Agents that interfere with thymic stromal lymphopoietin (TSLP) receptor signaling

Abstract

The present disclosure relates to antibodies (e.g., human antibodies) and antigen-binding fragments thereof that specifically bind to and neutralize (human) thymic stromal lymphopoietin (TSLP). Such molecules may increase antibody half-life, increase resistance to proteases, and / or enhance Fc activity. c A modified F that reduces its ability to interact with gamma receptors c Such molecules are useful for treating allergic and non-allergic inflammation associated with TSLP-associated disorders and diseases.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 62 / 873,051, filed July 11, 2019, which is incorporated by reference herein in its entirety.

[0002] Description of electronically submitted text files The contents of the text file submitted electronically attached hereto are incorporated by reference in their entirety: Computer-Readable Copy of the Sequence Listing (Filename: TABI_002_01WO_SeqList_ST25; Recorded Date, July 3, 2020; File Size, 49 KB). [Background technology]

[0003] Background of the Disclosure Thymic stromal lymphopoietin (TSLP) is an interleukin 7-like cytokine that plays a crucial role in regulating immune responses and differentiation of hematopoietic cells. TSLP is widely expressed in lung, skin and intestinal epithelial cells, Hassall's corpuscles in the thymic medulla, mucosa-associated lymphoid tissues and tonsils (Liu, Soumelis et al. 2007; Rochman and Leonard 2008; Sokol, Barton et al. 2008). High TSLP expression levels are found in the heart, liver, spleen and prostate compared to expression levels in the lung, skeletal muscle, kidney, spleen, ovary, small intestine and colon (Quentmeier, Drexler et al. 2001).

[0004] Epithelial cell-derived cytokines are produced in response to environmental and proinflammatory stimuli. It induces the release of T cell-attracting chemokines from monocytes and enhances the maturation of myeloid dendritic cells. Within the thymus, TSLP activates myeloid and plasmacytoid dendritic cells, leading to the production of regulatory T cells. TSLP is important for the regulation of type 2 (Th2, T2) immunity through its activity in dendritic cells, T and B cells, as well as the production of cytokines by antigen-specific Th2 cells (Ziegler, Roan et al. 2013).

[0005] TSLP signals through a heterodimeric receptor complex composed of the thymic stromal lymphopoietin receptor (TSLPR) and the IL-7R alpha chain (Verstraete, van Schie et al. 2014). Upon binding to its receptor complex, TSLP can activate multiple signal transduction pathways (Zhong, Sharma et al. 2014). Stimulation of the IL7R / TSLPR complex by TSLP induces phosphorylation and activation of Janus kinases (JAKs). Activated JAKs then regulate the activity of multiple transcription (STAT) factors, including STAT1, STAT3, STAT4, STAT5a, STAT5b, and STAT6. Several other proteins, such as AKT1, ERK1 / 2, JNK, ribosomal protein S6 kinase, and 4E-BP1, are also activated by TSLP stimulation. In addition, phosphoproteomic analysis reveals that TSLP can regulate the phosphorylation of 226 proteins, including kinases and protein phosphatases, such as SHP-1 and SHP-2.

[0006] TSLP is known to be involved in initiating inflammatory cascades and can contribute to the development of chronic diseases including allergic asthma, atopic dermatitis, allergic rhinitis, ulcerative colitis, and many chronic fibroproliferative disorders (Vannella, Ramalingam et al. 2016). Studies have also suggested that TSLP is involved in immune disorders including infectious diseases (Piliponsky et al. 2016), cancer (De Monte et al. 2011), autoimmunity (Moret et al. 2011); and innate and adaptive immune responses (Ziegler, Roan, 2013;Headley et al. 2009).

[0007] TSLP is a master regulator of allergic inflammation, particularly the type 2 inflammatory pathway, and has therefore become a promising target for the treatment of asthma. Asthma is a disorder characterized by inflammation of the bronchi due to increased production of mucus inside the bronchi. Asthma patients experience symptoms such as coughing, wheezing, shortness of breath, chest tightness, pain or pressure, typically due to chronic airway hyperresponsiveness due to eosinophil infiltration (Ohta, Nagase et al. 2017). Some patients with severe asthma frequently have exacerbations associated with persistent eosinophilic inflammation, despite continuous treatment with high-dose inhaled glucocorticoids with or without oral glucocorticoids. A certain percentage of refractile asthma patients who do not respond to conventional treatments have severe symptoms most likely due to non-eosinophilic, non-Th2 cell populations. TSLP expression is higher in the airways of asthmatics than in healthy controls, and levels correlate with Th2 cytokine and chemokine expression and disease severity (Gauvreau, O'Byrne et al. 2014). More recently, human anti-TSLP IgG, which specifically binds human TSLP, has been reported. 2 The antibody tezepelumab (AMG 157) is in clinical trials for uncontrolled asthma (Comeau, DESMEDT et al. 2006) (Comeau, Smothers et al. 2012) (Corren, Parnes et al. 2017).

[0008] Besides TSLP targeting, three mAbs directed against IL-5 have been approved for patients with severe asthma and eosinophilic phenotype: mepolizumab and reslizumab, which bind directly to IL-5, and benralizumab, which binds to the IL-5 receptor (Ortega, Liu et al. 2014) (Castro, Zangrilli et al. 2015) (Bleecker, FitzGerald et al. 2016). Dupilumab, an antibody that binds to the IL-4 receptor alpha chain and prevents binding of both IL-4 and IL-13, was approved for patients with moderate to severe eosinophilic asthma (Castro, Corren et al. 2018). Notably, all of these therapeutic antibodies appear to be more effective in patients with increasing levels of T2 biomarkers compared to those with lower levels.

[0009] The prevalence of TSLP in other nonallergic diseases and some fibrotic conditions should also be considered as a justification for targeted therapy (Ying, Zhang 2015). In the autoimmune disease systemic sclerosis, TSLP is highly expressed in immune cells in perivascular areas and skin. TSLP is upregulated in dermal epithelial cells, mast cells, and fibroblasts in systemic sclerosis patients (Usategui, Criado et al. 2013). TSLP and its receptor are strongly expressed in the lungs of patients with idiopathic pulmonary fibrosis, a severe fibrotic lung disease (Datta, Alexander et al. 2013). In fibrotic skin diseases, dermal fibroblasts produce high levels of TSLP in response to TGFβ in keloid tissue.

[0010] Further support for TSLP as a therapeutic target comes from the finding that TSLP has a role in the initiation and progression of a variety of tumors. Such tumors include solid tumors (such as breast, colon, and pancreas) as well as hematological tumors (such as B-cell acute lymphocytic leukemia (B-ALL)) (Corren, Ziegler 2019). Tumors that exhibit Th2-type responses generally have a worse prognosis than tumors with a predominantly Th-type response. Tumor cells secrete Il-1α and IL-1β, which are required for TSLP expression by cancer-associated fibroblasts, suggesting that the tumor and tumor microenvironment are important in the induction of TSLP. Studies with human cervical cancer cells, gastric and ovarian cancers also provide evidence that TSLP-mediated crosstalk exists between hematopoietic cells infiltrating the tumor and stroma as well as the tumor itself.

[0011] TSLP has an important role in cancer, but its function as a pro- or anti-tumor factor depends on the type of tumor. TSLP in metastatic breast cancer is an area of ​​intensive research, but conflicting findings have been found in mouse models and human breast tumor cell lines. Similarly, the role of TSLP in skin tumors is bifurcated. The role of TSLP in B-ALL is complicated by the presence of genetic lesions due to mutations in genes encoding components of the TSLP signaling pathway, which occur in 50-60% of patients with poor prognosis (Corren, Ziegler 2019).

[0012] These observations suggest an important role for TSLP in human disease, and disruption of TSLP and the TSLP receptor signaling pathway may have clinical benefit for diseases that lack effective treatments. For asthma, as an example, the mortality rate due to asthma-related conditions is around 250,000 per year worldwide, and this number is predicted to grow to over 100 million by 2025 (Pawankar 2014). Allergic asthma patients often have other conditions, such as diabetes, obesity, cardiovascular disease, and gastroesophageal disease, leading to more complicated and worse outcomes. Approximately 70% of asthma patients also have allergies. Allergic diseases include anaphylaxis, food allergies, certain forms of asthma, rhinitis, conjunctivitis, angioedema, eosinophilic disorders including urticaria, atopic dermatitis, eczema, eosinophilic esophagitis, and drug and insect allergies (Pawankar 2014) (Kay 2001). Undoubtedly, there is a significant need to reduce Th2 cytokine-associated inflammation due to high healthcare costs, morbidity, impact on quality of life, absenteeism, poorer work performance, and an increasing patient population with allergic and non-allergic diseases. Agents such as neutralizing antibody molecules that interfere with TSLP receptor binding and signaling and have desirable efficacy and pharmacokinetic profiles are an unmet need to inhibit symptoms (both T2-high and T2-low) associated with chronic inflammatory conditions. [Prior art documents] [Non-patent literature]

[0013] [Non-Patent Document 1] Corren, J. and Ziegler, S. F. Nature Immunology (2019) 20:1603-1609 [Non-Patent Document 2] Pawankar, R. World Allergy Organization Journal(2014)7(1):1~3 [Non-Patent Document 3] Kay, AB New England Journal of Medicine (2001) 344(1):30~37 Summary of the Invention [Means for solving the problem]

[0014] Summary of the Disclosure The present disclosure provides monoclonal antibodies and antigen-binding fragments thereof that specifically bind to thymic stromal lymphopoietin, TSLP, and neutralize, inhibit, block, abrogate, reduce or interfere with at least one activity thereof. Activities of TSLP that can be neutralized, inhibited, blocked, abrogated, reduced or interfered with by the antibodies or fragments thereof disclosed herein include, but are not limited to, neutralization of the activation of its receptor complex by TSLP, etc. In embodiments, the antibody is a monoclonal antibody, e.g., a murine monoclonal antibody or a humanized antibody. In embodiments, the antibody is an antigen-binding antibody fragment.

[0015] The present disclosure provides a murine monoclonal antibody against human TSLP, designated as TAVO202, comprising a heavy chain variable region sequence having the amino acid sequence of SEQ ID NO:1 and a light chain variable region sequence having the amino acid sequence of SEQ ID NO:2.

[0016] The present disclosure provides a heavy chain variable region of TAVO202 that contains three complementarity determining regions (CDRs), designated HCDR1, HCDR2 and HCDR3, having the amino acid sequences set forth as SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, respectively.

[0017] The present disclosure provides a light chain variable region of TAVO202 that comprises three CDRs, designated LCDR1, LCDR2 and LCDR3, having the amino acid sequences shown as SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, respectively.

[0018] The present disclosure provides one humanized heavy chain variable region of TAVO202, designated as 202H2, having the amino acid sequence shown as SEQ ID NO:9.

[0019] The present disclosure provides two humanized light chain variable regions of TAVO202, designated 202L3 and 202L4, having the amino acid sequences set forth as SEQ ID NO:10 and SEQ ID NO:11, respectively.

[0020] In embodiments, the disclosure provides a humanized antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 according to SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, respectively. In certain embodiments, the humanized antibody comprises a heavy chain variable region having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO:9. In certain embodiments, the humanized antibody comprises a light chain variable region having at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO:10 or SEQ ID NO:11.

[0021] As a non-limiting example, the present disclosure provides a first humanized antibody for TAVO202, designated as TAVO6264, comprising the heavy chain sequence shown as SEQ ID NO: 12 and the light chain sequence shown as SEQ ID NO: 13, comprising a humanized heavy chain variable region 202H2 and a humanized light chain variable region 202L3 with a human IgG2 Fc.

[0022] The present disclosure provides a second humanized antibody for TAVO202, designated as TAVO6265, comprising the heavy chain sequence shown as SEQ ID NO:12 and the light chain sequence shown as SEQ ID NO:14, comprising a humanized heavy chain variable region 202H2 and a humanized light chain variable region 202L4 with a human IgG2 Fc.

[0023] The present disclosure provides a third humanized antibody for TAVO202, designated as TAVO7264, comprising a humanized heavy chain variable region 202H2 and a humanized light chain variable region 202L3 with a human IgG1 Fc with L234F, L235E, D265A, F405L mutations, the heavy chain sequence of which is shown as SEQ ID NO:15 and the light chain sequence of which is shown as SEQ ID NO:13.

[0024] The present disclosure provides a fourth humanized antibody for TAVO202, designated as TAVO7265, comprising a heavy chain sequence shown as SEQ ID NO: 15 and a light chain sequence shown as SEQ ID NO: 14, the humanized heavy chain variable region 202H2 and a humanized light chain variable region 202L4 with human IgG1 Fc with L234F, L235E, D265A, F405L mutations.

