unrelated human IgG (Jackson, #009-000-003) and a blank condition without antibody were run as negative controls. 20 µl of PE-conjugated monovalent mouse anti-human IgG (BD Pharmingen, #555787) was added to each well. The beads were incubated for 1 hour and then washed. The beads were resuspended in 75 µl PBSA, and at least 100 events per bead cord were collected on a BioPlex instrument (BioRad).

[0271] The median fluorescence intensity (MFI) of the antibody pair without TSLP was subtracted from the signal of the corresponding reaction with TSLP. For antibodies that bound simultaneously and were therefore thought to be in different vials, the reaction values ​​should have met two criteria: 1) these values ​​should be twice that of the highest self-paired coated antibody, unrelated antibody, or blank; and 2) these values ​​should be greater than the signal of the detection antibody present with unrelated or blank coated beads.

[0272] The analysis of competition among the antibodies described above was complicated by the fact that there was a discrepancy between the performance of the antibodies as probes and their performance as blockers. However, when considering only the antibody bins that were clear (i.e., each antibody blocking the other when used as a reference), the minimum value among the eight bins was found as shown in Table 4 below.

[0273] [Table 4-2] It should be noted that some antibodies (such as A23 and A6) were found to be present in multiple bins. It was possible to determine other binning relationships, and the inclusion or exclusion of antibodies from these bins was biased towards exclusion.

[0274] The results of this assay determined which other antibodies cross-competed with the reference antibody for binding. "Cross-competing for binding" means that when used as a blocking antibody, the reference antibody can block the binding of other antibodies when used as probes, and vice versa. In other words, if a reference antibody could block other antibodies, but the other antibodies could not block that reference antibody, those antibodies were not said to cross-compete. A list of cross-competing antibodies is provided in Table 5.

[0275] [Table 5] Example 9: Epitope Mapping While epitopes are often thought to be linear sequences, antibodies more frequently recognize the surface of targets composed of discontinuous amino acids. These amino acids, though far removed from a linear sequence, can come into close proximity through target folding, and antibodies that recognize such epitopes are known as conformation-sensitive antibodies or simply conformational antibodies. This type of binding can be defined by the use of denatured Western blotting, in which the target is heated in the presence of detergent and reducing agents before electrophoresis on the gel to unfold it. The blot from this gel can then be probed with an antibody, and an antibody that can recognize the target after this treatment likely recognizes a linear epitope. While the epitopes of antibodies that bind linear sequences can be defined by binding to peptides (e.g., PepSpot), conformational antibodies are not expected to bind standard peptides with high affinity.

[0276] Reduced, heat-denatured, and purified parental TSLP protein was loaded onto a 10% Bistrice Nupage gel in MES SDS electrophoresis buffer. The protein was transferred to a PVDF membrane blocked with 5% defatted dried milk (NFDM) in PBS + 0.05% Tween (PBST) and incubated with TSLP antibody for 1 hour at RT. These blots were washed three times with PBST and then incubated with goat anti-huIgG secondary antibody for 1 hour at RT. These blots were washed again and incubated with anti-goat IgG:Alexa 680. After three washes with PBST, these blots were scanned on LiCo to visualize the bands.

[0277] Antibodies A2, A4, A5, A6, A7, A10, A21, A23, and A26 were characterized using this method. Antibodies A2, A4, and A5 bound to linear epitopes, as evidenced by strong bands on Western blot. All other antibodies were conformational, resulting in either no band or a very weak band on Western blot.

[0278] Epitopes can be further defined structurally or functionally. Functional epitopes are generally a subset of structural epitopes and consist of residues that directly contribute to the affinity of interactions (e.g., hydrogen bonding, ionic interactions). Structural epitopes are antibodies It can be thought of as a patch of the target area that is covered by it.

[0279] Systematic mutagenesis (scanning mutagenesis) is used to further define the epitopes bound by antibodies. Often, alanine systematic mutagenesis is used to define functional epitopes; alanine (methyl side chain) substitution is essentially a cleavage of the wild-type amino acid side chain and is extremely subtle. Interactions with the protein backbone (such as hydrogen bonding to amide links) are defined by alanine scanning mutagenesis. This may not be revealed by scanning. Instead, we used systematic mutagenesis of arginine and glutamate. These two side chains were chosen for their large stereovolume and charge, which allow for mutations that occur in structural epitopes and have a greater impact on antibody binding. Arginine was generally used unless the WT residue was arginine or lysine (in which case this residue was mutated to glutamate to switch the charge). In a few cases, the WT residue was mutated to both arginine and glutamate.

