Novel anti-TSLP antibodies

Abstract

Provided herein are anti-TSLP antibodies or antigen-binding fragments thereof, isolated polynucleotides encoding them, pharmaceutical compositions comprising them, and uses thereof.

Description

[Technical field]

[0001]

[0001] The present disclosure relates generally to novel anti-TSLP antibodies. [Background technology]

[0002]

[0002] Thymic stromal lymphopoietin (TSLP) is a protein belonging to the cytokine family. It is known to play an important role in the maturation of T cell populations through the activation of antigen-presenting cells. TSLP is produced primarily by non-hematopoietic cells, such as fibroblasts, epithelial cells, and various types of stromal or stromal-like cells.

[0003] TSLP is a multifunctional cytokine that can exert biological functions through the TSLP / IL-7Rα receptor on the surface of various immune cells, including DC cells, CD4 and CD8+ T cells, B cells, ILC2, sensory neurons, mast cells, basophils, eosinophils, and NKT cells. TSLP transmits signals through the JAK / STAT (JAK kinase-signal transducer and activator of transcription) pathway. TSLP binds to TSLPR on the cell membrane and then binds to IL-7Rα to form a stable TSLP-TSLPR-IL7Rα complex. The intracellular segment of the TSLPR receptor in the complex recruits and activates JAK2, which acts together with JAK1 recruited by IL7Rα to activate downstream signal molecules. Studies have shown that in human peripheral blood-derived CD11c+ DC cells, TSLP can activate STAT1, STAT3, STAT4, STAT5, and STAT6, and in particular, the STAT5 activation signal is important for promoting Th2 cell differentiation and secreting Th2 factors.

[0004]

[0004] TSLP is known to regulate type 2 inflammation through activation of dendritic cells, mast cells, and ILC2s, and is thus closely associated with the development of type II inflammatory diseases. Summary of the Invention [Problem to be solved by the invention]

[0005]

[0005] Therefore, blocking TSLP signaling may be an effective strategy for the treatment of asthma, allergic dermatitis, and other diseases associated with TSLP signaling. However, TSLP-related diseases have a high unmet medical need. Thus, there remains a need for novel anti-TSLP antibodies. [Means for solving the problem]

[0006] Summary of the Invention

[0006] Throughout this disclosure, the articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an antibody" means one antibody or more than one antibody.

[0007]

[0007] In one respect, the present disclosure provides an antibody or antigen-binding fragment thereof capable of specifically binding to human TSLP, the antibody or antigen-binding fragment thereof comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2 and HCDR3 contained within any one of the heavy chain variable region sequences of SEQ ID NO:1 or 3, and / or light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3 contained within any one of the heavy chain variable region sequences of SEQ ID NO:2 or 4.

[0008] In one aspect, the disclosure relates to an antibody or antigen-binding fragment thereof capable of specifically binding to human TSLP, comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, wherein (a) HCDR1 comprises the amino acid sequence of SEQ ID NO:11, HCDR2 comprises the amino acid sequence of IFPGDGX1T (SEQ ID NO:59), and HCDR3 comprises the amino acid sequence of ARX2GX3X4X5X6X7X8YAMDY (SEQ ID NO:60), wherein X1 is E or D, X2 is G or S, X3 is Y or F, X4 is V or D, X5 is N or Y, X6 is absent or G, X7 is F or Y, and X8 is L or F; or (b) HCDR1 comprises the amino acid sequence of SYWX9N (SEQ ID NO:61), and HCDR2 comprises the amino acid sequence of QIFPGDGX 10 TX 11 YNGX 12 and HCDR3 comprises the amino acid sequence of X 13 GX 14 X 15 X 16 X 17 X 18 X 19 X9 is M or I; 10 is E or D, and X 11 is N or T, and X 12 is K or N, and X 13 is G or S, and X 14 is Y or F, and X 15 is V or D, and X 16 is N or Y, and X 17 does not exist or is G, and X 18 is F or Y, and X 19 is L or F.

[0009] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain complementarity determining region 1 (LCDR1), an LCDR2 and an LCDR3, wherein (a) LCDR1 comprises the amino acid sequence of SEQ ID NO: 14, LCDR2 comprises the amino acid sequence of SEQ ID NO: 15, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 16, or (b) LCDR1 comprises the amino acid sequence of SEQ ID NO: 53 and LCDR2 comprises GTSX 20 LAS (SEQ ID NO: 64), and LCDR3 comprises the amino acid sequence of SEQ ID NO: 16, 20 is T or N.

[0010] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3, and / or a light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein (a) HCDR1 comprises the amino acid sequence of SEQ ID NO: 11, HCDR2 comprises the amino acid sequence of IFPGDGX1T (SEQ ID NO: 59), and HCDR3 comprises the amino acid sequence of ARX2GX3X4X5X6X7X8YAMDY (SEQ ID NO: 60), and LCDR1 comprises the amino acid sequence of SEQ ID NO: 1 X1 is E or D, X2 is G or S, X3 is Y or F, X4 is V or D, X5 is N or Y, X6 is absent or G, X7 is F or Y, and X8 is L or F; or (b) HCDR1 comprises the amino acid sequence of SYWX9N (SEQ ID NO: 61) and HCDR2 comprises the amino acid sequence of QIFPGDGX, 10 TX 11 YNGX 12 and HCDR3 comprises the amino acid sequence of X 13 GX 14 X 15 X 16 X 17 X 18 X 19 LCDR1 comprises the amino acid sequence of SEQ ID NO:53; LCDR2 comprises the amino acid sequence of GTSX20 LAS (SEQ ID NO: 64), and LCDR3 comprises the amino acid sequence of SEQ ID NO: 16, X9 is M or I, and X 10 is E or D, and X 11 is N or T, and X 12 is K or N, and X 13 is G or S, and X 14 is Y or F, and X 15 is V or D, and X 16 is N or Y, and X 17 does not exist or is G, and X 18 is F or Y, and X 19 is L or F, and X 20 is T or N.