[0025] The present disclosure provides a fifth humanized antibody for TAVO202, designated as TAVO9764, comprising a humanized heavy chain variable region 202H2 and a humanized light chain variable region 202L3 with a human IgG1 Fc with L234A, L235A, M428L, N434S mutations, the fifth humanized antibody for TAVO202, comprising a heavy chain sequence shown as SEQ ID NO: 16 and a light chain sequence shown as SEQ ID NO: 13.

[0026] The present disclosure provides a sixth humanized antibody for TAVO202, designated as TAVO9765, comprising a heavy chain sequence shown as SEQ ID NO:16 and a light chain sequence shown as SEQ ID NO:14, the humanized heavy chain variable region 202H2 and a humanized light chain variable region 202L4 with a human IgG1 Fc with L234A, L235A, M428L, N434S mutations.

[0027] The anti-TSLP monoclonal antibody is a full-length IgG 1 , IgG 2 , IgG 3 , IgG 4 F may be an antibody or specifically binds to (e.g., human) TSLP and neutralizes, inhibits, blocks, abrogates, reduces or interferes with at least one activity of TSLP. ab , F( ab’ )2 Alternatively, it may comprise only the antigen-binding portion comprising an scFv fragment. The antibody scaffold may be modified to affect functionality, for example to eliminate residual effector functions.

[0028] The present disclosure also provides anti-TSLP monoclonal antibodies having an extended half-life compared to a wild-type antibody. The extended half-life is determined by the C value of an antibody having any one set of mutations selected from M252Y / S254T / T256E, M428L / N434S, T250Q / M428L, N434A, and T307A / E380A / N434A compared to the parent wild-type antibody. H2 and C H3 This can be achieved by engineering the domain, with residue numbering according to the EU index.

[0029] The disclosure also provides anti-TSLP monoclonal antibodies that have enhanced resistance to proteolysis by proteases that cleave the wild-type antibody between or at residues 222-237 (EU numbering). Resistance to proteolysis can be achieved by engineering E233P / L234V / L235A mutations in the hinge region and a G236 deletion compared to the parent wild-type antibody, where residue numbering is according to the EU index.

[0030] The present disclosure also provides vectors comprising the polynucleotides of the present disclosure.

[0031] The present disclosure also provides a method of producing an anti-TSLP monoclonal antibody of the present disclosure, comprising culturing a host cell of the present disclosure under conditions in which the antibody is expressed, and purifying the antibody.

[0032] The present disclosure also provides a pharmaceutical composition comprising an anti-TSLP antibody, or antigen-binding fragment thereof, of the present disclosure and a pharma- ceutically acceptable carrier.

[0033] The disclosure also provides methods of detecting binding of anti-TSLP antibodies.

[0034] The present disclosure also provides a method of blocking the binding of TSLP to its receptor TLPR, comprising contacting TSLPR with any one of the anti-TSLP antibodies or antigen-binding fragments thereof provided herein.

[0035] The present disclosure also provides a method of treating asthma, comprising administering to a subject in need thereof a therapeutically effective amount of a provided antibody against TSLP.

[0036] The disclosure also provides a method of treating COPD and idiopathic pulmonary fibrosis, comprising administering to a subject in need thereof a therapeutically effective amount of a provided antibody against TSLP.

[0037] The disclosure also provides a method of treating symptoms of atopic dermatitis, comprising administering to a subject in need thereof a therapeutically effective amount of a provided antibody against TSLP.

[0038] The disclosure also provides a method of treating symptoms of eczema, comprising administering to a subject in need thereof a therapeutically effective amount of a provided antibody against TSLP.

[0039] The present disclosure also provides methods of treating symptoms of other allergic conditions, such as eosinophilic esophagitis, comprising administering to a subject in need thereof a therapeutically effective amount of a provided antibody against TSLP.

[0040] The disclosure also provides a method of treating a symptom of inflammatory bowel disease, comprising administering to a subject in need thereof a therapeutically effective amount of a provided antibody against TSLP.

[0041] The disclosure also provides a method of treating a fibrotic condition, such as systemic sclerosis, systemic idiopathic pulmonary fibrosis, and keloid disease, comprising administering to a subject in need thereof a therapeutically effective amount of a provided antibody against TSLP.

[0042] The present disclosure also provides a method of treating cancer, i.e., breast cancer, pancreatic cancer, colorectal cancer, lymphoblastic leukemia, head and neck cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a provided antibody against TSLP. In certain embodiments, for example, the following items are provided: (Item 1) An isolated antibody or antigen-binding fragment thereof that specifically binds to thymic stromal lymphopoietin (TSLP) and neutralizes, inhibits, blocks, abrogates, reduces or interferes with the activity of TSLP. (Item 2) 2. The antibody or antigen-binding fragment thereof according to item 1, which is a mouse monoclonal antibody or antibody fragment having a heavy chain variable region shown as SEQ ID NO:1 and a light chain variable region shown as SEQ ID NO:2. (Item 3) 2. The antibody or antigen-binding fragment according to item 1, comprising heavy chain complementarity determining region (CDR) sequences HCDR1, HCDR2, HCDR3 according to SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively. (Item 4) 2. The antibody or antigen-binding fragment according to item 1, comprising the light chain complementarity determining region (CDR) sequences LCDR1, LCDR2, LCDR3 according to SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively. (Item 5) 2. The antibody or antigen-binding fragment of item 1, which is a humanized antibody or antibody fragment. (Item 6) 6. The antibody or antigen-binding fragment of item 5, wherein the humanized antibody or fragment comprises a heavy chain variable region as shown in SEQ ID NO:9. (Item 7) 6. The antibody or antigen-binding fragment of item 5, wherein the humanized antibody or fragment comprises a light chain variable region as shown in SEQ ID NO: 10. (Item 8) 6. The antibody or antigen-binding fragment of item 5, wherein the humanized antibody or fragment comprises a light chain variable region as shown in SEQ ID NO:11. (Item 9) 6. The antibody or antigen-binding fragment of item 5, wherein the antibody or fragment comprises heavy chain complementarity determining region (CDR) sequences HCDR1, HCDR2, HCDR3 according to SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively, and light chain LCDR1, LCDR2, LCDR3 according to SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region having at least 80% sequence identity to SEQ ID NO: 9, and a light chain variable region having at least 80% sequence identity to SEQ ID NO: 10 or 11. (Item 10) 6. The antibody or antigen-binding fragment of item 5, wherein the humanized antibody comprises a human IgG heavy chain sequence as shown in SEQ ID NO: 12. (Item 11) 6. The antibody or antigen-binding fragment of item 5, wherein the humanized antibody comprises a human light chain sequence shown as SEQ ID NO: 13. (Item 12) 6. The antibody or antigen-binding fragment of item 5, wherein the humanized antibody comprises a human light chain sequence shown as SEQ ID NO: 14. (Item 13) 6. The antibody or antigen-binding fragment of item 5, wherein the humanized antibody comprises a human IgG heavy chain sequence as shown in SEQ ID NO: 15. (Item 14) 6. The antibody or antigen-binding fragment of item 5, wherein the humanized antibody comprises a human IgG heavy chain sequence as shown in SEQ ID NO: 16. (Item 15) The antibody is IgG 1 , IgG 2 , IgG 3 or IgG 4 15. The antibody or antigen-binding fragment according to items 1 to 14, which is an isotype. (Item 16) The antibody comprises one or more Fs that extend the half-life of the engineered antibody compared to the parent wild-type antibody. c 15. The antibody or antigen-binding fragment according to any one of items 1 to 14, having a mutation. (Item 17) The antibody comprises one or more Fs that enhance the resistance of the engineered antibody to proteolysis by proteases compared to the parent wild-type antibody. c 15. The antibody or antigen-binding fragment according to any one of items 1 to 14, having a mutation. (Item 18) The antibody has one or more Fs that reduce or eliminate an effector function of the engineered antibody compared to the parent wild-type antibody. c 15. The antibody or antigen-binding fragment according to any one of items 1 to 14, having a mutation. (Item 19) L234A, L235A, M428L and N434S F, which extend the half-life and reduce effector function of the engineered antibody compared to the parent wild-type antibody. c 15. The antibody or antigen-binding fragment according to items 1 to 14, which has a mutation and wherein the residue numbering is according to the EU index. (Item 20) 6. The antibody or antigen-binding fragment of item 5, wherein the humanized antibody or fragment comprises a human IgG1 heavy chain with L234A, L235A, M428L, N434S Fc mutations as shown in SEQ ID NO: 16. (Item 21) 21. The antibody or antigen-binding fragment of items 1 to 20, which blocks binding of human TSLP to its receptor complex. (Item 22) 21. The antibody or antigen-binding fragment according to items 1 to 20, which neutralizes, reduces or interferes with the functional activity of TSLP towards its receptor complex. (Item 23) 21. The antibody or antigen-binding fragment of items 1 to 20, which neutralizes TSLP-driven reporter gene activation in a STAT5 reporter gene assay. (Item 24) 21. The antibody or antigen-binding fragment of items 1 to 20, which neutralizes TSLP-driven proliferation of cells expressing the TSLP receptor complex in a cell proliferation assay. (Item 25) 21. The antibody or antigen-binding fragment of items 1 to 20, which inhibits TSLP-driven cytokine release from human dendritic cells in a cytokine release assay. (Item 26) 21. The antibody or antigen-binding fragment of items 1 to 20, which inhibits TSLP-driven asthma and pulmonary inflammation in an animal asthma model. (Item 27) An isolated polynucleotide encoding the anti-TSLP IgG antibody according to any one of items 1 to 20. (Item 28) 28. A vector comprising the polynucleotide according to item 27. (Item 29) 29. The vector according to item 28, which is an expression vector. (Item 30) 30. A host cell comprising the vector according to item 28 or 29. (Item 31) A method for producing an anti-TSLP IgG antibody or fragment thereof according to any one of items 1 to 20, comprising the steps of culturing the host cell according to item 29 under conditions in which the anti-TSLP IgG antibody or fragment is expressed, and isolating the anti-TSLP IgG antibody or fragment. (Item 32) A method for measuring the half-life of an anti-TSLP IgG antibody or fragment thereof according to any one of items 1 to 20. (Item 33) A method for measuring resistance to proteolysis of an anti-TSLP IgG antibody or fragment thereof according to items 1 to 20. (Item 34) A method for measuring an effector function of an anti-TSLP IgG antibody or fragment thereof according to any one of items 1 to 20. (Item 35) 21. A method for treating a TSLP-mediated disease or disorder in a subject in need of such treatment, comprising administering to the subject an effective amount of an anti-TSLP IgG antibody or fragment thereof according to any one of claims 1 to 20. (Item 36) 36. The method of claim 35, wherein the TSLP-mediated disease or disorder is asthma, including moderate to severe uncontrolled asthma. (Item 37) 36. The method of claim 35, wherein the TSLP-mediated disease or disorder is a fibrotic disease and related condition, including systemic sclerosis, systemic idiopathic pulmonary fibrosis, and keloid disease. (Item 38) 36. The method of claim 35, wherein the TSLP-mediated disease or disorder is chronic obstructive pulmonary disease, such as emphysema, chronic bronchitis, and refractory asthma. (Item 39) 36. The method of claim 35, wherein the TSLP-mediated disease or disorder is an inflammatory disease, including rhinitis, atopic dermatitis, eczema, systemic sclerosis, food allergy, and inflammatory bowel disease. (Item 40) The method of claim 35, wherein the TSLP-mediated disease or disorder is certain cancers, including lung cancer, breast cancer, pancreatic cancer, colorectal cancer, lymphoblastic leukemia, and head and neck cancer. (Item 41) 41. The method of any one of items 35 to 40, further comprising administering a second agent to the subject in need of treatment. (Item 42) 42. The method of claim 41, wherein the second agent is selected from the group consisting of corticosteroids, bronchodilators, antihistamines, anti-leukotrienes, PDE-4 inhibitors; anti-cancer drugs, immunomodulatory drugs, and cytokine therapy drugs. [Brief description of the drawings]

[0043] [Figure 1] Figure 1: Binding to (A) human and (B) mouse TSLP by the mouse monoclonal anti-human TSLP antibody TAVO202.

[0044] [Diagram 2]Figure 2: (A) Cell proliferation response upon stimulation with human TSLP in BAF cells transfected with the human TSLP receptor complex. (B) Dose-dependent neutralization of human TSLP-driven cell proliferation by TAVO202.