[0280] Ninety-five amino acids distributed throughout the TSLP were selected for mutations to arginine or glutamic acid. Since hydrophobic residues are generally found within the folded core of a protein, the selection was biased towards charged or polar amino acids to reduce the likelihood of mutations resulting in misfolded proteins. Because no crystal structure exists, these residues were selected essentially randomly, and they were distributed throughout the TSLP. The TSLP containing the mutated furin cleavage site was used as described in Example 8.

[0281] BIOPLEX TM The binding assay was used to measure the binding of anti-TSLP antibodies to mutant TSLP. Biotinylated Penta-His Ab (Qiagen, lot number: 130163339) was bound to streptavidin-coated beads (Luminex, #L100-L1XX-01, where XX specifies the bead code) of 100 bead codes. These were used to capture his-tagged proteins. The 100 bead codes allowed for multiplexing of all 85 mutants, 3 parent controls, an unrelated protein, and 12 blanks. Antibody binding to mutant proteins was compared to antibody binding to the parent proteins.

[0282] 100 μl of 1:5 dilutions of TSLP mutant and parent proteins from the supernatant, along with 1 μg / mL purified TSLP WT, 1 μg / mL of unrelated protein, or no protein, were conjugated to coated beads at RT for 1 hour with vigorous shaking. These beads were washed and divided equally into 96-well filter plates (Millipore). 100 μl of 4-fold dilution of anti-TSLP antibody was added to triple wells, incubated at RT for 0.5 hours, and washed. 100 μl of 1:250 dilution of PE-conjugated anti-human IgG Fc (Jackson, #109-116-170) was added to each well, incubated at RT for 0.5 hours, and washed. The beads were resuspended in 75 μL, shaken for at least 3 minutes, and BIOPLEX sterilized. TM I read it above.

[0283] When mutations to arginine or glutamate disrupted antibody binding, the residue was considered part of a structural epitope ("hit"). This was observed as a shift in EC50 or a reduction in maximum signal compared to antibody binding to the parental TSLP.

[0284] Statistical analysis of antibody binding curves to parental and mutant antibodies was used to identify statistically significant EC50 shifts. This analysis took into account variability in assays and curve fitting.

[0285] The EC50 of the mutant and parental binding curves was compared. Statistically significant differences were identified as hits for further consideration. Curves with the "nofit" or "badfit" flag were excluded from this analysis.

[0286] Two sources of variability were considered in the comparison of EC50 estimates: variability from curve fitting and bead-bead variability. Since the parent and mutants were coupled to different beads, their differences were complicated by the bead-bead differences. Curve-fit variability was estimated using the standard error of the log-EC50 estimate. Bead-bead variability was determined experimentally using experiments in which the parent controls were each coupled to a single bead. Bead-bead variability was estimated using the bead variability in the EC50 estimate of the parent coupling curve.

[0287] A comparison of two EC50s (on a logarithmic scale) was performed using Student's t-test. The t-statistic is calculated as the ratio between δ (the absolute difference between the EC50 estimates) and the standard deviation of δ. The variance of δ is estimated by the sum of three components: the variance estimates of EC50 for the mutant and parent curves in the nonlinear regression, and twice the bead-bead variance estimated from separate experiments. The multiple, two-way bead-bead variances are due to the assumption that both the mutant and parent beads have the same variance.

[0288] The degrees of freedom of the standard deviation of δ were calculated using the Satterthwaite (1946) approximation. Individual p-values ​​and confidence intervals (95% and 99%) were obtained based on Student's t-distribution for each comparison. In the case of multiple parent controls, a conservative approach was taken by selecting the parent control that most closely resembled the mutant (i.e., the one with the largest p-value).

[0289] While running numerous tests simultaneously, multiplicity adjustment was crucial to control for false positives. Two forms of multiplicity adjustment were performed for this analysis: family wise error (FWE) control and false discovery rate (FDR) control. The FWE approach controls the probability that one or more hits are absent; the FDR approach controls the expected proportion of false positives among selected hits. The former approach is more conservative and less robust than the latter. For this analysis, many methods were available for both approaches, and we selected the Hochberg (1988) method for FWE analysis and the Benjamini-Hochberg (1995) FDR method for FDR analysis. Adjusted p-values ​​for both approaches were calculated for either each antibody or the entire assay.