[0011]

[0011] In some embodiments, HCDR1 comprises the amino acid sequence of SEQ ID NO: 11, HCDR2 comprises the amino acid sequence of SEQ ID NO: 12 or 25, HCDR3 comprises the amino acid sequence of SEQ ID NO: 13 or 26, LCDR1 comprises the amino acid sequence of SEQ ID NO: 14, LCDR2 comprises the amino acid sequence of SEQ ID NO: 15, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 16.

[0012]

[0012] In some embodiments, HCDR1 comprises the amino acid sequence of SEQ ID NO: 50 or 55, HCDR2 comprises the amino acid sequence of SEQ ID NO: 51 or 56, HCDR3 comprises the amino acid sequence of SEQ ID NO: 52 or 57, LCDR1 comprises the amino acid sequence of SEQ ID NO: 53, LCDR2 comprises the amino acid sequence of SEQ ID NO: 54 or 58, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 16.

[0013] In some embodiments, (a) HCDR1 comprises the amino acid sequence of SEQ ID NO:11, HCDR2 comprises the amino acid sequence of SEQ ID NO:12, and HCDR3 comprises the amino acid sequence of SEQ ID NO:13, LCDR1 comprises the amino acid sequence of SEQ ID NO:14, LCDR2 comprises the amino acid sequence of SEQ ID NO:15, and LCDR3 comprises the amino acid sequence of SEQ ID NO:16; (b) HCDR1 comprises the amino acid sequence of SEQ ID NO:11, HCDR2 comprises the amino acid sequence of SEQ ID NO:25, and HCDR3 comprises the amino acid sequence of SEQ ID NO:26, LCDR1 comprises the amino acid sequence of SEQ ID NO:14, LCDR2 comprises the amino acid sequence of SEQ ID NO:15, and LCDR3 comprises the amino acid sequence of SEQ ID NO:16. (c) HCDR1 comprises the amino acid sequence of SEQ ID NO:50, HCDR2 comprises the amino acid sequence of SEQ ID NO:51, HCDR3 comprises the amino acid sequence of SEQ ID NO:52, LCDR1 comprises the amino acid sequence of SEQ ID NO:53, LCDR2 comprises the amino acid sequence of SEQ ID NO:54, and LCDR3 comprises the amino acid sequence of SEQ ID NO:16; (d) HCDR1 comprises the amino acid sequence of SEQ ID NO:55, HCDR2 comprises the amino acid sequence of SEQ ID NO:56, HCDR3 comprises the amino acid sequence of SEQ ID NO:57, LCDR1 comprises the amino acid sequence of SEQ ID NO:53, LCDR2 comprises the amino acid sequence of SEQ ID NO:58, and LCDR3 comprises the amino acid sequence of SEQ ID NO:16.

[0014] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein further comprise one or more of heavy chain framework region 1 (HFR1), HFR2, HFR3, and HFR4, and / or one or more of light chain framework region 1 (LFR1), LFR2, LFR3, and LFR4, wherein HFR1 is selected from the group consisting of SEQ ID NO:17, SEQ ID NO:27, or X. 21 VQLVQSGAEVKKPGX 22 SX 23 KX 24 SCKX 25 S (SEQ ID NO: 65) or a homologous sequence having at least 85% sequence identity thereto, and HFR2 comprises SEQ ID NO: 18 or WVRQX 27 PGX 28GLEWMG (SEQ ID NO: 66) or a homologous sequence having at least 85% sequence identity thereto; and HFR3 is SEQ ID NO: 19, SEQ ID NO: 28, or X 31 VTX 32 X 33 X 34 DX 35 SX 36 STX 37 YX 38 X 39 X 40 SSLX 41 X 42 X 43 DTAX 44 YYC (SEQ ID NO:67) or a homologous sequence having at least 85% sequence identity thereto, HFR4 comprises the amino acid sequence of SEQ ID NO:20 or SEQ ID NO:35 or a homologous sequence having at least 85% sequence identity thereto, LFR1 comprises the amino acid sequence of SEQ ID NO:21, SEQ ID NO:29, or SEQ ID NO:36 or a homologous sequence having at least 85% sequence identity thereto, and LFR2 comprises SEQ ID NO:22, SEQ ID NO:30, or WYQQKPGQSPRPWIX. 45 (SEQ ID NO:68) or a homologous sequence having at least 85% sequence identity thereto, LFR3 comprises the amino acid sequence of SEQ ID NO:23, SEQ ID NO:31, or SEQ ID NO:38 or a homologous sequence having at least 85% sequence identity thereto, and LFR4 comprises the amino acid sequence of SEQ ID NO:24 or SEQ ID NO:39 or a homologous sequence having at least 85% sequence identity thereto, 21 is E or Q, and X 22 is E, S, or A, and X 23 is L or V, and X 24 is I or V, and X 25 is S or A, and X 27 is M or A, and X 28 is K or Q, and X 31 is Q or R, and X 32 is I or M, and X 33 is S or T, and X 34 is A or R, and X 35 is K or T, and X 36 is I or T, and X37 is A or V, and X 38 is L or M, and X 39 is Q or E, and X 40 is W or L, and X 41 is K or R, and X 42 is A or S, and X 43 is S or E, and X 44 is M or V, and X 45 is Y or F.

[0015]

[0015] In some embodiments, HFR1 comprises a sequence selected from the group consisting of SEQ ID NOs: 17, 27, 32, 40, 42, 47, and 87, HFR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 18, 33, 43, and 48, HFR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 19, 28, 34, 41, 44, and 49, HFR4 comprises a sequence of SEQ ID NOs: 20 and 35, LFR1 comprises a sequence of SEQ ID NOs: 21, 29, and 36, LFR2 comprises a sequence selected from the group consisting of SEQ ID NOs: 22, 30, 37, and 45, LFR3 comprises a sequence selected from the group consisting of SEQ ID NOs: 23, 31, and 38, and LFR4 comprises a sequence selected from the group consisting of SEQ ID NOs: 24 and 39.