[0045] [Diagram 3] Figure 3: (A) Dose-dependent STAT5 reporter gene expression upon stimulation with human TSLP in a luciferase reporter assay. (B) Dose-dependent neutralization of reporter gene expression by TAVO202 in a human TSLP-driven reporter gene expression assay.

[0046] [Figure 4] Figure 4: Sequence alignment of the heavy and light chain variable regions of TAVO202 with humanized VH and VL variants. Humanized antibodies can be generated by pairing a humanized VH variant (202H2) and two humanized VL variants (202L3 and 202L4) with different IgG Fc.

[0047] [Diagram 5] Figure 5: (A) SDS-PAGE analysis of an example humanized anti-TSLP IgG antibody under non-reducing and (B) reducing conditions. (C) Size-exclusion chromatography (SEC) analysis of an example anti-TSLP IgG antibody.

[0048] [Figure 6] Figure 6: Binding to (A) human and (B) cynomolgus TSLP by an example humanized anti-TSLP IgG antibody.

[0049] [Figure 7] Figure 7: Neutralization of human TSLP-driven reporter gene activation by an example humanized anti-TSLP IgG antibody in a STAT5 reporter gene expression assay.

[0050] [Figure 8]Figure 8: Neutralization of human TSLP-driven CCL17 release from dendritic cells isolated from two donors by humanized anti-TSLP IgG antibodies.

[0051] [Figure 9] Figure 9: Binding to mouse FcRn at pH 6.0 by humanized anti-TSLP IgG antibodies TAVO9765, which have half-life extending Fc mutations, and TAVO7265, which lacks such mutations.

[0052] [Figure 10] Figure 10: PK profile of anti-TSLP antibody in cynomolgus monkeys. Anti-TSLP antibody with Fc mutations for half-life extension (TAVO9765) was administered as a 4 mg / kg intravenous dose to cynomolgus monkeys. Plasma concentrations are shown up to 35 days post-dosing. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] Detailed Description of the Disclosure definition The terminology used herein is for the purpose of describing the embodiments only and is not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In describing and claiming this disclosure, the following terminology will be used:

[0054] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes combinations of two or more cells, etc.

[0055] "Antibody" is intended in a broad sense to include immunoglobulin molecules including monoclonal antibodies, including murine, human, humanized and chimeric monoclonal antibodies, antibody fragments, bispecific or multispecific antibodies, dimeric, tetrameric or multimeric antibodies, single chain antibodies, domain antibodies, and any other modified configuration of immunoglobulin molecule that contains an antigen binding site of the required specificity. A "full-length antibody molecule" is composed of two heavy chains (HC) and two light chains interconnected by disulfide bonds, and multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (V H ) and the heavy chain constant region (domain C H1 , Hinge, C H2 and C H3 Each light chain is composed of a light chain variable region (V L ) and the light chain constant region (C L ) is composed of V H and V L The region can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with framework regions (FRs). H and V L is composed of three CDR and four FR segments, which are arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.

[0056] A "complementarity determining region (CDR)" is an "antigen binding site" in an antibody. CDRs can be defined using various terms: (i) a complementarity determining region (CDR) is made up of three V H (HCDR1, HCDR2, HCDR3), and three are V L (ii) "hypervariable region," "HVR," or "HV" refers to the three V H (H1, H2, H3), and three are V L(L1, L2, L3) and refer to the regions of an antibody variable domain that are hypervariable in structure as defined by Chothia and Lesk (Chothia and Lesk 1987). The International ImMunoGeneTics (IMGT) database (http: / / www_imgt_org) provides a standardized numbering and definition of antigen-binding sites. The agreement between CDR, HV and IMGT delineations is described in Lefranc et al. (Lefranc, Pommie et al. 2003). The terms "CDR", "HCDR1", "HCDR2", "HCDR3", "LCDR1", "LCDR2" and "LCDR3" as used herein include CDRs defined by any of the above methods, Kabat, Chothia or IMGT, unless otherwise expressly stated herein.

[0057] Immunoglobulins can be assigned to five major classes, IgA, IgD, IgE, IgG, and IgM, depending on the heavy chain constant region amino acid sequence. IgA and IgG are classified as isotype IgA 1 , IgA 2 , IgG 1 , IgG 2 , IgG 3 and IgG 4 The antibody light chains of any vertebrate species can be assigned to one of two clearly distinct types, namely kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0058] An "antibody fragment" refers to an antibody fragment that contains heavy chain complementarity determining regions (HCDRs) 1, 2, and 3, light chain complementarity determining regions (LCDRs) 1, 2, and 3, a heavy chain variable region (V H ), or the light chain variable region (V L Antibody fragments refer to portions of immunoglobulin molecules that retain the heavy and / or light chain antigen-binding sites, such as the well-known F ab , F( ab’ )2, F d and F v Fragment, plus one V HDomain antibodies (dAbs) are made up of domains. H and V L The domains are linked together via synthetic linkers to form the V H / V L Various types of single chain antibody designs can be created in which the domains can pair intramolecularly or V H and V L When the domains are expressed as separate single chain antibody constructs, they can pair intermolecularly to form monovalent antigen-binding sites, such as single chain Fvs (scFvs) or diabodies, e.g., as described in International Patent Publication Nos. WO1998 / 44001, WO1988 / 01649, WO1994 / 13804 and WO1992 / 01047.

[0059] "Monoclonal antibody" refers to an antibody population having a single amino acid composition in each heavy and light chain, except for possible known modifications such as removal of the C-terminal lysine from the antibody heavy chain. A monoclonal antibody typically binds to one antigen epitope, except that a bispecific monoclonal antibody binds to two distinct antigen epitopes. A monoclonal antibody can have heterogeneous glycosylation within the antibody population. A monoclonal antibody can be monospecific or polyspecific, or monovalent, bivalent or polyvalent. Bispecific antibodies are included within the term monoclonal antibody.

[0060] An "isolated antibody" refers to an antibody or antibody fragment that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that, for example, specifically binds to TSLP, is substantially free of antibodies that specifically bind to antigens other than TSLP).

[0061] An "isolated antibody" encompasses antibodies that have been isolated to greater purity, such as antibodies that are 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% pure.

[0062] "Humanized antibody" refers to an antibody in which the antigen binding site is derived from a non-human species and the variable region framework is derived from a human immunoglobulin sequence. Humanized antibodies can contain substitutions in the framework, and the framework need not be an exact copy of an expressed human immunoglobulin or human immunoglobulin germline gene sequence.

[0063] "Human antibody" refers to an antibody having heavy and light chain variable regions in which both the framework and antigen binding sites are derived from sequences of human origin, and is optimized to have a minimal immune response when administered to a human subject. If the antibody contains a constant region or a portion of a constant region, the constant region is also derived from sequences of human origin.

[0064] Numbering of amino acid residues in antibody constant regions throughout this specification is according to the EU index as set forth in Kabat et al. (Kabat, National Institutes of et al. 1991), unless expressly stated otherwise.

[0065] Conventional one-letter and three-letter amino acid codes are used herein, as shown in Table 1. Table 1. [Table 1]

[0066] A "variant" of a polypeptide (e.g., an antigen binding protein or antibody) comprises an amino acid sequence in which one or more amino acid residues have been inserted, deleted and / or substituted into the amino acid sequence compared to another polypeptide sequence. Variants include antibodies and fragments thereof having the recited percent identity to the antibodies or fragments provided herein, or to the antibodies or fragments having the recited DNA or amino acid sequences.

[0067] The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. "Percent identity", "percent homology", "sequence identity" or "sequence homology" etc. refer to the percent of identical residues between amino acids or nucleotides in the compared molecules, which is calculated based on the smallest size of the molecules being compared. For such calculations, gaps in the alignment (if any) are preferably addressed by a specific mathematical model or computer program (i.e., "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, AM, ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, DW, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, AM, and Griffin, HG, eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073. In calculating percent identity, the sequences being compared are typically aligned in a way that gives the maximum match between the sequences.

[0068] The constant region sequence of a mammalian IgG heavy chain is H1 - Hinge-C H2 -C H3 The "hinge", "hinge region" or "hinge domain" of IgG is generally designated as the hinge of human IgG according to the EU index. 1 The F chain is generally defined as including Glu216 and terminating at Pro230, but functionally, the flexible portion of the chain can be considered to include additional residues designated the upper and lower hinge regions, such as Glu216 to Gly237, with the lower hinge generally being F. c F responsible for γR binding c The hinge region of other IgG isotypes is designated residues 233-239 of the IgG hinge region by placing the first and last cysteine ​​residues that form inter-heavy chain S-S bonds. 1 The boundaries, numbered according to the EU index, may vary slightly, but are aligned with the C H1 The domain is V H domain and adjacent to the amino terminus of the hinge region of the immunoglobulin heavy chain molecule and includes the first (most amino terminal) constant region of the immunoglobulin heavy chain, e.g., about EU positions 118-215. c The domain extends from amino acid 231 to amino acid 447; H2 The domain consists of a C H3 extends from about Gly341 or Gln342 to Lys447. H1 The IgG heavy chain constant region residues of the region end with Lys. c The domain-containing molecule comprises at least C H2 Domain and C H3 domain and thus includes at least about the region from Ala231 to Lys447 of the IgG heavy chain constant region. c The domain-containing molecule can optionally include at least a portion of a hinge region.

[0069] "Epitope" refers to the portion of an antigen to which an antibody specifically binds. Epitopes typically consist of chemically active (such as polar, non-polar or hydrophobic) surface groupings of moieties such as amino acids or polysaccharide side chains and can have specific charge characteristics as well as specific three-dimensional structural features. Epitopes can be composed of contiguous and / or discontinuous amino acids that form a conformational spatial unit. For discontinuous epitopes, amino acids from different parts of the linear sequence of the antigen are brought into close proximity in three-dimensional space due to folding of the protein molecule. Antibody "epitopes" depend on the methodology used to identify the epitope.

[0070] "Leader sequence" as used herein includes any signal peptide that can be processed by a mammalian cell, including the human B2M leader. Such sequences are well known in the art.

[0071] The terms "peptide", "polypeptide" and "protein" are used interchangeably herein and refer to polymeric forms of amino acids of any length, which may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. These terms also include polypeptides with co-translational (e.g., signal peptide cleavage) and post-translational modifications of the polypeptide, such as, for example, disulfide bond formation, glycosylation, acetylation, phosphorylation, proteolytic cleavage, and others. Furthermore, as used herein, "polypeptide" refers to proteins that contain modifications, such as deletions, additions and substitutions (generally conservative in nature, as known to those skilled in the art) relative to the native sequence, provided that the protein maintains a desired activity. These modifications may be deliberate, such as by site-directed mutagenesis, or may be accidental, such as by mutations of the host that produces the protein or errors due to PCR amplification or other recombinant DNA methods.

[0072] The term "recombinant," as used herein to describe a nucleic acid molecule, means a polynucleotide of genomic, cDNA, viral, semisynthetic and / or synthetic origin that, by its origin or manipulation, is not associated with all or part of an associated polynucleotide sequence in nature. The term "recombinant," when used in reference to a host cell or virus, refers to the host cell or virus into which a recombinant polynucleotide has been introduced. Recombinant is also used herein in reference to a material (e.g., a cell, nucleic acid, protein, or vector) that has been altered by the introduction of heterologous material (e.g., a cell, nucleic acid, protein, or vector).

[0073] The terms "polynucleotide," "oligonucleotide," "nucleic acid," and "nucleic acid molecule" are used interchangeably herein to include polymeric forms of nucleotides, either ribonucleotides or deoxyribonucleotides. The terms refer only to the primary structure of the molecule.

[0074] "Vector" refers to a polynucleotide that can be replicated in a biological system or can be transferred between such systems.Vector polynucleotides typically contain elements such as origins of replication, polyadenylation signals or selection markers that function to facilitate replication or maintenance of such polynucleotides in biological systems, such as cells, viruses, animals, plants and reconstituted biological systems, utilizing biological components that can replicate vectors.Vector polynucleotides can be single-stranded or double-stranded DNA or RNA molecules, cDNA, or hybrids thereof.

[0075] An "expression vector" refers to a vector that can be utilized in a biological system or reconstituted biological system to direct translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

[0076] "Valent" refers to the presence of a specified number of binding sites specific for an antigen in a molecule. Thus, the terms "monovalent," "bivalent," "tetravalent," and "hexavalent" refer to the presence of 1, 2, 4, and 6, respectively, binding sites specific for an antigen in a molecule.