[0290] Mutations in which the EC50 differed significantly from the parent (i.e., having an FWE-corrected p-value of less than 0.01 for each antibody, or having a maximum signal less than 50% of the parent) were considered part of the structural epitope (Table 6). Mutations that were significant either by an EC50 shift (shirt) or a reduction in the maximum signal for all antibodies were considered misfolded. These mutations were Y15R, T55R, T74R, and A77R.

[0291] [Table 6] Several mutations (particularly K73E, K21E, and D22R) disrupted the binding of multiple antibodies. Mutagenesis helps validate the data generated by binning and further refine the epitope space. Mutations within TSLP appear to affect the clusters of antibodies that enter the bin together.

[0292] Example 10: Toxicology Antibodies that still bind to human TSLP and cross-react with TSLP from other species enable toxicological testing in those species. In this example, antibodies that cross-react with cynomolgus monkey TSLP were administered to cynomolgus monkeys. These monkeys were then observed for toxic effects.

[0293] A single-dose safety pharmacology study in cynomolgus monkeys showed that a single intravenous dose of 300 mg / kg of the antibody had no cardiovascular, respiratory, thermoregulatory, or neurobehavioral effects.

[0294] Cynomolgus monkeys (5 per sex / group) were administered subcutaneously at doses of 30, 100, or 300 mg / kg once a week for 4 weeks. No adverse toxicological effects were observed at any dose. The antibodies did not affect clinical findings, body weight, ophthalmology, ECG, clinicopathology, or anatomical pathology.

[0295] In a separate study, four telemeterized cynomolgus monkeys were administered a single intravenous dose of vehicle (day 1) and 300 mg / kg of antibody (day 3). No effects were observed on cardiovascular, respiratory, or neurological function over the 4-day observation period.

[0296] Further testing of the above antibodies was conducted in accordance with the FDA guidelines "Points to Consist der in the Manufacture and Testing of Monoclonal Antibody Products for Human Use” (FDA Center for Biologics Evaluation and As recommended in Research (February 28, 1997), we determined the cross-reactivity with normal human and cynomolgus monkey tissues. No staining of normal tissues was observed at either 1 ug / mL or 50 ug / mL.

[0297] The results above suggest that this antibody is not expected to produce toxic effects in humans.

Claims

1. below: a. below: i. A light chain CDR3 sequence selected from the group consisting of light chain CDR3 sequences A1 to A27, differing in only two amino acid additions, substitutions, and / or deletions; ii. QQAX ８ SFPLT (Sequence ID 251); A light chain CDR3 sequence selected from the group consisting of, b. below: i. A heavy chain CDR3 sequence selected from the group consisting of heavy chain CDR3 sequences A1 to A27, which differs in only three amino acids or less in addition, substitution, and / or deletion; ii. GGGIX １２ VADYYX １３ YGMDV (Sequence ID 255) and; iii. DX ２１ GX ２２ SGWPLFX ２３ Y (Sequence ID 259) and; A heavy chain CDR3 sequence selected from the group consisting of the following: An isolated antigen-binding protein comprising an amino acid sequence selected from the group consisting of, X ８ is an N residue or a D residue; X １２ is a P residue or an A residue; X １３ is a Y residue or an F residue; X ２１ is a G residue or an R residue; X ２２ is an S residue or a T residue; X ２３ is an A residue or a D residue, and The antigen-binding protein specifically binds to TSLP. Antigen-binding protein.