[0016] In some embodiments, (a) HFR1 comprises the amino acid sequence of SEQ ID NO:17, HFR2 comprises the amino acid sequence of SEQ ID NO:18, HFR3 comprises the amino acid sequence of SEQ ID NO:19, HFR4 comprises the amino acid sequence of SEQ ID NO:20, LFR1 comprises the amino acid sequence of SEQ ID NO:21, LFR2 comprises the amino acid sequence of SEQ ID NO:22, LFR3 comprises the amino acid sequence of SEQ ID NO:23, and LFR4 comprises the amino acid sequence of SEQ ID NO:24; or (b) HFR1 comprises the amino acid sequence of SEQ ID NO:27, HFR2 comprises the amino acid sequence of SEQ ID NO:18, HFR3 comprises the amino acid sequence of SEQ ID NO:25, and LFR4 comprises the amino acid sequence of SEQ ID NO:26. comprises the amino acid sequence of SEQ ID NO:28, HFR4 comprises the amino acid sequence of SEQ ID NO:20, LFR1 comprises the amino acid sequence of SEQ ID NO:29, LFR2 comprises the amino acid sequence of SEQ ID NO:30, LFR3 comprises the amino acid sequence of SEQ ID NO:31, and LFR4 comprises the amino acid sequence of SEQ ID NO:24, or (c) HFR1 comprises the amino acid sequence of SEQ ID NO:32, HFR2 comprises the amino acid sequence of SEQ ID NO:33, HFR3 comprises the amino acid sequence of SEQ ID NO:34, HFR4 comprises the amino acid sequence of SEQ ID NO:35, LFR1 comprises the amino acid sequence of SEQ ID NO:36, and LFR2 comprises the amino acid sequence of SEQ ID NO:24. (d) HFR1 comprises the amino acid sequence of SEQ ID NO: 40, HFR2 comprises the amino acid sequence of SEQ ID NO: 33, HFR3 comprises the amino acid sequence of SEQ ID NO: 41, HFR4 comprises the amino acid sequence of SEQ ID NO: 35, LFR1 comprises the amino acid sequence of SEQ ID NO: 36, LFR2 comprises the amino acid sequence of SEQ ID NO: 37, LFR3 comprises the amino acid sequence of SEQ ID NO: 38, and LFR4 comprises the amino acid sequence of SEQ ID NO: 39; or (e) HFR1 comprises the amino acid sequence of SEQ ID NO: 40, HFR2 comprises the amino acid sequence of SEQ ID NO: 33, HFR3 comprises the amino acid sequence of SEQ ID NO: 41, HFR4 comprises the amino acid sequence of SEQ ID NO: 35, LFR1 comprises the amino acid sequence of SEQ ID NO: 36, LFR2 comprises the amino acid sequence of SEQ ID NO: 37, LFR3 comprises the amino acid sequence of SEQ ID NO: 38, and LFR4 comprises the amino acid sequence of SEQ ID NO: 39. ) HFR1 comprises the amino acid sequence of SEQ ID NO: 42, HFR2 comprises the amino acid sequence of SEQ ID NO: 43, HFR3 comprises the amino acid sequence of SEQ ID NO: 44, HFR4 comprises the amino acid sequence of SEQ ID NO: 35, LFR1 comprises the amino acid sequence of SEQ ID NO: 36, LFR2 comprises the amino acid sequence of SEQ ID NO: 45, LFR3 comprises the amino acid sequence of SEQ ID NO: 38, and LFR4 comprises the amino acid sequence of SEQ ID NO: 39, or (f) HFR1 comprises the amino acid sequence of SEQ ID NO: 87, HFR2 comprises the amino acid sequence of SEQ ID NO: 33, and HFR3 comprises the amino acid sequence of SEQ ID NO: 41;HFR4 comprises the amino acid sequence of SEQ ID NO:35, LFR1 comprises the amino acid sequence of SEQ ID NO:36, LFR2 comprises the amino acid sequence of SEQ ID NO:45, LFR3 comprises the amino acid sequence of SEQ ID NO:38, and LFR3 comprises the amino acid sequence of SEQ ID NO:39, or (g) HFR1 comprises the amino acid sequence of SEQ ID NO:47, HFR2 comprises the amino acid sequence of SEQ ID NO:48, HFR3 comprises the amino acid sequence of SEQ ID NO:49, HFR4 comprises the amino acid sequence of SEQ ID NO:35, LFR1 comprises the amino acid sequence of SEQ ID NO:36, LFR2 comprises the amino acid sequence of SEQ ID NO:45, LFR3 comprises the amino acid sequence of SEQ ID NO:38, and LFR4 comprises the amino acid sequence of SEQ ID NO:39.

[0017]

[0017] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment thereof provided herein comprises a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 8, 10 and 86, as well as homologous sequences thereof having at least 80% sequence identity and still retaining specific binding affinity for human TSLP.

[0018]

[0018] In some embodiments, the light chain variable region of the antibody or antigen-binding fragment thereof provided herein comprises a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6 and 9, and homologous sequences thereof having at least 80% sequence identity and still retaining specific binding affinity for human TSLP.

[0019]

[0019] In some embodiments, in the antibody or antigen-binding fragment thereof provided herein, the heavy chain variable region comprises the sequence of SEQ ID NO:1 and the light chain variable region comprises the sequence of SEQ ID NO:2, or the heavy chain variable region comprises the sequence of SEQ ID NO:3 and the light chain variable region comprises the sequence of SEQ ID NO:4, or the heavy chain variable region comprises the sequence of SEQ ID NO:5 and the light chain variable region comprises the sequence of SEQ ID NO:6, or the heavy chain variable region comprises the sequence of SEQ ID NO:7 and the light chain variable region comprises the sequence of SEQ ID NO:6, or the heavy chain variable region comprises the sequence of SEQ ID NO:86 and the light chain variable region comprises the sequence of SEQ ID NO:9, or the heavy chain variable region comprises the sequence of SEQ ID NO:8 and the light chain variable region comprises the sequence of SEQ ID NO:9, or the heavy chain variable region comprises the sequence of SEQ ID NO:10 and the light chain variable region comprises the sequence of SEQ ID NO:9.