[0077] As used herein, the term "heterologous" used in reference to a nucleic acid sequence, protein or polypeptide means that these molecules do not naturally occur in the cell from which the heterologous nucleic acid sequence, protein or polypeptide is obtained. For example, a nucleic acid sequence encoding a human polypeptide inserted into a cell that is not a human cell is a heterologous nucleic acid sequence in this context. While heterologous nucleic acids can be derived from different organisms or animal species, such nucleic acids do not necessarily originate from separate organism species to be heterologous. For example, in some instances, a synthetic nucleic acid sequence or its encoded polypeptide may be heterologous to the cell into which it is introduced, in that the cell into which it is introduced did not previously contain the synthetic nucleic acid. Thus, for example, a synthetic nucleic acid sequence or its encoded polypeptide can be considered heterologous to a human cell, even if one or more components of the synthetic nucleic acid sequence or its encoded polypeptide are originally obtained from a human cell.

[0078] "Host cell" as used herein refers to an in vivo or in vitro eukaryotic cell, or a cell derived from a multicellular organism cultured as a single-cell entity (e.g., a cell line), and such eukaryotic cells can be or have been used as recipients of nucleic acids (e.g., expression vectors containing nucleotide sequences encoding the multimeric polypeptides of the present disclosure), including the progeny of the original cell genetically modified by the nucleic acid. It is understood that the progeny of a single cell may not necessarily be completely identical in morphology or genomic or total DNA content (complement) to the original parent due to natural, accidental, or deliberate mutations.

[0079] A "recombinant host cell" (also referred to as a "genetically modified host cell") is a host cell into which a heterologous nucleic acid, e.g., an expression vector, has been introduced. For example, a genetically modified eukaryotic host cell is genetically modified by the introduction into a suitable eukaryotic host cell of a heterologous nucleic acid, e.g., an exogenous nucleic acid that is foreign to the eukaryotic host cell or a recombinant nucleic acid that is not normally found in the eukaryotic host cell.

[0080] "Specific binding" or "specifically binds" or "binds" refers to antibody binding to a specific antigen with greater affinity than to other antigens. Typically, the equilibrium dissociation constant for binding (K D ) is about 1 × 10 -8 M or less, e.g., about 1 x 10 -9 M or less, approximately 1 x 10 -10 M or less, approximately 1 x 10 -11 M or less or about 1 × 10 -12 M or less, typically with respect to its K for binding to a non-specific antigen (e.g., BSA, casein). D At most 1 / 100 of K D An antibody "specifically binds" when D can be measured using standard procedures.

[0081] As used herein, the terms "treatment," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic, in terms of a partial or complete cure of the disease and / or adverse effects that may result from the disease.

[0082] "Treatment", as used herein, covers any treatment of a disease in a mammal, e.g., a human, and includes (a) preventing the disease from occurring in a subject who may be predisposed to the disease, but has not yet been diagnosed as having the disease; (b) inhibiting the disease, i.e., arresting its development; and (c) alleviating the disease, i.e., causing regression of the disease.

[0083] The terms "individual," "subject," "host," and "patient," used interchangeably herein, refer to mammals, including but not limited to murines (e.g., rats, mice), lagomorphs (e.g., rabbits), non-human primates, humans, dogs, cats, ungulates (e.g., horses, cows, sheep, pigs, goats), and the like.

[0084] "Therapeutically effective amount" or "effective amount" refers to the amount of an agent, or the combined amount of two agents, that when administered to a mammal or other subject for treating a disease, is sufficient to affect such treatment for the disease. A "therapeutically effective amount" will vary depending on the agent, the disease and its severity, and the age, weight, etc., of the subject to be treated.

[0085] Before the present disclosure is further described, it is to be understood that the present disclosure is not limited to the described embodiments, as they may, of course, vary. The scope of the present disclosure will be limited only by the appended claims, and it is also to be understood that the terminology used herein is for the purpose of describing embodiments only and is not intended to be limiting.

[0086] Composition of anti-TSLP antibodies and antigen-binding fragments Described herein are monoclonal antibodies and antigen-binding fragments thereof that specifically bind to TSLP and neutralize, inhibit, block, abrogate, reduce or interfere with at least one activity of TSLP, such as the functional activity of TSLP on its receptor complex. Anti-TSLP antibodies and antigen-binding fragments can be administered therapeutically to a subject to treat a TSLP-mediated disease.

[0087] The present disclosure provides a murine monoclonal antibody, designated as TAVO202, identified from a murine hybridoma screen that specifically binds to and neutralizes, inhibits, blocks, abrogates, reduces or interferes with at least one activity of human thymic stromal lymphopoietin (TSLP). TAVO202 comprises a heavy chain variable region sequence having the amino acid sequence of SEQ ID NO:1, and a light chain variable region sequence having the amino acid sequence of SEQ ID NO:2. The heavy chain variable region of TAVO202 comprises three complementarity determining regions, designated as HCDR1, HCDR2 and HCDR3, having the amino acid sequences shown as SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, respectively. The light chain variable region of TAVO202 comprises three complementarity determining regions, designated as LCDR1, LCDR2 and LCDR3, having the amino acid sequences shown as SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, respectively.

[0088] Heavy chain variable region sequence of anti-human TSLP mouse monoclonal antibody TAVO202 (SEQ ID NO:1) [ka] The sequences of the three CDRs of the heavy chain are underlined.

[0089] Light chain variable region sequence of anti-human TSLP mouse monoclonal antibody TAVO202 (SEQ ID NO:2) [ka] The sequences of the three CDRs of the light chain are underlined.

[0090] HCDR1 sequence of anti-human TSLP mouse monoclonal antibody TAVO202 (SEQ ID NO:3) [ka]

[0091] HCDR2 sequence of anti-human TSLP mouse monoclonal antibody TAVO202 (SEQ ID NO:4) [ka]

[0092] HCDR3 sequence of anti-human TSLP mouse monoclonal antibody TAVO202 (SEQ ID NO:5) [ka]

[0093] LCDR1 sequence of anti-human TSLP mouse monoclonal antibody TAVO202 (SEQ ID NO:6) [ka]

[0094] LCDR2 sequence of anti-human TSLP mouse monoclonal antibody TAVO202 (SEQ ID NO:7) [ka]

[0095] LCDR3 sequence of anti-human TSLP mouse monoclonal antibody TAVO202 (SEQ ID NO:8) [ka]

[0096] The murine anti-human TSLP antibody TAVO202 can be humanized by grafting the murine CDRs onto a human germline scaffold. Several critical murine residues are preserved by backmutation to achieve higher stability and better expression while minimizing immunogenicity. For TAVO202, one humanized VH variant (202H2) was designed to target the IGHV1-69 * Based on IGKV1-9, two humanized VL variants were designed and * Based on 01, 202L3, IGKV6-21 * It was designed by 202L4 based on 01, with two back mutations present in the framework.

[0097] Based on the foregoing, the present disclosure provides one humanized heavy chain variable region of TAVO202, designated as 202H2, having the amino acid sequence set forth as SEQ ID NO:9.

[0098] Humanized heavy chain variable region 202H2 sequence (SEQ ID NO:9) [ka] The sequences of the three CDRs of the humanized heavy chain are underlined.

[0099] The present disclosure provides two humanized light chain variable regions of TAVO202, designated 202L3 and 202L4, having the amino acid sequences set forth as SEQ ID NO:10 and SEQ ID NO:11, respectively.

[0100] Humanized light chain variable region 202L3 sequence (SEQ ID NO: 10) [ka] The sequences of the three CDRs of the humanized light chain are underlined.

[0101] Humanized light chain variable region 202L4 sequence (SEQ ID NO:11) [ka] The sequences of the three CDRs of the humanized light chain are underlined.

[0102] As a non-limiting example, six humanized antibodies for TAVO202 can be generated by pairing the humanized 202H2 heavy chain variable region and two humanized light chain variable regions, 202L3 and 202L4, with different IgG Fc (Figure 4, Table 2). Table 2. Exemplary antibodies [Table 2-1] [Table 2-2]

[0103] The present disclosure provides a first humanized antibody for TAVO202, designated as TAVO6264, comprising a humanized heavy chain variable region 202H2 and a humanized light chain variable region 202L3 with a human IgG2 Fc, comprising the heavy chain sequence shown as SEQ ID NO: 12 and the light chain sequence shown as SEQ ID NO: 13.

[0104] The present disclosure provides a second humanized antibody for TAVO202, designated as TAVO6265, comprising the heavy chain sequence shown as SEQ ID NO:12 and the light chain sequence shown as SEQ ID NO:14, comprising a humanized heavy chain variable region 202H2 and a humanized light chain variable region 202L4 with a human IgG2 Fc.

[0105] The present disclosure provides a third humanized antibody for TAVO202, designated as TAVO7264, comprising a humanized heavy chain variable region 202H2 and a humanized light chain variable region 202L3 with a human IgG1 Fc with L234F, L235E, D265A, F405L mutations, the heavy chain sequence of which is shown as SEQ ID NO:15 and the light chain sequence of which is shown as SEQ ID NO:13.

[0106] The present disclosure provides a fourth humanized antibody for TAVO202, designated as TAVO7265, comprising a heavy chain sequence shown as SEQ ID NO: 15 and a light chain sequence shown as SEQ ID NO: 14, the humanized heavy chain variable region 202H2 and a humanized light chain variable region 202L4 with human IgG1 Fc with L234F, L235E, D265A, F405L mutations.

[0107] The present disclosure provides a fifth humanized antibody for TAVO202, designated as TAVO9764, comprising a humanized heavy chain variable region 202H2 and a humanized light chain variable region 202L3 with a human IgG1 Fc with L234A, L235A, M428L, N434S mutations, the fifth humanized antibody for TAVO202, comprising a heavy chain sequence shown as SEQ ID NO: 16 and a light chain sequence shown as SEQ ID NO: 13.

[0108] The present disclosure provides a sixth humanized antibody for TAVO202, designated as TAVO9765, comprising a heavy chain sequence shown as SEQ ID NO:16 and a light chain sequence shown as SEQ ID NO:14, the humanized heavy chain variable region 202H2 and a humanized light chain variable region 202L4 with a human IgG1 Fc with L234A, L235A, M428L, N434S mutations.

[0109] Anti-TSLP humanized antibody heavy chain based on 202H2 and IgG2 Fc (SEQ ID NO: 12) [ka] The sequence of the heavy chain variable domain is underlined.

[0110] Anti-TSLP humanized antibody light chain based on 202L3 (SEQ ID NO: 13) [ka] The sequence of the light chain variable domain is underlined.

[0111] Anti-TSLP humanized antibody light chain based on 202L4 (SEQ ID NO: 14) [ka] The sequence of the light chain variable domain is underlined.

[0112] Anti-TSLP humanized antibody heavy chain based on IgG1 Fc with 202H2 and L234F, L235E, D265A, F405L mutations (SEQ ID NO: 15) [ka] The heavy chain variable domain sequence is underlined, and the L234F, L235E, D265A, and F405L mutations are shown in bold.

[0113] Anti-TSLP humanized antibody heavy chain based on IgG1 Fc with 202H2 and L234A, L235A, M428L, N434S mutations (SEQ ID NO: 16) [ka] The heavy chain variable domain sequence is underlined, and the L234A, L235A, M428L, and N434S mutations are shown in bold.

[0114] The present disclosure also provides bispecific or multispecific antibody preparations that may include engagement of any two, three or four TSLP epitopes by having Fab or scFv domains that include one humanized heavy chain variable region of TAVO202, designated as 202H2, having the amino acid sequence shown as SEQ ID NO:9, and one of two humanized light chain variable regions of TAVO202, designated as 202L3 and 202L4, having the amino acid sequences shown as SEQ ID NO:10 and SEQ ID NO:11, respectively. For example, this may be a bispecific antibody with Fab or scFv domains 202H2 and 202L3 and 202H2 and 202L4, or any other domain permutation, that can engage two, three or four epitopes.

[0115] The TSLP-binding antibodies and fragments of the present disclosure include antigen-binding fragments that retain full ability to specifically bind TSLP. TSLP-binding fragments, as used herein, can comprise any three or more consecutive amino acids of an antibody (e.g., four or more, five or more, six or more, eight or more, or even ten or more consecutive amino acids), and include Fab, Fab', F(ab')2, and F(v) fragments, or individual light or heavy chain variable regions or portions thereof. These fragments are similar to the F(v) fragments of an intact antibody. c These fragments lack the fragments, are cleared from the circulation more rapidly than intact antibodies, and can have less non-specific tissue binding. These fragments can be produced from intact antibodies using well-known methods, for example, by proteolytic cleavage with enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).