2. Furthermore, the following: a. below: i. Light chain CDR1 sequences that differ from the light chain CDR1 sequences A1 to A27 by only three amino acids or less added, substituted, and / or deleted; ii. RSSQSLX １ YSDGX ２ TYLN (Sequence ID 246) and; iii. RASQX ４ X ５ SSWLA (Sequence ID 249) and; A light chain CDR1 sequence selected from the group consisting of the following: b. below: i. Light chain CDR2 sequences that differ from CDR2 sequences A1 to A27 by only two amino acids or less added, substituted, and / or deleted; ii. KVSX ３ WDS (Sequence ID 247) and; iii. X ６ X ７ SSLQS (Sequence ID 250) and; iv. QDX ９ KRPS (Sequence ID 252) and; A light chain CDR2 sequence selected from the group consisting of the following, c. below: i. Heavy chain CDR1 sequences that differ from CDR1 sequences A1 to A27 by only two amino acids or less added, substituted, and / or deleted; ii. X １０ YGMH (SEQ ID NO: 253) and; iii. X １５ X １６ YMX １７ (Sequence number 257) and; A heavy chain CDR1 sequence selected from the group consisting of, d. below: i. Heavy chain CDR2 sequences that differ from CDR2 sequences A1 to A27 by only three amino acids or less added, substituted, and / or deleted; ii. VIWX １１ DGSNKYYADSVKG (Sequence ID 254) and; iii. VISYDGSX １４ KYYADSVKG (Sequence ID 256) and; iv. WINPNSGGTNX １８ X １９ X ２０ KFQG (Sequence ID 258) and; A heavy chain CDR2 sequence selected from the group consisting of the following: An isolated antigen-binding protein according to claim 1, further comprising an amino acid sequence selected from the group consisting of, X １ is a V residue or an I residue; X ２ is an N residue or a D residue; X ３ is a Y residue or an N residue; X ４ is a G residue or an S residue; X ５ is an L residue or an I residue; X ６ is an N residue or a T residue; X ７ is a T residue or an A residue; X ９ is a K residue or an N residue; X １０ is an S residue or an N residue; X １１ is a Y residue or an F residue; X １４ is a Y residue or an N residue; X １５ is a D residue or a G residue; X １６ is a Y residue or a D residue; X １７ is a Y residue or an H residue; X １８ is a Y residue or an H residue; X １９ is a V residue or an A residue; X ２０ is a Q residue or an R residue, and The antigen-binding protein specifically binds to TSLP. Antigen-binding protein.

3. below: a. below: i. Light chain CDR1 sequence selected from A1 to A27; ii. Light chain CDR2 sequence selected from A1 to A27; iii. Light chain CDR3 sequence selected from A1 to A27, Light chain variable domains including, or b. below: i. A heavy chain CDR1 sequence selected from A1 to A27; ii. Heavy chain CDR2 sequences selected from A1 to A27; and iii. Heavy chain CDR3 sequence selected from A1 to A27, Heavy chain variable domains including, or c. The light chain variable domain of (a) and the heavy chain variable domain of (b), The isolated antigen-binding protein according to claim 1, comprising any of the following:

4. below: a. below: i. Amino acids having a sequence that is at least 80% identical to the light chain variable domain sequence selected from L1 to L27; ii. A sequence of amino acids encoded by a polynucleotide sequence that is at least 80% identical to the polynucleotide sequence encoding the L1-L27 light chain variable domain sequence; iii. The amino acid sequence encoded by a polynucleotide sequence that hybridizes under moderately stringent conditions to the complementary chain of a polynucleotide consisting of L1-L27 light chain variable domain sequences; A light chain variable domain sequence selected from the group consisting of the following, b. below: i. Amino acids that are at least 80% identical to the heavy chain variable domain sequences of H1-H27 array; ii. A sequence of amino acids encoded by a polynucleotide sequence that is at least 80% identical to the polynucleotide sequence encoding the heavy chain variable domain sequences of H1 to H27; iii. The amino acid sequence encoded by a polynucleotide sequence that hybridizes under moderately stringent conditions to the complementary chain of a polynucleotide consisting of heavy chain variable domain sequences H1 to H27. A heavy chain variable domain sequence selected from the group consisting of, c. The light chain variable domain of (a) and the heavy chain variable domain of (b), An isolated antigen-binding protein according to claim 1, comprising any of the above, wherein the antigen-binding protein specifically binds to TSLP.

5. below: a. Light chain variable domain sequence selected from the group consisting of L1 to L27 b. A heavy chain variable domain sequence selected from the group consisting of H1 to H27, or c. Light chain variable domain of (a) and heavy chain variable domain of (b) An isolated antigen-binding protein comprising any of the following, wherein the antigen-binding protein specifically binds to TSLP.