[0020] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein further comprise one or more amino acid residue substitutions or modifications, while still retaining specific binding affinity for human TSLP. In some embodiments, at least one of the substitutions or modifications is in one or more of the CDR sequences of the heavy chain variable region or the light chain variable region, and / or in one or more of the non-CDR sequences. In some embodiments, at least one of the substitutions is a conservative substitution.

[0021] In some embodiments, the antibody or antigen-binding fragment thereof provided herein further comprises an Fc region, optionally an Fc region of a human immunoglobulin (Ig), or optionally an Fc region of a human IgG. In some embodiments, the Fc region is derived from human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, or IgM. In some embodiments, the Fc region derived from human IgG4 comprises a mutation of S228P, F234A, L235A, M252Y, S254T, T256E, or K447del (PAA-YTE). In some embodiments, the Fc region derived from human IgG1 comprises one or more mutations selected from the group consisting of L234A, L235A, M252Y, S254T, and T256E. In some embodiments, the Fc region derived from human IgG1 comprises the following mutations: L234A, L235A, M252Y, S254T, and T256E (LALA-YTE).

[0022]

[0022] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein further comprise a signal peptide at the N-terminus of the heavy chain variable region and / or a signal peptide at the N-terminus of the light chain variable region.

[0023] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are humanized. In some embodiments, the antibodies or antigen-binding fragments thereof provided herein are monoclonal antibodies, bispecific antibodies, multispecific antibodies, recombinant antibodies, chimeric antibodies, labeled antibodies, bivalent antibodies, anti-idiotypic antibodies, or fusion proteins.

[0024]

[0024] In some embodiments, the antibody or antigen-binding fragment thereof provided herein is a diabody, Fab, Fab', F(ab')2, Fd, Fv fragment, disulfide stabilized Fv fragment (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide stabilized diabody (ds diabody), single chain antibody molecule (scFv), scFv dimer (bivalent diabody), multispecific antibody, camelized single chain domain antibody, nanobody, domain antibody, or bivalent domain antibody.

[0025]

[0025] In some embodiments, the antibody or antigen-binding fragment thereof provided herein has one or more properties selected from the group consisting of: a) having the ability to specifically bind to human TSLP, b) having the ability to block the binding between TSLP and TSLPR, c) having the ability to block the binding between TSLP and cells expressing TSLPR and IL7R, d) having the ability to inhibit TSLP-dependent proliferation of BaF3 cells, e) having the ability to inhibit TSLP-dependent TARC secretion from PBMCs, or f) having the ability to inhibit TSLP-dependent STAT5 activation in cells expressing TSLPR and IL7R.

[0026]

[0026] In some embodiments, the antibody or antigen-binding fragment thereof does not specifically bind to monkey, rat or mouse TSLP.

[0027] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to long form of human TSLP and does not bind to short form of human TSLP, where long form of human TSLP has the amino acid sequence set forth in SEQ ID NO:73 and short form of human TSLP has the amino acid sequence set forth in SEQ ID NO:74.

[0027]

[0028] In some embodiments, the antibody or antigen-binding fragment thereof is capable of inhibiting human TSLP-dependent proliferation of a human TSLPR-expressing cell. In some embodiments, the cell further expresses human IL7R.

[0028]

[0029] In some embodiments, the antibody or antigen-binding fragment thereof binds to a different epitope than that bound by a reference antibody, wherein the amino acid sequence of the heavy chain of the reference antibody is set forth as SEQ ID NO: 77 and the amino acid sequence of the light chain of the reference antibody is set forth as SEQ ID NO: 78.

[0029]

[0030] In some embodiments, the antibody or antigen-binding fragment thereof is linked to one or more conjugate moieties, which in some embodiments comprise an agent for detection or isolation, such as a clearance modifier, a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme substrate label, a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binder, or other anti-cancer drug.

[0030]

[0031] In another aspect, the present disclosure provides an isolated polynucleotide encoding an antibody or antigen-binding fragment thereof disclosed herein. In another aspect, the present disclosure provides a vector comprising an isolated polynucleotide disclosed herein. In another aspect, the present disclosure provides a host cell comprising a vector disclosed herein.

[0031]

[0032] In another aspect, the disclosure provides a pharmaceutical composition comprising (i) an antibody or antigen-binding fragment thereof disclosed herein, or a polynucleotide encoding the antibody or antigen-binding fragment thereof, and (ii) one or more pharma- ceutically acceptable carriers, diluents, buffers, or excipients.

[0032]

[0033] In some embodiments, the pharmaceutical composition further comprises an additional therapeutic agent. In some embodiments, the additional therapeutic agent is an agent for treating inflammatory disease, autoimmune disease, and cancer. In some embodiments, the additional therapeutic agent is an agent that targets IL-33, IL-25, IL-4, IL-5, IL-4R, or IL-13.

[0033]

[0034] In another aspect, the disclosure provides a method of expressing an antibody or antigen-binding fragment thereof disclosed herein, comprising culturing a host cell disclosed herein under conditions in which a vector disclosed herein is expressed.

[0034]

[0035] In another aspect, the disclosure provides a method of treating, preventing, or ameliorating a TSLP-related disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof capable of binding to long human TSLP having the amino acid sequence set forth in SEQ ID NO:73 and digested forms of TSLP.

[0035]

[0036] In another aspect, the present disclosure provides a method for treating, preventing, or alleviating a TSLP-related disease or disorder in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or its antigen-binding fragment, a polynucleotide encoding the antibody or its antigen-binding fragment, and / or a pharmaceutical composition disclosed herein. In some embodiments, the disease or disorder is associated with dysregulation of TSLPR-mediated signaling compared to a control level. In some embodiments, the dysregulation of TSLP-mediated signaling comprises dysregulation of STAT5 activation by phosphorylation. In some embodiments, the dysregulation of TSLP-mediated signaling comprises dysregulation of proliferation of TSLPR-expressing cells.