[0116] The TSLP-binding antibodies and fragments of the present disclosure may also encompass diabodies, which are bivalent antibodies in which the VH and VL domains are expressed on a single polypeptide chain but use a linker that is too short to allow pairing between the two domains on the same chain, thereby allowing the domains to pair with complementary domains on another chain and create two antigen-binding sites.

[0117] The TSLP-binding antibodies and fragments of the present disclosure can also include single chain antibody fragments (scFv) that bind TSLP. scFvs are fragments of an antibody light chain variable region (V L operably linked to an antibody heavy chain variable region (V H), where the heavy and light chain variable regions together or individually form a binding site that binds TSLP. Such TSLP-binding fragments can be prepared by methods known in the art, such as, for example, synthesis or PCR-mediated amplification of the variable portions of the heavy and light chains of an antibody molecule and a flexible protein linker composed of the amino acids Gly and Ser. The resulting DNA fragment is cloned for expression in E. coli or mammalian cells. The expressed TSLP-binding fragment is purified from the host cells.

[0118] The TSLP-binding antibodies and fragments of the present disclosure include full-length antibodies comprising two heavy chains and two light chains. Exemplary human or humanized antibodies include IgG, IgM, IgE, IgA and IgD antibodies. The antibodies may be of any class (IgG, IgM, IgE, IgGA, IgD, etc.) or isotype. For example, a human antibody may comprise an IgG Fc domain, such as at least one of the isotypes IgG1, IgG2, IgG3 or IgG4.

[0119] In some instances, IgG F c The domain is IgG as SEQ ID NO: 17. 1 F c These include amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity to the sequence.

[0120] In some instances, IgG F c The domain is IgG as SEQ ID NO: 18. 2 F c These include amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity to the sequence.

[0121] In some instances, IgG F cThe domain is IgG as SEQ ID NO: 19. 3 F c These include amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity to the sequence.

[0122] In some instances, IgG F c The domain is IgG as SEQ ID NO: 20 4 F c These include amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% amino acid sequence identity to the sequence.

[0123] The S228P mutation was engineered into an IgG4 antibody to 4 The stability can be enhanced.

[0124] IgG 1 F c (SEQ ID NO:17): [ka]

[0125] IgG 2 F c (SEQ ID NO:18): [ka]

[0126] IgG 3 F c (SEQ ID NO:19): [ka]

[0127] IgG 4 F c (SEQ ID NO:20) [ka]

[0128] The anti-TSLP antibody is a modified F c The modified F c The region is the wild type F c In some embodiments, the anti-TSLP antibody comprises at least one amino acid modification relative to the modified F region. c In this case, the naturally occurring F c The region is altered to increase the half-life of the antibody in a biological environment relative to the parent wild-type antibody, eg, serum half-life or half-life as measured by an in vitro assay.

[0129] Exemplary mutations that can be made alone or in combination are T250Q, M252Y, I253A, S254T, T256E, P257I, T307A, D376V, E380A, M428L, H433K, N434S, N434A, N434H, N434F, H435A and H435R mutations, or the fusion of an albumin-binding peptide to the C-terminus of the Fc) that have enhanced circulating antibody half-life compared to a control lacking said Fc mutations.

[0130] In certain embodiments, the half-life extension is achieved by administering to the subject IgG1 F as SEQ ID NO:21. c This can be achieved by engineering the M252Y / S254T / T256E mutations in , where residue numbering is according to the EU index (Dall'Acqua, Kiener et al. 2006).

[0131] In certain embodiments, the half-life extension is achieved by administering to the IgG 1 F c This can also be achieved by engineering the M428L / N434S mutation in (Zalevsky, Chamberlain et al. 2010).

[0132] In certain embodiments, the half-life extension is achieved by administering to the IgG 1 F c This can also be achieved by engineering the T250Q / M428L mutation in (Hinton, Xiong et al. 2006).

[0133] In certain embodiments, the half-life extension is achieved by administering to the IgG 1 F c This can also be achieved by engineering the N434A mutation in (Shields, Namenuk et al. 2001).

[0134] In certain embodiments, the half-life extension is achieved by administering to the IgG 1 F c This can also be achieved by engineering the T307A / E380A / N434A mutations in (Petkova, Akilesh et al. 2006).

[0135] F in antibody half-life extension c The effect of the manipulation was compared with that of native IgG F c The antibody can be evaluated in a PK study in mice in comparison to an antibody having the

[0136] IgG with M252Y / S254T / T256E mutations 1 F c (SEQ ID NO:21) [ka]

[0137] IgG with M428L / N434S mutation 1 F c (SEQ ID NO:22) [ka]

[0138] IgG with T250Q / M428L mutation 1 Fc (SEQ ID NO:23) [ka]

[0139] IgG with N434A mutation 1 F c (SEQ ID NO:24) [ka]

[0140] IgG with T307A / E380A / N434A mutations 1 F c (SEQ ID NO:25) [ka]

[0141] In some embodiments, the anti-TSLP antibody comprises a modified F c In this case, the naturally occurring F c The region has been altered to enhance resistance of the antibody to proteolysis by proteases that cleave the wild-type antibody between or at residues 222-237 (EU numbering).

[0142] In certain embodiments, the antibody or antigen-binding fragment thereof further comprises a mutation in the lower hinge region of the Fc (e.g., equivalent to residues 222-237 of IgG1 Fc) that allows for increased protease resistance (e.g., resistance to degradation by MMP-3, MMP-7, MMP-12, MMP-13, cathepsin G, pepsin, IdeS, or GluV8) relative to a native IgG1 antibody in an environment with endogenous proteolytic activity.

[0143] In certain embodiments, resistance to proteolysis can be achieved by engineering E233P / L234V / L235A mutations and a G236 deletion in the hinge region compared to the parent wild-type antibody as SEQ ID NO:26, residue numbering according to the EU index (Kinder, Greenplate et al. 2013).

[0144] IgG with E233P / L234V / L235A mutations and deletion of G236 1 F c (SEQ ID NO:26) [ka]

[0145] When effector functionality is not desired, the antibodies of the present disclosure may be modified to include activated F c γ receptor (F c γR) and / or inhibit F-dependent cytotoxicity, such as C1q binding, complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC) or phagocytosis (ADCP). c At least one mutation that reduces effector function was present in antibody F c can be further manipulated to introduce

[0146] activation F c F can be mutated to reduce binding of the antibody to γR and subsequently reduce effector function. c The positions are those described, for example, in (Xu, Alegre et al. 2000)(Vafa, Gilliland et al. 2014)(Bolt, Routledge et al. 1993)(Chu, Vostiar et al. 2008)(Shields, Namenuk et al. 2001). Fc mutations with minimal ADCC, ADCP, CDC, Fc-mediated cell activation were also described as sigma mutations for IgG1, IgG2 and IgG4 (Tam, McCarthy et al. 2017).

[0147] Exemplary mutations that can be made alone or in combination include 1 , IgG 2 , IgG 3 or IgG 4 These mutations are K214T, E233P, L234V, L234A, G236 deletion, V234A, F234A, L235A, G237A, P238A, P238S, D265A, S267E, H268A, H268Q, Q268A, N297A, A327Q, P329A, D270A, Q295A, V309L, A327S, L328F, A330S and P331S.

[0148] Exemplary combination mutations that can be made to reduce ADCC include IgG 1 L234A / L235A in IgG 2 V234A / G237A / P238S / H268A / V309L / A330S / P331S in IgG 4 F234A / L235A in IgG 4 S228P / F234A / L235A in IgG 1 , IgG 2 , IgG 3 or IgG 4 N297A, IgG 2 V234A / G237A in IgG 1 K214T / E233P / L234V / L235A / G236 deletion / A327G / P331A / D365E / L358M in IgG 2 H268Q / V309L / A330S / P331S in IgG 1 S267E / L328F in IgG 1 L234F / L235E / D265A in IgG 1 L234A / L235A / G237A / P238S / H268A / A330S / P331S in IgG 4 S228P / F234A / L235A / G237A / P238S in IgG 4 S228P / F234A / L235A / G236 deletion / G237A / P238S in IgG 2Residues 117–260 from and IgG 4 F with residues 261-447 derived from c etc., Hybrid IgG 2 / 4 F c You can also use a domain.

[0149] In some embodiments, the anti-TSLP antibody comprises a modified F c In this case, the naturally occurring F c The area is F c It has been engineered to facilitate the creation of bispecific antibodies by heterodimerization.

[0150] In certain embodiments, Fc heterodimerization can be achieved by engineering F405L and K409R mutations in two parent antibodies and creating a bispecific antibody in a process known as Fab arm exchange (Labrijn, Meesters et al. 2014).

[0151] In certain embodiments, Fc heterodimerization can also be achieved by Fc mutations to facilitate a knob-in-hole strategy (see, e.g., International Publication No. WO 2006 / 028936). An amino acid with a small side chain (hole) is attached to one Fc. c The amino acids with large side chains (knobs) are introduced into the F domain to form the other F c After co-expression of two heavy chains, heterodimers are formed as a result of preferential interaction between the heavy chain with the "knob" and the heavy chain with the "hole" (Ridgway, Presta et al. 1996).

[0152] An exemplary F forming knob and hole c The mutation pairs are T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S and T366W / T366S / L368A / Y407V.

[0153] In certain embodiments, Fc heterodimerization can also be achieved by Fc mutations to facilitate electrostatically matched interaction strategies (Gunasekaran, Pentony et al. 2010). Mutations can be engineered to create positively charged residues in one Fc domain and negatively charged residues in the other Fc domain, as described in US Patent Publication No. US2010 / 0015133; US Patent Publication No. US2009 / 0182127; US Patent Publication No. US2010 / 028637 or US Patent Publication No. US2011 / 0123532. Heavy chain heterodimerization can be formed by electrostatically matched interactions between two mutated Fcs.

[0154] Antibodies of the present disclosure that further include conservative modifications are within the scope of the present disclosure.

[0155] "Conservative modifications" refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Conservative modifications include amino acid substitutions, additions and deletions. A conservative substitution is one in which an amino acid is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been well defined and include amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), basic side chains (e.g., lysine, arginine, histidine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), uncharged polar side chains (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine, tryptophan), aromatic side chains (e.g., phenylalanine, tryptophan, histidine, tyrosine), aliphatic side chains (e.g., glycine, alanine, valine, leucine, isoleucine, serine, threonine), amides (e.g., asparagine, glutamine), beta-branched side chains (e.g., threonine, valine, isoleucine) and sulfur-containing side chains (cysteine, methionine). Additionally, any native residue in the polypeptide can be substituted with alanine, as previously described for alanine scanning mutagenesis ((MacLennan, Rice et al. 1998); (Sasaki and Sutoh 1998)). Amino acid substitutions to the antibodies of the present disclosure can be made by known methods, for example, by PCR mutagenesis (U.S. Pat. No. 4,683,195). Alternatively, libraries of variants can be generated, for example, using random (NNK) or non-random codons, for example, the DVK codons that code for 11 amino acids (Ala, Cys, Asp, Glu, Gly, Lys, Asn, Arg, Ser, Tyr, Trp). The resulting antibody variants can be tested for their characteristics using the assays described herein.

[0156] The antibodies of the present disclosure can be post-translationally modified by processes such as glycosylation, isomerization, deglycosylation, or non-naturally occurring covalent modifications, such as the addition of polyethylene glycol moieties (PEGylation) and lipidation. Such modifications can occur in vivo or in vitro. For example, the antibodies of the present disclosure can be conjugated to polyethylene glycol (PEGylation) to improve their pharmacokinetic profile. Conjugation can be performed by techniques known to those skilled in the art. Conjugation of therapeutic antibodies with PEG has been shown to enhance pharmacodynamics without interfering with function (Leong, DeForge et al. 2001; Yang, Basu et al. 2003; Knight, Jordan et al. 2004).