6. L1H1, L2H2, L3H3, L4H4, L5H5, L6H6, L7H7, L8H8, L9H9, L10H10, L11H11, L12H12, L13.1H13, L1 3.2H13, L14.1H14, L14.2H14, L15.1H15, L15.2H15, L16.1H16, L16.2H16, L17H17, L18.1H18, L The isolated antigen-binding protein according to claim 5, comprising a light chain variable domain sequence and a heavy chain variable domain sequence selected from the group consisting of 18.2H18, L19.1H19, L19.2H19, L20.1H20, L20.2H20, L21H21, L22H22, L23H23, L24H24, L25H25, L26H26, and L27H27.

7. The isolated antigen-binding protein according to claim 1 or 5, wherein the binding protein binds to TSLP with a Kd substantially the same as that of a reference antibody selected from the group consisting of antibodies A2, A3, A4, and A5.

8. The isolated antigen-binding protein according to claim 1 or 5, wherein the binding protein inhibits TSLP activity with the same IC50 as a reference antibody selected from the group of antibodies consisting of A2, A3, A4, and A5 according to a primary cell OPG assay.

9. The isolated antigen-binding protein according to claim 1, wherein the antigen-binding protein is selected from the group consisting of human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, antigen-binding antibody fragments, single-chain antibodies, monomeric antibodies, diabodies, triabodies, tetrabodies, Fab fragments, F(fa')x fragments, domain antibodies, IgD antibodies, IgE antibodies, and IgM antibodies, and IgG1 antibodies, and IgG2 antibodies, and IgG3 antibodies, and IgG4 antibodies, and IgG4 antibodies having at least one mutation in the hinge region that reduces the tendency to form intra-H chain disulfide bonds.

10. The isolated antigen-binding protein according to claim 1, wherein the antigen-binding protein is a human antibody.

11. A pharmaceutical composition comprising the antibody described in claim 9 or 10.

12. An isolated nucleic acid comprising a polynucleotide sequence encoding a light chain variable domain, a heavy chain variable domain, or both of the antigen-binding factor described in claim 5.

13. The isolated nucleic acid according to claim 12, wherein the sequence is selected from L1 to L27, H1 to H27 or both.

14. A recombinant expression vector comprising the nucleic acid described in claim 12.

15. A host cell comprising the vector according to claim 14.

16. A hybridoma capable of producing the antibody described in claim 10.

17. A method for producing the antibody according to claim 11, the method comprising the step of incubating the host cell according to claim 15 under conditions that enable the cell to express the antibody.

18. A method for treating a TSLP-associated inflammatory condition in a subject requiring treatment, the method comprising the step of administering a therapeutically effective amount of the composition according to claim 11 to the subject.

19. The method according to claim 18, wherein the inflammatory condition is selected from the group consisting of allergic asthma, allergic rhinosinusitis, allergic conjunctivitis, and atopic dermatitis.

20. A method for treating TSLP-associated fibrosis in a subject requiring treatment for TSLP-associated fibrosis, the method comprising the step of administering a therapeutically effective amount of the composition according to claim 11 to the subject.

21. The method according to claim 20, wherein the fibrotic disorder is selected from the group consisting of scleroderma, interstitial lung disease, idiopathic pulmonary fibrosis, fibrosis resulting from chronic hepatitis B or chronic hepatitis C, radiation-induced fibrosis, and fibrosis resulting from wound healing.

22. An isolated antigen-binding protein that cross-competes with an antibody selected from the group consisting of A1 to A27 for binding to TSLP.

23. The isolated antigen-binding protein according to claim 22, wherein the antigen-binding protein comprises a heavy chain variable region and a light chain variable region of an antibody.

24. An isolated antigen-binding protein that binds to wild-type TSLP with wild-type affinity, wherein the antigen-binding protein binds to any of a group of mutated TSLPs with a higher affinity than wild-type affinity, and the group of mutated TSLPs includes a mutation selected from the group consisting of K67E, K97E, K98E, R100E, K101E, and K103E.

25. The isolated antigen-binding protein according to claim 24, which has a higher binding affinity to any two or more members of the group of mutated TSLPs than the wild-type affinity.

26. The isolated antigen-binding protein according to claim 25, which has a higher binding affinity to all members of the group of mutated TSLPs than the wild-type affinity.