[0036]

[0037] In some embodiments, the disease or disorder is selected from the group consisting of an inflammatory disease, an autoimmune disease, and cancer.

[0038] In some embodiments, the disease or disorder is asthma (including severe asthma), idiopathic pulmonary fibrosis, atopic dermatitis (AD), allergic conjunctivitis, allergic rhinitis (AR), Netherton syndrome (NS), eosinophilic esophagitis (EoE), food allergy, allergic diarrhea, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis (ABPA), allergic fungal sinusitis, chronic pruritus, cancer, rheumatoid arthritis, chronic obstructive pulmonary disease (COPD), systemic sclerosis, and / or chronic pulmonary fibrosis. In one embodiment, the present invention relates to a method for treating eosinophilic bowel disease, aortic ulcers, multiple sclerosis, keloids, ulcerative colitis, chronic rhinosinusitis (CRS), polyposis, chronic eosinophilic pneumonia, eosinophilic bronchitis, allergic bronchopulmonary aspergillosis, celiac disease, eosinophilic gastroenteritis, Churg-Strauss disease, eosinophilic myalgia syndrome, hypereosinophilic syndrome, eosinophilic granulomatosis with polyangiitis, eosinophilic esophagitis, inflammatory bowel disease, fibrotic disorders, inflammatory bowel disease, Hodgkin's lymphoma, and systemic lupus erythematosus.

[0037]

[0039] In some embodiments, the cancer is selected from the group consisting of breast cancer, pancreatic cancer, colon cancer, lung cancer, ovarian cancer, prostate cancer, and B-cell acute lymphoblastic leukemia.

[0040] In some embodiments, the fibrotic disorder is selected from the group consisting of systemic and localized scleroderma, keloids and hypertrophic scars, interstitial lung disease (ILD), idiopathic pulmonary fibrosis (IPF), liver fibrosis due to chronic hepatitis B or C infection, radiation-induced fibrosis, and fibrosis resulting from wound healing, atherosclerosis, restenosis, lung inflammation and fibrosis, cirrhosis of the liver, kidney disease, heart disease due to scar tissue, and eye diseases such as macular degeneration, and retinal and vitreous retinopathy, fibrosis due to chemotherapy drugs, and trauma and burns.

[0038]

[0041] In some embodiments, the disease or disorder is selected from the group consisting of asthma, polypoid rhinosinusitis, COPD, urticaria, EoE, and atopic dermatitis.

[0042] In another aspect, the disclosure provides a method for detecting the presence or amount of TSLP in a sample, comprising contacting the sample with an antibody or antigen-binding fragment thereof disclosed herein and determining the presence or amount of TSLP in the sample. In some embodiments, the method further comprises determining whether TSLP is overexpressed in cells in the sample.

[0039]

[0043] In another aspect, the disclosure provides for the use of an antibody or antigen-binding fragment thereof, pharmaceutical composition, and / or a polynucleotide encoding an antibody or antigen-binding fragment thereof disclosed herein in the manufacture of a medicament for treating, preventing, or ameliorating a disease or disorder associated with TSLP.

[0040]

[0044] In another aspect, the present disclosure provides a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, and a TCR signaling domain, wherein the antigen-binding domain specifically binds TSLP and comprises an antigen-binding fragment disclosed herein. In another aspect, the present disclosure provides a nucleic acid sequence encoding the chimeric antigen receptor (CAR) disclosed herein. In another aspect, the present disclosure provides a cell comprising a nucleic acid sequence disclosed herein. In another aspect, the present disclosure provides a vector comprising a nucleic acid sequence disclosed herein. In another aspect, the present disclosure provides a method for stimulating a T cell-mediated immune response to a TSLP-rich environment or tissue in a mammal, comprising administering to the mammal an effective amount of a cell genetically modified to express a CAR disclosed herein.

[0041]

[0045] In another aspect, the present disclosure provides a method for treating a mammal having a disease or disorder responsive to TSLP inhibition, comprising administering to the mammal an effective amount of a cell disclosed herein, thereby treating the mammal. In some embodiments, the cell is an autologous T cell. In some embodiments, the mammal is a human subject. In some embodiments, the mammal has been identified as having a TSLP-positive cell or a cell in which TSLP signaling is upregulated. [Brief description of the drawings]

[0042] [Figure 1]

[0046] FIG. 1 shows binding of antibodies Chi39H11 and BMK to TSLP as measured by ELISA. [Diagram 2]

[0047] FIG. 2 shows binding of antibodies Chi39H11, BMK, and controls to a fusion protein of human long TSLP expressed on the cell membrane, as measured by FACS. [Diagram 3]

[0048] FIG. 3 shows the activity of antibodies Chi39H11, BMK, and controls to block binding of human long TSLP to TSLPR-expressing cells. [Figure 4]

[0049] Figures 4a and 4b show cellular binding of antibodies Chi39H11, 39H11Z07 (Figure 4a), 39H11Z11 (Figure 4b), BMK, and controls to human long TSLP expressed on the cell membrane. [Diagram 5]

[0050] Figures 5a and 5b show the activity of antibodies Chi39H11, 39H11Z07 (Figure 5a), 39H11Z11 (Figure 5b), BMK, and a control in blocking binding of human long TSLP to TSLPR-expressing cells. [Figure 6]

[0051] FIG. 6 shows the activity of antibodies Chi39H11, 39H11Z07, 39H11Z11, BMK, and controls in inhibiting recombinant long TSLP-dependent BaF3 proliferation. [Figure 7]

[0052] FIG. 7 shows cellular binding of antibodies Chi35G12, BMK, and controls to human long form of TSLP expressed on the cell membrane. [Figure 8]

[0053] FIG. 8 shows the activity of antibodies Chi35G12, BMK, and controls in inhibiting recombinant long TSLP-dependent BaF3 proliferation. [Figure 9]