[0157] The antibodies of the present disclosure can be modified to improve stability, selectivity, cross-reactivity, affinity, immunogenicity or other desirable biological or biophysical properties, and this is within the scope of the present disclosure. Antibody stability is influenced by several factors, including (1) the core packing of individual domains, which affects its intrinsic stability; (2) protein / protein interface interactions, which affect HC and LC pairing; (3) burial of polar and charged residues; (4) H-bond networks for polar and charged residues; and (5) surface charge and polar residue distribution, among other intra- and intermolecular forces (Worn and Pluckthun 2001). Potential structure-destabilizing residues can be identified based on the crystal structure of the antibody or, in certain cases, by molecular modeling, and the effect of the residues on antibody stability can be examined by making and evaluating variants with mutations at the identified residues. One way to increase antibody stability is to increase the thermal transition midpoint (T m In general, protein T mIt correlates with its stability and inversely correlates with its susceptibility to unfolding and denaturation and degradation processes in solution, which depends on the tendency of the protein to unfold (Remmele and Gombotz 2000). Several studies have found correlations between the ranking of formulations' physical stability measured as thermal stability by DSC and physical stability measured by other methods (Maa and Hsu 1996; Remmele, Nightlinger et al. 1997; Gupta and Kaisheva 2003; Bedu-Addo, Johnson et al. 2004; Zhang, Roy et al. 2004). Formulation studies have shown that Fab T m This suggests that the variability in the mAb response may have implications for the long-term physical stability of the corresponding mAbs.

[0158] The antibodies of the present disclosure have the following properties: c The IgG can have amino acid substitutions in the 1 An example for is H224S (or H224Q) in the hinge 221-DKTHTC-226 (Eu numbering), which blocks radical-induced cleavage (Yates, Gunasekaran et al. 2010); 4 In , the S228P mutation blocks half-antibody exchange (Angal, King et al. 1993; Labrijn, Buijsse et al. 2009).

[0159] Expression and purification of anti-TSLP antibodies The anti-TSLP antibodies and fragments of the disclosure can be encoded by a single nucleic acid (e.g., a single nucleic acid that includes nucleotide sequences encoding the light and heavy chain polypeptides of the antibody) or by two or more separate nucleic acids, each encoding a different portion of the antibody or antibody fragment.

[0160] As non-limiting examples, the disclosure provides a nucleic acid sequence as SEQ ID NO: 27 encoding an anti-TSLP humanized antibody heavy chain based on 202H2 and IgG2 Fc, a nucleic acid sequence as SEQ ID NO: 28 encoding an anti-TSLP antibody light chain sequence based on 202L3, and a nucleic acid sequence as SEQ ID NO: 29 encoding an anti-TSLP antibody light chain sequence based on 202L4.

[0161] Nucleotide sequence for anti-TSLP humanized antibody heavy chain based on 202H2 and IgG2 Fc (SEQ ID NO:27) [ka] [ka]

[0162] Nucleotide sequence for the anti-TSLP humanized antibody light chain based on 202L3 (SEQ ID NO:28) [ka]

[0163] Nucleotide sequence for the anti-TSLP humanized antibody light chain based on 202L4 (SEQ ID NO:29) [ka] [ka]

[0164] The nucleic acid can be inserted into a vector, for example, a nucleic acid expression vector and / or a targeting vector. Such vectors can be used in a variety of ways, for example, for the expression of anti-TSLP-binding antibodies or antibody fragments in cells or transgenic animals. The vector is typically selected to be functional in the host cell in which it will be used. The nucleic acid molecule encoding the anti-TSLP-binding antibody or fragment can be amplified / expressed in prokaryotic, yeast, insect (baculovirus system) and / or eukaryotic host cells. The choice of host cell will depend, in part, on whether the anti-TSLP-binding antibody or fragment is to be post-translationally modified (e.g., glycosylated and / or phosphorylated). If so, yeast, insect or mammalian host cells are preferred. Expression vectors typically contain one or more of the following components: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a leader sequence for secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting a nucleic acid encoding a polypeptide to be expressed, and a selectable marker element.

[0165] In most cases, a leader or signal sequence is engineered at the N-terminus of the anti-TSLP antibody or fragment to guide its secretion. Secretion of the anti-TSLP antibody or fragment from a host cell will result in removal of the signal peptide from the antibody or fragment. Thus, the mature antibody or fragment will lack any leader or signal sequence. In some cases, such as when glycosylation is desired in a eukaryotic host cell expression system, various presequences can be engineered to improve glycosylation or yield. For example, the peptidase cleavage site of the signal peptide can be altered, or prosequences can be added that can also affect glycosylation.

[0166] The present disclosure further provides a cell (e.g., an isolated or purified cell) comprising the nucleic acid or vector of the present disclosure. The cell may be any type of cell that can be transformed by the nucleic acid or vector of the present disclosure to produce the polypeptide encoded thereby. To express the anti-TSLP-binding antibody or fragment, the DNA encoding the partial or full-length light and heavy chains obtained as described above is inserted into an expression vector so that the gene is operably linked to the transcriptional and translational control sequences.

[0167] Methods for introducing nucleic acids and vectors into isolated cells, and for culturing and selecting transformed host cells in vitro, are known in the art and include the use of calcium chloride-mediated transformation, transduction, conjugation, triparental mating, DEAE, dextran-mediated transfection, infection, membrane fusion with liposomes, high-velocity bombardment with DNA-coated microprojectiles, direct microinjection into single cells, and electroporation.

[0168] After introducing the nucleic acid or vector of this disclosure into the cells, the cells are cultured under conditions suitable for expression of the encoded sequence. The antibody, antigen-binding fragment or portion of the antibody can then be isolated from the cells.

[0169] In certain embodiments, two or more vectors that together encode anti-TSLP-binding antibodies or antigen-binding fragments thereof can be introduced into a cell.

[0170] Purification of anti-TSLP binding antibodies or fragments secreted into the cell culture medium can be accomplished using a variety of techniques, including affinity, immunoaffinity or ion exchange chromatography, molecular sieve chromatography, preparative gel electrophoresis or isoelectric focusing, chromatofocusing, and high pressure liquid chromatography. c The antibody containing the region is F cIt can be purified by affinity chromatography with Protein A, which selectively binds to the region.

[0171] Modified forms of antibodies or antigen-binding fragments can be prepared with affinity tags, such as hexahistidine or other small peptides such as FLAG (Eastman Kodak Co., New Haven, Conn.) or myc (Invitrogen), at either their carboxyl or amino termini and purified by one-step affinity columns. For example, polyhistidine binds to nickel with excellent affinity and specificity, and thus nickel affinity columns (such as Qiagen® nickel columns) can be used to purify polyhistidine-tagged selective binding agents. In some instances, two or more purification steps can be used.

[0172] Modified forms of antibodies or antigen-binding fragments can be prepared with affinity tags, such as hexahistidine or other small peptides such as FLAG (Eastman Kodak Co., New Haven, Conn.) or myc (Invitrogen), at either their carboxyl or amino termini and purified by one-step affinity columns. For example, polyhistidine binds to nickel with excellent affinity and specificity, and thus nickel affinity columns (such as Qiagen® nickel columns) can be used to purify polyhistidine-tagged selective binding agents. In some instances, two or more purification steps can be used.

[0173] TSLP Thymic stromal lymphopoietin, TSLP, is an epithelial cell-derived protein that belongs to a family of cytokines implicated in allergic inflammation (Cianferoni and Spergel 2014). It is produced primarily by non-hematopoietic cells such as fibroblasts, epithelial cells, and different epithelial-like cells, and plays a key role in the maturation of T cell populations by activation of antigen-presenting cells. TSLP is produced in response to pro-inflammatory stimuli and drives allergic inflammatory responses, mainly through its effects on dendritic cells for T helper (Th)2 cytokine production.

[0174] Human TSLP has two isoforms. The long form of TSLP, lfTSLP, is expressed in several tissues including the heart, liver and prostate, and induces the release of T cell-attracting chemokines from monocytes and enhances maturation of CD11c-dendritic cells. It can induce allergic inflammation by directly activating mast cells (Reche, Soumelis et al. 2001, Zhang and Zhou 2012). Isoform 2 is the short form of TSLP, sfTSLP, which is the predominant form in oral mucosa, skin keratinocytes and salivary glands and can function as an antimicrobial peptide (Bjerkan, Sonesson et al. 2016). Human TSLP maps to chromosome 5q22.1 (Quentmeier, Drexler et al. 2001).

[0175] The amino acid sequence of lfTSLP is provided as SEQ ID NO:30, of which the first 28 amino acids constitute the signal peptide and Y29 to Q159 are in the mature form. The amino acid sequence of sfTSLP is provided as SEQ ID NO:31.

[0176] Amino acid sequence for the long form of TSLP, lfTSLP (SEQ ID NO:30) [ka]

[0177] Amino acid sequence for short form TSLP, sfTSLP (SEQ ID NO:31) [ka]

[0178] The TSLP receptor (TSLPR) binds TSLP with low affinity and is a member of the hematopoietin receptor family (Park, Martin et al. 2000). The combination of the IL-7 receptor alpha chain (IL-7Rα) and the TSLPR is required for high affinity binding of TSLP to form a ternary complex (Verstraete, van Schie et al. 2014). The ternary complex is required for cell proliferation and signal transduction.

[0179] Following TSLP binding, STAT5 phosphorylation is induced, leading to expression of downstream transcription factors. Early results suggest that TSLP engagement in the TSLPR complex activates STAT5 without detectable JAK activation, but recent results demonstrate that JAK1 and JAK2, which bind to IL-7Rα and the TSLPR chain, respectively, are required for TSLP-mediated STAT5 activation (Rochman, Kashyap et al. 2010). Other signaling pathways stimulated by many cytokines, such as activation of ERK1, 2, and p70S6K, are not involved in TSLP activity (Quentmeier, Drexler et al. 2001).

[0180] The homology between mouse and human TSLP is 43% and 39% for TSLPR, with no cross-reactivity between species. Despite the poor amino acid sequence identity between mouse and human TSLP and between mouse and human TSLPR, the fact that high affinity binding of TSLP to its receptor in both species requires IL-7Rα suggests that human TSLP and TLPLR are orthologues of mouse TSLP and TSLPR. Thus, mouse models have been used for various biological studies, including the use of chimeric human TSLP and human TSLPR transgenic mouse lines (Francis, Milford et al. 2016).

[0181] TSLP expression is associated with many pathologies, including asthma (Ying, O'Connor et al. 2005), inflammatory arthritis (Koyama, Ozawa et al. 2007), inflammatory bowel disease (Park, Jeong et al. 2017), atopic dermatitis (Ebner, Nguyen et al. 2007), eczema, eosinophilic esophagitis and other allergic conditions (Soumelis and Liu 2004), (Soumelis, Reche et al. 2002). Understanding the mechanisms of TSLP production and potential agents that block production can enable better ways to prevent or treat these conditions. Thus, targeting TSLP can inhibit multiple biological pathways involved in asthma and Th2 cytokine-mediated inflammatory disorders (Gauvreau, O'Byrne et al. 2014).

[0182] Binding and functional activity of anti-TSLP antibodies The present disclosure encompasses anti-TSLP monoclonal antibodies or antibody fragments that selectively bind TSLP with greater affinity than to other antigens. The anti-TSLP monoclonal antibodies and fragments can selectively bind human TSLP, but also detectably bind non-human TSLP. Alternatively or additionally, the TSLP-binding antibodies and antibody fragments can have the same or substantially the same potency for recombinant human TSLP and endogenous human TSLP.

[0183] In vitro and cell-based assays are well described in the art for use in determining the binding of TSLP antibodies to their targets. For example, the binding of TSLP to its receptor can be determined by immobilizing a TSLP-binding antibody, sequestering TSLP with the immobilized antibody, determining whether TSLP is bound to the antibody, contacting a soluble form of the receptor with the bound TSLP / antibody complex, and determining whether the soluble receptor is bound to the complex. The protocol can also include a step of contacting the soluble receptor with the immobilized antibody prior to contact with the TSLP antibody to ensure that the soluble receptor does not bind to the immobilized antibody. This protocol can be performed using a Biacore® instrument for kinetic analysis of binding interactions. Such protocols can also be used to determine whether an antibody or other molecule allows or blocks the binding of TSLP to its receptor.

[0184] For other TSLP / receptor binding assays, the permissiveness or blocking of TSLP binding to its receptor can be determined by comparing the binding of TSLP to its receptor in the presence or absence of a TSLP antibody or a TSLP-binding fragment thereof. Blocking is identified in the assay readout as a specified reduction in TSLP binding to its receptor in the presence of an anti-TSLP antibody or a TSLP-binding fragment thereof, compared to a control sample containing the corresponding buffer or diluent but not the TSLP antibody or a TSLP-binding fragment thereof. The assay readout can be viewed qualitatively as an indication of the presence or absence of blocking, or can be viewed quantitatively as an indication of the percent or fold reduction in binding due to the presence of the antibody or fragment. If a TSLP-binding antibody or TSLP-binding fragment substantially blocks TSLP binding to its receptor, then TSLP binding to its receptor is reduced by at least 10-fold, alternatively at least about 20-fold, alternatively at least about 50-fold, alternatively at least about 100-fold, alternatively at least about 1000-fold, alternatively at least about 10,000-fold or even more compared to binding of the same concentration of TSLP to its receptor in the absence of the antibody or fragment.