27. An isolated antigen-binding protein that binds to wild-type TSLP with wild-type affinity, wherein the antigen-binding protein binds to any of the mutant TSLP groups with a lower affinity than the wild-type affinity, and the mutant TSLP groups are K21E, T25R, S28R, An antigen-binding protein containing a mutated TSLP, which includes a mutation selected from the group consisting of S64R and K73E.

28. The isolated antigen-binding protein according to claim 27, which has a lower binding affinity to any two or more members of the group of mutated TSLPs than the wild-type affinity.

29. The isolated antigen-binding protein according to claim 28, having a binding affinity lower than that of the wild type for all members of the group of mutated TSLPs.

30. The isolated antigen-binding protein according to claim 27, wherein the antigen-binding protein binds to any of a second group of mutated TSLPs with a higher affinity than that of the wild type, and the second group of mutated TSLPs includes a mutation selected from the group consisting of K67E, K97E, K98E, R100E, K101E, and K103E.

31. An isolated antigen-binding protein that binds to wild-type TSLP with wild-type affinity, wherein the antigen-binding protein binds to any of a group of mutated TSLPs with a higher affinity than wild-type affinity, and the group of mutated TSLPs includes a mutation selected from the group consisting of K97E, K98E, R100E, K101E, and K103E.

32. The isolated antigen-binding protein according to claim 31, which has a higher binding affinity to any two or more members of the group of mutated TSLPs than the wild-type affinity.

33. The isolated antigen-binding protein according to claim 32, which has a higher binding affinity to all members of the group of mutated TSLPs than the wild-type affinity.

34. An isolated antigen-binding protein that binds to wild-type TSLP with wild-type affinity, wherein the antigen-binding protein binds to any of a group of mutated TSLPs with an affinity lower than that of wild-type, and the group of mutated TSLPs includes mutations selected from the group consisting of K10E, A14R, K21E, D22R, K73E, K75E, and A76R, and comprises a mutated TSLP.

35. The isolated antigen-binding protein according to claim 34, which has a lower binding affinity to any two or more members of the group of mutated TSLPs than the wild-type affinity.

36. The isolated antigen-binding protein according to claim 35, which has a lower binding affinity to all members of the group of mutated TSLPs than the wild-type affinity.

37. The isolated antigen-binding protein according to claim 34, wherein the antigen-binding protein binds to any of a second group of mutated TSLPs with a higher affinity than that of the wild type, and the second group of mutated TSLPs comprises mutated TSLPs, wherein the mutations are selected from the group consisting of K97E, K98E, R100E, K101E, and K103E.

38. An isolated antigen-binding protein that binds to wild-type TSLP with wild-type affinity, wherein the antigen-binding protein binds to any of a group of mutated TSLPs with an affinity lower than that of wild-type, and the group of mutated TSLPs includes a mutation selected from the group consisting of K12E, D22R, S40R, R122E, N124E, R125E, and K129E.

39. The wild-type affinity for any two or more members of the aforementioned mutated TSLP group. The isolated antigen-binding protein according to claim 38, having a lower binding affinity than the isolated antigen-binding protein.

40. The isolated antigen-binding protein according to claim 39, which has a lower binding affinity to all members of the group of mutated TSLPs than the wild-type affinity.

41. An isolated antigen-binding protein that binds to wild-type TSLP with wild-type affinity, wherein the antigen-binding protein binds to any of a group of mutated TSLPs with an affinity lower than that of wild-type, and the group of mutated TSLPs includes mutations selected from the group consisting of S40R, S42R, H46R, R122E, and K129E.

42. The isolated antigen-binding protein according to claim 41, which has a lower binding affinity to any two or more members of the group of mutated TSLPs than the wild-type affinity.

43. The isolated antigen-binding protein according to claim 42, having a lower binding affinity to all members of the group of mutated TSLPs than the wild-type affinity.

44. An isolated antigen-binding protein that binds to wild-type TSLP with wild-type affinity, wherein the antigen-binding protein binds to any of a group of mutated TSLPs with an affinity lower than that of wild-type, and the group of mutated TSLPs includes a mutation selected from the group consisting of D2R, T4R, D7R, S42R, H46R, T49R, E50R, Q112R, R122E, R125E, and K129E.

45. The isolated antigen-binding protein according to claim 44, having a binding affinity lower than the wild-type affinity for any two or more members of the group of mutated TSLPs.