[0054] FIG. 9 shows cell binding of antibodies Chi35G12, 35G12Z01, 35G12Z02, 35G12Z03, BMK, and controls to human long TSLP expressed on the cell membrane. [Figure 10]

[0055] FIG. 10 shows the activity of antibodies Chi35G12, 35G12Z01, 35G12Z02, 35G12Z03, BMK, and controls in blocking binding of human long TSLP to TSLPR-expressing cells. [Figure 11]

[0056] FIG. 11 shows the activity of antibodies Chi35G12, 35G12Z01, 35G12Z02, 35G12Z03, BMK, and controls in inhibiting recombinant TSLP-dependent BaF3 proliferation. [Figure 12]

[0057] FIG. 12 shows protein binding of antibodies mAb35G12, mAb39H11, BMK, and controls to TSLP. [Figure 13-1]

[0058] Figures 13a-d show protein binding of antibodies mAb35G12, mAb39H11, BMK, and a control to human TSLP protein (Figure 13a), cynomolgus monkey TSLP (Figure 13b), mouse TSLP (Figure 13c), and rat TSLP (Figure 13d). [Figure 13-2]

[0058] Figures 13a-d show protein binding of antibodies mAb35G12, mAb39H11, BMK, and a control to human TSLP protein (Figure 13a), cynomolgus monkey TSLP (Figure 13b), mouse TSLP (Figure 13c), and rat TSLP (Figure 13d). [Figure 14]

[0059] FIG. 14 shows the results of epitope analysis of antibodies 39H11, 35G12, and BMK. [Figure 15]

[0060] FIG. 15 shows the activity of antibodies mAb35G12, mAb39H11, BMK, and controls in blocking binding of human long TSLP to TSLPR-expressing cells. [Figure 16]

[0061] FIG. 16 shows cell binding of antibodies mAb35G12, mAb39H11, BMK, and controls to human short-chain TSLP expressed on the cell membrane. [Figure 17]

[0062] FIG. 17 shows that TSLP proteins 52Ha (ie, long mutant TSLP), 52Hb (long native TSLP), and 52Hb-d (ie, a digestion product of 52Hb) were immunoblotted with anti-his-HRP. [Figure 18-1]

[0063] Figures 18a and 18b show protein binding of antibodies 35G12Z03-IgG2, 39H11Z07-IgG2, and BMK to digested TSLP. [Figure 18-2]

[0063] Figures 18a and 18b show protein binding of antibodies 35G12Z03-IgG2, 39H11Z07-IgG2, and BMK to digested TSLP. [Figure 19]

[0064] FIG. 19 shows inhibition of TSLP-dependent STAT5 activation by antibodies mAb35G12, mAb35G12_IgG1, mAb39H11, BMK, and controls. [Figure 20]

[0065] FIG. 20 shows inhibition of native TSLP-dependent TARC secretion by antibodies mAb35G12, mAb39H11, BMK, and controls. [Figure 21]

[0066] FIG. 21 shows inhibition of TSLP-dependent BaF3 proliferation by antibodies mAb35G12, mAb35G12_IgG1, mAb39H11, BMK, and controls. [Figure 22]

[0067] FIG. 22 shows inhibition of native TSLP-dependent BaF3 proliferation by antibodies mAb35G12, mAb39H11, BMK, and controls. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043]

[0068] Detailed Description of the Invention

[0069] The following description of the present disclosure is intended only to describe various embodiments of the present disclosure. Therefore, the specific modifications discussed should not be interpreted as limiting the scope of the present disclosure. It is clear to those skilled in the art that various equivalents, modifications and alterations can be made without departing from the scope of the present disclosure, and it is understood that such equivalent embodiments should be included in this specification. All references cited in this specification, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0044]

[0070] definition

[0071] The term "antibody" as used herein includes any immunoglobulin, monoclonal, polyclonal, polyvalent, bivalent, monovalent, multispecific, or bispecific antibody that binds to a specific antigen. A natural intact antibody comprises two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as alpha, delta, epsilon, gamma, and mu, each of which consists of a variable region (VH) and a first, second, third, and optionally a fourth constant region (CH1, CH2, CH3, CH4, respectively). Mammalian light chains are classified as lambda or kappa, each of which consists of a variable region (VL) and a constant region. Antibodies have a "Y" shape, with the axis of the Y consisting of the second and third constant regions of two heavy chains bound together via disulfide bonds. Each arm of the Y comprises the variable region and first constant region of one heavy chain bound to the variable and constant regions of one light chain. The variable regions of the light and heavy chains are involved in antigen binding. The variable regions of both chains generally contain three highly variable loops called complementarity determining regions (CDRs) (light chain CDRs including LCDR1, LCDR2 and LCDR3, heavy chain CDRs including HCDR1, HCDR2 and HCDR3).The boundaries of the CDRs of the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the rules of Kabat, IMGT, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, AM, J. Mol. Biol., 273(4), 927(1997); Chothia, C. et al., J Mol Biol. Dec 5; 186(3):651-63(1985); Chothia, C. and Lesk, AM, J. Mol. Biol., 196, 901(1987); Chothia, C. et al., Nature. Dec 21-28; 342(6252):877-83(1989); Kabat EA et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991); Marie-Paule Lefranc et al., Developmental and Comparative Immunology, 27:55-77 (2003); Marie-Paule Lefranc et al., Immunome Research, 1(3), (2005); Marie-Paule Lefranc, Molecular Biology of B cells (second edition), chapter 26, 481-514, (2015)). The three CDRs are inserted between adjacent sections known as framework regions (FRs) (light chain FRs including LFR1, LFR2, LFR3 and LFR4, and heavy chain FRs including HFR1, HFR2, HFR3 and HFR4), which are more highly conserved than the CDRs and form a scaffold supporting the highly variable loops. The constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions. Antibodies are assigned to a class based on the amino acid sequence of the constant region of their heavy chains.The five main classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of alpha, delta, epsilon, gamma, and mu heavy chains, respectively. Some of the major antibody classes are divided into subclasses, such as IgG1 (gamma 1 heavy chain), IgG2 (gamma 2 heavy chain), IgG3 (gamma 3 heavy chain), IgG4 (gamma 4 heavy chain), IgA1 (alpha 1 heavy chain), or IgA2 (alpha 2 heavy chain).