[0185] Preferred anti-TSLP antibodies for use in accordance with the present disclosure generally bind human TSLP with high affinity (e.g., as determined by BIACORE), e.g., an equilibrium binding dissociation constant (KD) for TSLP of about 10 nM or less, about 5 nM or less, about 1 nM or less, about 500 pM or less, or more preferably about 250 pM or less, about 100 pM or less, about 50 pM or less, about 25 pM or less, about 10 pM or less, about 5 pM or less, about 3 pM or less, about 1 pM or less, about 0.75 pM or less, about 0.5 pM or less, or about 0.3 pM or less.

[0186] The antibodies or fragments of the disclosure can bind to TSLP with an EC50 of about 10 nM or less, about 5 nM or less, about 2 nM or less, about 1 nM or less, about 0.75 nM or less, about 0.5 nM or less, about 0.4 nM or less, about 0.3 nM or less, or even about 0.2 nM or less, as determined, for example, by enzyme-linked immunosorbent assay (ELISA).

[0187] Preferably, the antibodies or antibody fragments of the present disclosure do not cross-react with any targets other than TSLP. For example, the present antibodies and fragments can bind to TSLP but do not detectably bind to other proteins, or have at least about 100-fold (e.g., at least about 150-fold, at least about 200-fold, or even at least about 250-fold) greater selectivity in their binding to TSLP compared to their binding to other proteins.

[0188] The critical amino acid residues (epitopes) bound by the TSLP-binding antibodies or fragments described in this disclosure can be determined using peptide arrays, such as, for example, PepSpot™ peptide arrays (JPT Peptide Technologies, Berlin, Germany), in which a scan of 12 amino acid peptides spanning the entire TSLP amino acid sequence, with each peptide overlapping the previous one by 11 amino acids, are synthesized directly on a membrane. Alternatively or additionally, antibody competition experiments can be performed and such assays are well known in the art.

[0189] The present disclosure also encompasses neutralizing antibodies or neutralizing fragments thereof that bind to and neutralize the biological activity of TSLP. Neutralization of TSLP biological activity can be assessed by assaying one or more indicators of biological activity, such as TSLP-stimulated reporter gene expression in a reporter assay. Neutralization of TSLP biological activity can also be assessed in vivo by animal models of asthma or atopic dermatitis. Preferably, the TSLP-binding antibodies and fragments of the present disclosure neutralize the biological activity of TSLP that leads to the signaling function of its receptor bound by TSLP.

[0190] The antibody or fragment may be a neutralizing antibody or fragment that specifically binds to a TSLP epitope that affects the biological activity of TSLP. The antibody or fragment may bind to a neutralization-sensitive epitope of TSLP. When a neutralization-sensitive epitope of TSLP is bound by one of the antibodies or fragments, the result is a loss of biological activity that contains the epitope.

[0191] In certain embodiments, the antibodies or fragments thereof of the present disclosure can neutralize, inhibit, block, abrogate, reduce or interfere with the activity of TSLP by binding to an epitope of TSLP that is directly involved in the targeted activity of TSLP. In another embodiment, the antibodies or fragments thereof of the present disclosure can neutralize, inhibit, block, abrogate, reduce or interfere with the activity of TSLP by binding to an epitope of TSLP that is not directly involved in the targeted activity of TSLP, but the antibodies or fragments which bind thereto sterically or conformationally inhibit, block, abrogate, reduce or interfere with the targeted activity of TSLP. In yet another embodiment, the antibodies or fragments thereof of the present disclosure can bind to an epitope of TSLP that is not directly involved in the targeted activity of TSLP (i.e., a non-blocking antibody), but the antibodies or fragments which bind thereto result in enhanced clearance of TSLP.

[0192] In certain embodiments, the antibody or antigen-binding fragment thereof a) inhibits binding of TSLP and TSLPR; b) reduces expression levels of TSLP-dependent genes; c) inhibits TSLP-induced cell proliferation; d) inhibits TSLP-induced STAT5 activation; and / or e) inhibits TSLP-induced CCL17 production from primary human dendritic cells.

[0193] Pharmaceutical Compositions The TSLP-binding antibodies and antibody fragments for use according to the present disclosure can be formulated in compositions, particularly pharmaceutical compositions, for use in the methods herein. Such compositions contain a therapeutically or prophylactically effective amount of the TSLP-binding antibodies or antibody fragments of the present disclosure in a mixture with a suitable carrier, such as a pharma- ceutically acceptable agent. Typically, the TSLP-binding antibodies and antibody fragments of the present disclosure are sufficiently purified for administration to animals before formulation in a pharmaceutical composition.

[0194] Pharmaceutically acceptable agents include carriers, excipients, diluents, antioxidants, preservatives, colorants, flavorings and diluting agents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, tonicity agents, co-solvents, wetting agents, complexing agents, buffering agents, antimicrobials and surfactants.

[0195] The compositions may be in liquid form or in lyophilized or freeze-dried form and may contain one or more lyoprotectants, excipients, surfactants, high molecular weight structural additives and / or bulking agents.

[0196] The composition can be suitable for parenteral administration. Exemplary compositions are suitable for injection or infusion into animals by any route available to those skilled in the art, such as intra-articular, subcutaneous, intravenous, intramuscular, intraperitoneal, intracerebral (intracemall), intraventricular, intramuscular, intraocular, intraarterial, intralesional, intrarectal, transdermal, oral and inhalation routes.

[0197] The pharmaceutical compositions described herein can be formulated for controlled or sustained delivery in a manner that results in local concentration of the product (e.g., bolus, depot effect), sustained release and / or increased stability or half-life in a particular local environment.

[0198] How to use The antibodies and fragments are useful in the prevention and treatment of TSLP-mediated diseases or medical conditions, such as asthma, allergic rhinitis, atopic dermatitis, allergies, and certain cancers.

[0199] One aspect of the present disclosure provides a method of treating moderate to severe uncontrolled asthma, including asthma that is not Th2-driven, by administering to a subject in need thereof a therapeutically effective amount of any one of the subject antibodies or antigen-binding fragments thereof, and the subject pharmaceutical compositions.

[0200] Another aspect of the present disclosure provides methods of treating fibrotic diseases and related conditions, including systemic sclerosis, systemic idiopathic pulmonary fibrosis, and keloid diseases, by administering to a subject in need thereof a therapeutically effective amount of any one of the subject antibodies or antigen-binding fragments thereof, and the subject pharmaceutical compositions.

[0201] Another aspect of the present disclosure provides methods of treating chronic obstructive pulmonary disease, such as emphysema, chronic bronchitis, and refractory asthma, by administering to a subject in need thereof a therapeutically effective amount of any one of the subject antibodies or antigen-binding fragments thereof, and the subject pharmaceutical compositions.

[0202] Another aspect of the present disclosure provides methods of treating disorders associated with inflammatory diseases, including rhinitis, atopic dermatitis, eczema, food allergies and inflammatory bowel disease, comprising administering to a subject in need thereof a therapeutically effective amount of any one of a subject antibody or antigen-binding fragment thereof, and a subject pharmaceutical composition.

[0203] Another aspect of the present disclosure provides methods of treating certain cancers, including lung cancer, breast cancer, pancreatic cancer, colorectal cancer, lymphoblastic leukemia, and head and neck cancer, comprising administering to a subject in need thereof a therapeutically effective amount of any one of the subject antibodies or antigen-binding fragments thereof, and the subject pharmaceutical compositions.

[0204] Certain aspects of the present disclosure also provide for the use of any of the antibodies or antigen-binding fragments thereof provided herein in the manufacture of a medicament for blocking the binding of TSLP to its receptor TLPR and / or for the prevention and treatment of a TSLP-mediated disease or medical condition, such as those provided herein. Certain aspects of the present disclosure also provide for the use of the antibodies or antigen-binding fragments thereof provided herein in a method for blocking the binding of TSLP to its receptor TLPR and / or for the prevention and treatment of a TSLP-mediated disease or medical condition, such as those provided herein. For example, in embodiments, the present disclosure provides for the use of the antibodies provided herein in a method for treating asthma, COPD, idiopathic pulmonary fibrosis, atopic dermatitis, eczema, eosinophilic esophagitis, inflammatory bowel disease, systemic sclerosis, systemic idiopathic pulmonary fibrosis, keloid disease, or cancer.

[0205] In addition to therapeutic uses, the present antibodies and fragments can be used in diagnostic methods to detect TSLP (e.g., in biological samples such as serum or plasma) using conventional immunoassays such as enzyme-linked immunosorbent assay (Rizos CV), radioimmunoassay (RIA) or tissue immunohistochemistry.

[0206] Methods for detecting TSLP in a biological sample can include contacting the biological sample with one or more of the present antibodies or fragments and detecting either the antibody or fragment that is bound to TSLP or the antibody or fragment that is not bound, thereby detecting TSLP in the biological sample. The antibody or fragment can be directly or indirectly labeled with a detectable substance to facilitate detection of the bound or unbound antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials. EXAMPLES

[0207] The following examples are provided to further describe the present disclosure and are intended to be illustrative and not limiting of the disclosure.

[0208] Example 1 TSLP binding affinity for mouse anti-TSLP antibody TAVO202 TAVO202 was identified as a mouse anti-human TSLP antibody by hybridoma screening. An ELISA-based binding assay was used to assess TAVO202 binding to recombinant human TSLP. In this assay, 1 μg / mL recombinant human TSLP (R&D Systems) was coated onto an ELISA plate. Increasing concentrations of TAVO202 antibody were added to the plate, and its binding to recombinant human TSLP was detected by an HRP-conjugated anti-mouse secondary antibody. It was observed that TAVO202 bound to recombinant human TSLP in a dose-dependent manner with an EC50 at 2.4 ng / mL (Figure 1A).

[0209] We also assessed the binding of TAVO202 to mouse TSLP in a similar ELISA assay by coating plates with mouse TSLP (R&D Systems). TAVO202 showed no significant binding affinity for mouse TSLP (Figure 1B).

[0210] Example 2 In vitro assay for TSLP neutralization by TAVO202 A TSLP-mediated cell proliferation assay was developed to evaluate the functional activity of TAVO202. In this assay, human IL-7 receptor alpha (IL-7Rα) and TSLP receptor (TSLPR) were co-transfected into BAF3 mouse pro-B cells. Recombinant human TSLP was added to the transfected cells, and cell proliferation was quantified two days later by cell proliferation assay. It was observed that recombinant human TSLP was able to stimulate proliferation of the transfected BAF3 cell line in a dose-dependent manner with an EC50 at 0.18 ng / mL (Figure 2A).

[0211] To assess the functional activity of TAVO202, increasing concentrations of TAVO202 were added together with 0.5 ng / mL human TSLP to BAF3 cells co-transfected with IL-7Rα and TSLPR. It was observed that TAVO202 was able to dose-dependently neutralize TSLP activity in stimulating transfected BAF3 cell proliferation with an IC50 of 6.6 ng / mL (Figure 2B).

[0212] Besides the cell proliferation assay, a TSLP-driven reporter gene expression assay was also developed to evaluate the functional activity of TAVO202. STAT5 activation is a downstream event that occurs after TSLP binds to and activates its receptor complex (TSLPR:IL-7Rα). In this assay, HEK293T cells were transiently transfected with plasmids expressing TSLPR, IL-7Rα, and a STAT5 luciferase reporter construct. One day after transfection, recombinant human TSLP was added, and TSLP-driven luciferase reporter gene expression was quantified 24 hours later. It was observed that human TSLP was able to drive reporter gene expression in a dose-dependent manner with an EC50 at 1.4 ng / mL (Figure 3A).

[0213] To assess the functional activity of TAVO202, increasing concentrations of TAVO202 were added together with 10 ng / mL human TSLP to HEK293T cells co-transfected with IL-7Rα, TSLPR and STAT5 luciferase reporter genes. It was observed that TAVO202 dose-dependently neutralized TSLP activity in stimulating reporter gene expression with an IC50 of 20 ng / mL (Figure 3B).