46. The isolated antigen-binding protein according to claim 45, having a binding affinity lower than that of the wild type for all members of the group of mutated TSLPs.

47. The isolated antigen-binding protein according to claim 44, which binds to mutated TSLP containing mutant K101E with a higher affinity than that of the wild type.

48. An isolated antigen-binding protein that binds to wild-type TSLP with wild-type affinity, wherein the antigen-binding protein binds to any of a group of mutated TSLPs with an affinity lower than that of wild-type, and the group of mutated TSLPs includes a mutation selected from the group consisting of N5R, S17R, T18R, K21E, D22R, T25R, T33R, H46R, A63R, S64R, A66R, E68R, K73E, K75E, A76R, A92R, T93R, Q94R, and A95R.

49. The isolated antigen-binding protein according to claim 48, having a binding affinity lower than the wild-type affinity for any two or more members of the group of mutated TSLPs.

50. The isolated antigen-binding protein according to claim 49, which has a lower binding affinity to all members of the group of mutated TSLPs than the wild-type affinity.

51. The isolated antigen-binding protein according to claim 48, wherein the antigen-binding protein binds to any of a second group of mutated TSLPs with a higher affinity than that of the wild type, and the second group of mutated TSLPs comprises mutated TSLPs, the mutations of which are selected from the group consisting of K97E, K98E, R100E, K101E, and K103E.

52. An isolated antigen-binding protein that binds to wild-type TSLP with wild-type affinity, wherein the antigen-binding protein binds to any of a group of mutated TSLPs with an affinity lower than that of wild-type, and the group of mutated TSLPs includes a mutation selected from the group consisting of K21E, K21R, D22R, T25R, T33R, S64R, K73E, K75E, E111R, and S114R.

53. The isolated antigen-binding protein according to claim 52, which has a lower binding affinity to any two or more members of the group of mutated TSLPs than the wild-type affinity.

54. The isolated antigen-binding protein according to claim 53, which has a lower binding affinity to all members of the group of mutated TSLPs than the wild-type affinity.

55. The isolated antigen-binding protein according to claim 52, wherein the antigen-binding protein binds to any of a second group of mutated TSLPs with a higher affinity than that of the wild type, and the second group of mutated TSLPs comprises mutated TSLPs, the mutations being selected from the group consisting of K97E, K98E, R100E, K101E, and K103E.

56. An isolated antigen-binding protein that binds to wild-type TSLP with wild-type affinity, wherein the antigen-binding protein binds to any of a group of mutated TSLPs with an affinity lower than that of wild-type, and the group of mutated TSLPs includes a mutation selected from the group consisting of E9R, K10E, K12E, A13R, S17R, S20R, K21E, K21R, K73E, K75E, N124E, and R125E.

57. The isolated antigen-binding protein according to claim 56, which has a lower binding affinity to any two or more members of the group of mutated TSLPs than the wild-type affinity.

58. The isolated antigen-binding protein according to claim 57, having a binding affinity lower than that of the wild type for all members of the group of mutated TSLPs.

59. The isolated antigen-binding protein according to claim 56, wherein the antigen-binding protein binds to any of a second group of mutated TSLPs with a higher affinity than that of the wild type, and the second group of mutated TSLPs includes a mutation selected from the group consisting of K67E, K97E, K98E, R100E, K101E, and K103E.

60. An isolated antigen-binding protein that binds to wild-type TSLP with wild-type affinity, wherein the antigen-binding protein binds to any of a group of mutated TSLPs with an affinity lower than that of wild-type, and the group of mutated TSLPs includes a mutation selected from the group consisting of A14R, K21E, D22R, A63R, S64R, K67E, K73E, A76R, A92R, and A95R.

61. The isolated antigen-binding protein according to claim 60, which has a lower binding affinity to any two or more members of the group of mutated TSLPs than the wild-type affinity.

62. The isolated antigen-binding protein according to claim 61, which has a lower binding affinity to all members of the group of mutated TSLPs than the wild-type affinity.

63. The antigen-binding protein binds to one of the second group of mutated TSLPs with a higher affinity than the wild-type affinity, and the second group of mutated TSLPs is K97E, K98 The isolated antigen-binding protein according to claim 60, comprising a mutated TSLP containing a mutation selected from the group consisting of E, R100E, K101E, and K103E.

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