[0045]

[0072] In certain embodiments, the antibodies provided herein encompass any antigen-binding fragment thereof. As used herein, the term "antigen-binding fragment" refers to an antibody fragment formed from a portion of an antibody that includes one or more CDRs, or any other antibody fragment that binds to an antigen but does not include an intact native antibody structure. Examples of antigen-binding fragments include, without limitation, diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), bispecific antibodies, multispecific antibodies, camelized single-chain domain antibodies, nanobodies, domain antibodies, or bivalent domain antibodies. An antigen-binding fragment can bind to the same antigen that the parent antibody binds.

[0046]

[0073] "Fab," with respect to an antibody, means the portion of an antibody that consists of a single light chain (both variable and constant regions) linked by disulfide bonds to the variable region and first constant region of a single heavy chain.

[0047]

[0074] "Fab'" refers to a Fab fragment that includes part of the hinge region.

[0075] "F(ab')2" means a Fab' dimer.

[0076] "Fc" refers to the portion of an antibody (e.g., of an IgG, IgA, or IgD isotype) that consists of the second and third constant domains of a first heavy chain linked via disulfide bonds to the second and third constant domains of a second heavy chain. For antibodies of the IgM and IgE isotypes, the Fc further comprises a fourth constant domain. The Fc portion of an antibody is involved in various effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), but does not function in antigen binding.

[0048]

[0077] "Fv" refers to the minimum fragment of an antibody that contains a complete antigen-binding site. The Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain.

[0078] A "single-chain Fv antibody" or "scFv" refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region linked together directly or via a peptide linker sequence (Huston JS et al. Proc Natl Acad Sci USA, 85:5879 (1988)).

[0049]

[0079] By "single chain Fv-Fc antibody" or "scFv-Fc" is meant an engineered antibody consisting of an scFv linked to the Fc region of an antibody.

[0080] "Camelized single domain antibodies", "heavy chain antibodies", or "HCAbs" are antibodies that consist of two V HIt refers to antibodies that contain heavy chains and do not contain light chains (Riechmann L. and Muyldermans S., J Immunol Methods. Dec 10; 231(1-2):25-38(1999); Muyldermans S., J Biotechnol. Jun; 74(4):277-302(2001); WO94 / 04678; WO94 / 25591; U.S. Patent No. 6,005,079). Heavy chain antibodies are originally derived from the Camelidae family (camels, dromedaries, and llamas). Camelized antibodies have no light chains but have bona fide antigen-binding capacity (Hamers-Casterman C. et al., Nature. Jun 3; 363(6428): 446-8 (1993); Nguyen VK. et al. Immunogenetics. Apr; 54(1): 39-47 (2002); Nguyen VK. et al. Immunology. May; 109(1): 93-101 (2003)). The variable domain of heavy chain antibodies (VHH domain) represents the smallest known antigen-binding unit generated by the adaptive immune response (Koch-Nolte F. et al., FASEB J. Nov; 21(13): 3490-8. Epub 2007 Jun 15 (2007)).

[0050]

[0081] "Nanobody" refers to an antibody fragment consisting of a VHH domain and two constant domains, CH2 and CH3, of a heavy chain antibody.

[0082] A "diabody" or "dAb" refers to a small antibody fragment with two antigen-binding sites, arranged in the same polypeptide chain as the V L V linked to domain H Domain Included (V H -V L or V L -V H) (see, e.g., Holliger P. et al., Proc Natl Acad Sci USA. Jul 15; 90(14):6444-8 (1993); EP404097; WO93 / 11161). By using a linker that is too short to allow pairing between the two domains in the same chain, the domains are forced to pair with complementary domains on another chain, thereby creating two antigen-binding sites. The antigen-binding sites may target the same or different antigens (or epitopes). In certain embodiments, a "bispecific ds diabody" is a diabody that targets two different antigens (or epitopes).

[0051]

[0083] "Domain antibody" refers to an antibody fragment that contains only the variable region of a heavy chain or the variable region of a light chain. In some instances, two or more V H The domains are covalently joined with a peptide linker to create a bivalent or multivalent domain antibody. H The domains may target the same or different antigens.

[0052]

[0084] As used herein, the term "valency" refers to the presence of a specific number of antigen-binding sites in a given molecule. The term "monovalent" refers to an antibody or antigen-binding fragment that has only one single antigen-binding site, and the term "multivalent" refers to an antibody or antigen-binding fragment that has multiple antigen-binding sites. Thus, the terms "bivalent," "tetravalent," and "hexavalent" refer to the presence of two, four, and six binding sites, respectively, in an antigen-binding molecule. In some embodiments, an antibody or antigen-binding fragment thereof is bivalent.

[0053]

[0085] As used herein, a "bispecific" antibody refers to an artificial antibody that has fragments derived from two different monoclonal antibodies and is capable of binding to two different epitopes, which may be on the same antigen or on two different antigens.

[0054]

[0086] In certain embodiments, an "scFv dimer" is a dimer of another V H -V L Dimerized with the V moiety (dimerized via a peptide linker) H -V L and a bivalent diabody or bispecific scFv (BsFv) comprising one part V H V in other parts L In another embodiment, an "scFv dimer" is a combination of a V (linked by a peptide linker) and a V (linked by a peptide linker) to form two binding sites that can target the same antigen (or epitope) or different antigens (or epitopes). L1 -V H2 (also linked by a peptide linker) V H1 -V L2 and a bispecific diabody comprising V H1 and V L1 In cooperation with V H2 and V L2 cooperate, with each cooperated pair having a different antigen specificity.