[0214] Example 3 Humanization of the mouse anti-human TSLP antibody TAVO202 The murine anti-human TSLP antibody TAVO202 was humanized by grafting the murine CDRs onto a human germline scaffold. Several key murine residues were preserved by backmutation to achieve higher stability and better expression while minimizing immunogenicity. * 1 humanized V based on 02 H A variant (202H2) was designed and constructed to have two reversion mutations, IGKV1-9 * 01 and IGKV6-21 * Based on 01, two humanized V L Two humanized variants were designed (Figure 4). L Variants and humanized V HBy combining variants, two humanized TAVO202 antibodies with IgG2 Fc, designated TAVO6264 and TAVO6265, were generated with the 202H2 variant paired with 202L3 and 202L4, respectively (Figure 4). Similarly, two humanized TAVO202 antibodies with IgG1 Fc with L234F, L235E, D265A, F405L mutations, designated TAVO7264 and TAVO7265, were also generated with the 202H2 variant paired with 202L3 and 202L4, respectively. Two other humanized TAVO202 antibodies, designated TAVO9764 and TAVO9765, with IgG1 Fc bearing L234A, L235A, M428L, N434S mutations, were also generated with the 202H2 variant paired with 202L3 and 202L4, respectively (Figure 4).

[0215] Example 4 Expression and purification of humanized TAVO202 Plasmids encoding the heavy and light chains of TAVO6264, TAVO6265, TAVO7264, TAVO7265, TAVO9764, and TAVO9765 were co-transfected into Expi293F cells according to the transfection kit instructions (Thermo Scientific). Five days after transfection, cells were spun down and the supernatant was passed through a 0.2 μm filter. Purification of the expressed antibodies in the supernatant was carried out by affinity chromatography on a Protein A agarose column (GE Healthcare Life Sciences). The purified antibodies were buffer exchanged into DPBS, pH 7.2 by dialysis, and protein concentration was determined by UV absorbance at 280 nm.

[0216] The purified humanized TAVO202 antibodies were subjected to SDS-PAGE analysis, and examples of TAVO7264 and TAVO7265 are shown in Figures 5A and 5B. Under reducing conditions, all four antibodies showed heavy and light chains with the expected molecular weights. Under non-reducing conditions, all four antibodies migrated as a major protein band with a molecular weight of around 150 kDa. The purified humanized TAVO202 antibodies were also evaluated by size exclusion chromatography (SEC). Figure 5C revealed the SEC profiles of the example antibodies TAVO7264 and TAVO7265.

[0217] Example 5 Binding to TSLP by humanized TAVO202 An ELISA-based binding assay was used to evaluate the humanized TAVO202 antibody binding to recombinant human TSLP antigen. In this assay, 1 μg / mL recombinant human TSLP (R&D Systems) was coated onto an ELISA plate. Increasing concentrations of humanized TAVO202 antibody were added to the plate, and its binding to recombinant human TSLP was detected by an HRP-conjugated anti-mouse secondary antibody. As an example, it was observed that TAVO6264 and TAVO6265 bound to recombinant human TSLP in a dose-dependent manner with comparable potency similar to the mouse antibody TAVO202 (Figure 6A).

[0218] We also assessed binding of the humanized TAVO202 antibody to cynomolgus monkey TSLP in a similar ELISA assay by coating plates with cynomolgus monkey TSLP. Neither the TAVO7264 nor the TAVO7265 antibodies were able to bind to monkey TSLP (FIG. 6B).

[0219] To further measure the binding of the humanized antibodies to immobilized recombinant TSLP, a surface plasmon resonance (SPR) binding assay will be performed using Biacore. This assay can measure not only the binding affinity, but also the kinetic rate constants and thermodynamics of binding.

[0220] Example 6 In vitro assay for TSLP neutralization by humanized TAVO202 antibody The functional activity of the humanized TAVO202 antibody was evaluated using a TSLP-driven STAT5 luciferase reporter gene expression assay. Increasing concentrations of TAVO6264 and TAVO6265 were added along with 10 ng / mL human TSLP to HEK293T cells co-transfected with IL-7Rα, TSLPR and STAT5 luciferase reporter genes. It was observed that both TAVO6264 and TAVO6265 were able to dose-dependently neutralize TSLP activity in stimulating reporter gene expression with IC50s comparable to TAVO202 (Figure 7).

[0221] Example 7 Inhibition of TSLP-mediated dendritic cell activation by humanized TAVO202 TSLP inhibits primary human CD1c T cells by increasing secretion of the CCL17 chemokine. + Anti-CD1c can stimulate the activation of blood dendritic cells. An ex vivo assay was set up to evaluate the humanized TAVO202 antibody in neutralizing TSLP-driven CCL17 release from activated dendritic cells. + CD1c antibodies were isolated from donor peripheral blood mononuclear cells (PBMCs) by antibody-coated magnetic bead selection (Miltenyi Biotec). + Dendritic cells were isolated. The isolated cells were incubated with recombinant human TSLP and the release of CCL17 was quantified by ELISA. It was observed that 15ng / mL recombinant human TSLP could stimulate several-fold enhanced CCL17 release from activated dendritic cells after 48 hours of incubation. Both TAVO7264 and TAVO7265 could block TSLP-driven CCL17 release from activated dendritic cells when added at 1ug / mL with TSLP. Figure 8 showed the results of the assay with PBMCs isolated from two different donors.

[0222] Example 8 F of humanized antibodies for extended half-life and reduced effector function c operation To improve the PK profile of humanized antibodies, c Mutations can be introduced into IgG1 antibodies to extend antibody half-life. Specifically, the M428L / N434S mutations were demonstrated to extend antibody half-life by increasing FcRn binding affinity (Booth, Ramakrishnan et al. 2018). Additionally, the L234A / L235A F c Mutations can abolish ADCC and CDC effector functions of IgG1 antibodies (Hezareh, Hessell et al. 2001). Therefore, a humanized IgG1 heavy chain based on 202H1 with L234A / L235A / M428L / N434S (AALS) mutations was generated, comprising the sequence shown as SEQ ID NO: 16. By pairing this Fc-engineered 202H1 heavy chain with either humanized 202L3 or 202L4 light chain, two humanized TAVO202 antibodies with extended half-life and reduced effector functions were generated, designated TAVO9764 and TAVO9765, respectively (Figure 4).

[0223] F c To study whether the engineered antibodies have improved FcRn binding affinity, binding by TAVO9765 and TAVO7265 to mouse FcRn was evaluated in an ELISA-based binding assay. 1 μg / mL recombinant mouse FcRn (R&D Systems) was coated onto an ELISA plate. Increasing concentrations of TAVO9765 and TAVO7265 antibodies were added to the plate, and their binding to recombinant FcRn at pH 6.0 was detected by an HRP-conjugated anti-human secondary antibody. It was observed that TAVO9765, which has Fc M428L / N434S mutations, can bind to FcRn with approximately 10-fold greater potency than TAVO7265, which lacks such half-life extending mutations ( FIG. 9 ).

[0224] TAVO9765 was tested in a cynomolgus monkey PK model to determine whether the M428L / N434S mutations could extend the circulating half-life of anti-TSLP antibodies. TAVO9765 was administered to male naive cynomolgus monkeys as an intravenous infusion of 4 mg / kg in a volume of 1.0 mL / kg based on body weight on day 0 for 3 minutes. Whole blood was collected into EDTA-K2 collection tubes prior to dosing and at 1 hour, 2 hours and days 2, 5, 9, 12, 16, 21, 24, 26, 29, 32, 35 after dosing. Plasma was separated by centrifugation at 3500×g for 10 minutes at 4° C. and then transferred to microcentrifuge tubes for storage at −80° C. Plasma samples were measured by standard ELISA methods to detect human IgG. Goat anti-human IgG was the capture antibody and HRP-conjugated goat anti-human IgG (H+L) was the detection antibody. The dilution factors of capture and detection antibodies were 13,333 and 30,000, respectively. PK data were analyzed using Winnonlin 6.4 software as half-life; 0 The PK profile of plasma concentration versus time of TAVO9765 is shown in Figure 10. The PK parameters of TAVO9765 were as follows: 0 :131405ng mL -1 , Half-life: 26.131 days, AUC 0-t :1268621ng·day·mL -1 , AUC 0-inf :2128728ng·day·mL -1 , Clearance: 1.879 mL kg -1 ·day -1 , Vd: 71.049 mL kg -1 .

[0225] References [ka] [ka] [ka] [ka]

change

change

change

change

change

Claims

1. An isolated antibody or antigen-binding fragment thereof that specifically binds to thymic stromal lymphopoietin (TSLP) and neutralizes, inhibits, blocks, abrogates, reduces or interferes with the activity of TSLP, the antibody or antigen-binding fragment thereof comprising heavy chain complementarity determining region (HCDR) sequences HCDR1, HCDR2, HCDR3 of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, respectively, and light chain complementarity determining region (LCDR) sequences LCDR1, LCDR2, LCDR3 of SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, respectively.

2. a) a murine monoclonal antibody or antibody fragment thereof having a heavy chain variable region shown as SEQ ID NO:1 and a light chain variable region shown as SEQ ID NO:2; or b) a humanized antibody or antibody fragment thereof comprising a heavy chain variable region set forth as SEQ ID NO: 9 or having at least 95% sequence identity to SEQ ID NO: 9, and a light chain variable region set forth as SEQ ID NO: 10 or SEQ ID NO: 11 or having at least 95% sequence identity to SEQ ID NO: 10 or SEQ ID NO: 11; The antibody or antigen-binding fragment thereof described in claim 1.

3. The antibody or antigen-binding fragment thereof according to claim 2, wherein the humanized antibody comprises a human IgG heavy chain sequence shown as any one of SEQ ID NOs: 12 or 15-16.

4. The antibody or antigen-binding fragment thereof described in claim 2, wherein the humanized antibody comprises a light chain sequence shown as any one of SEQ ID NOs: 13 to 14.

5. The antibody is IgG １ , IgG ２ , IgG ３ or IgG ４ The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which is an isotype.

6. The antibody, a) increasing the half-life of the engineered antibody compared to the parent wild-type antibody; b) enhances the resistance of the engineered antibody to proteolysis by proteases compared to the parent wild-type antibody; or c) reducing or eliminating effector functions of the engineered antibody compared to the parent wild-type antibody; One or more F ｃ The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which has a mutation.

7. L234A, L235A, M428L and N434S F, which antibodies extend the half-life and reduce effector function of the engineered antibody compared to the parent wild-type antibody. ｃ An antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, having a mutation and wherein residue numbering is according to the EU index.

8. 5. The antibody or antigen-binding fragment thereof of claim 4, wherein the humanized antibody or fragment comprises a human IgG1 heavy chain having the following Fc mutations: L234A, L235A, M428L, N434S, as shown in SEQ ID NO:

16.

9. a) blocks the binding of human TSLP to its receptor complex; b) neutralizing, reducing or interfering with the functional activity of TSLP on its receptor complex; c) neutralize TSLP-driven reporter gene activation in a STAT5 reporter gene assay; d) neutralizing TSLP-driven proliferation of cells expressing the TSLP receptor complex in a cell proliferation assay; e) inhibiting TSLP-driven cytokine release from human dendritic cells in a cytokine release assay; or f) inhibiting TSLP-driven asthma and pulmonary inflammation in animal asthma models; The antibody or antigen-binding fragment thereof according to claims 1 to 8.

10. An isolated polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

11. A vector comprising the polynucleotide of claim 10, optionally an expression vector.

12. A host cell comprising the vector of claim 11.

13. 13. A method for producing an antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, comprising culturing a host cell according to claim 12 under conditions in which the antibody or antigen-binding fragment thereof is expressed, and isolating the antibody or antigen-binding fragment thereof.

14. 10. A composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 9 for use in a method for treating a TSLP mediated disease or disorder in a subject in need thereof.

15. The TSLP-mediated disease or disorder is a) moderate to severe asthma, including uncontrolled asthma; b) optionally, fibrotic diseases and associated conditions selected from systemic sclerosis, systemic idiopathic pulmonary fibrosis and keloid diseases; c) Chronic obstructive pulmonary disease such as emphysema, chronic bronchitis, and intractable asthma. d) optionally an inflammatory disease selected from rhinitis, atopic dermatitis, eczema, systemic sclerosis, food allergy and inflammatory bowel disease, or e) optionally, the cancer is selected from lung cancer, breast cancer, pancreatic cancer, colorectal cancer, lymphoblastic leukemia, and head and neck cancer; A composition for use according to claim 14.

16. 16. The composition for use according to any one of claims 14 to 15, administered to the subject in need of treatment in combination with a second agent, optionally selected from the group consisting of corticosteroids, bronchodilators, antihistamines, anti-leukotrienes, PDE-4 inhibitors; anti-cancer drugs, immunomodulatory drugs and cytokine therapy drugs.

Images