[0055]

[0087] "dsFv" refers to a disulfide-stabilized Fv fragment in which the link between the variable region of a single light chain and the variable region of a single heavy chain is a disulfide bond. In some embodiments, a "(dsFv)2" or "(dsFv-dsFv')" is a Fv fragment that is composed of three peptide chains, i.e., linked by a peptide linker (e.g., a long flexible linker), and each of the two Vs is linked through a disulfide bridge. L Two V's attached to the part H In some embodiments, dsFv-dsFv' are bispecific, in which the respective disulfide-paired heavy and light chains have different antigen specificities.

[0056]

[0088] As used herein, the term "chimeric" refers to an antibody or antigen-binding fragment having a portion of a heavy and / or light chain derived from one species and the remainder of the heavy and / or light chain derived from a different species. In an illustrative example, a chimeric antibody may contain a constant region derived from a human and a variable region derived from a non-human animal, such as a mouse. In some embodiments, the non-human animal is a mammal, such as a mouse, rat, rabbit, goat, sheep, guinea pig, or hamster.

[0057]

[0089] As used herein, the term "humanized" means that the antibody or antigen-binding fragment contains CDRs derived from a non-human animal, FR regions derived from a human, and, where applicable, a constant region derived from a human.

[0058]

[0090] As used herein, the term "affinity" refers to the strength of the non-covalent interactions between an immunoglobulin molecule (i.e., an antibody) or a fragment thereof and an antigen.

[0091] As used herein, the term "specific binding" or "specifically binds" refers to a non-random binding reaction between two molecules, such as an antibody and an antigen. Specific binding is, for example, the K D value, i.e., the ratio of the dissociation rate to the association rate (k off / k on The binding affinity of the ligand can be characterized by its binding affinity, which is expressed by K D may be determined using any conventional method known in the art, including, but not limited to, surface plasmon resonance, microscale thermophoresis, HPLC-MS, and flow cytometry (such as FACS). -6 M or less (for example, 5×10 -7 Under M, 2x10 -7 M or less, 10 -7 M or less, 5×10 -8 Under M, 2x10 -8 M or less, 10 -8 M or less, 5×10 -9 M or less, 4x10 -9 M or less, 3×10 -9 Under M, 2x10-9 M or less, or 10 -9 M or less) K D The value can indicate specific binding between an antibody or antigen-binding fragment thereof and TSLP (eg, human TSLP).

[0059]

[0092] As used herein, the ability to "compete for binding to human TSLP" refers to the ability of a first antibody or antigen-binding fragment to inhibit, to any detectable extent, the binding interaction between human TSLP and a second anti-TSLP antibody. In certain embodiments, an antibody or antigen-binding fragment that competes for binding to human TSLP inhibits the binding interaction between human TSLP and a second anti-human TSLP antibody by at least 85%, or at least 90%. In certain embodiments, this inhibition may be greater than 95%, or greater than 99%.

[0060]

[0093] As used herein, the term "epitope" refers to a specific group of atoms or amino acids on an antigen to which an antibody binds. If two antibodies exhibit competitive binding to an antigen, they may bind to the same or closely related epitopes in the antigen. Epitopes may be linear or conformational (i.e., involving separated amino acid residues). For example, an antibody or antigen-binding fragment may be considered to bind to the same / closely related epitope as the reference antibody if it blocks at least 85%, or at least 90%, or at least 95% of the binding of the reference antibody to the antigen.

[0061]

[0094] As used herein, the term "amino acid" refers to an organic compound containing an amine (-NH2) and a carboxyl (-COOH) functional group along with a side chain characteristic of each amino acid. In this disclosure, the names of amino acids may also be represented as standard one-letter or three-letter codes, which are summarized below.

[0062] [Table 1]

[0063]

[0095] "Conservative substitution" in relation to amino acid sequence refers to replacing an amino acid residue with a different amino acid residue having a side chain with similar physicochemical properties. For example, conservative substitution can be made between amino acid residues with hydrophobic side chains (e.g., Met, Ala, Val, Leu, and Ile), between amino acid residues with neutral hydrophilic side chains (e.g., Cys, Ser, Thr, Asn, and Gln), between amino acid residues with acidic side chains (e.g., Asp, Glu), between amino acid residues with basic side chains (e.g., His, Lys, and Arg), or between amino acid residues with aromatic side chains (e.g., Trp, Tyr, and Phe). As is known in the art, conservative substitution usually does not cause significant changes in the conformational structure of a protein, and therefore can retain the biological activity of the protein.

[0064]

[0096] As used herein, the term "homologous" refers to a nucleic acid sequence (or its complementary strand) or amino acid sequence that has at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity with another sequence when optimally aligned.

[0065]

[0097] "Percent sequence identity" with respect to an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences and, if necessary, introducing gaps to maximize the number of identical amino acids (or nucleic acids). In other words, the percent sequence identity of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of identical amino acid residues (or bases) with respect to the reference sequence being compared by the total number of amino acid residues (or bases) in either the candidate or reference sequence, whichever is shorter. Conservative substitutions of amino acid residues may or may not be considered identical residues. Alignment for calculating percent amino acid (or nucleic acid) sequence identity can be accomplished using publicly available tools such as, for example, BLASTN, BLASTp (available from the US National Center for Biotechnology Information (NCBI) website, see also Altschul SF et al., J. Mol. Biol., 215:403-410 (1990); Stephen F. et al., Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available from the European Bioinformatics Institute website, see also Higgins DG et al., Methods in Enzymology, 266:383-402 (1996); Larkin MA et al., Bioinformatics (Oxford, England), 23(21):2947-8 (2007)), and ALIGN or Megalign (DNASTAR) software. One skilled in the art may use the default parameters provided by the tool or may customize appropriate parameters for the alignment, for example by selecting a suitable algorithm.

[0066]

[0098] As used herein, "effector function" refers to the biological activity attributable to the binding of the Fc region of an antibody to its effector, such as the C1 complex and Fc receptor. Exemplary effector functions include complement-dependent cytotoxicity (CDC), mediated by the interaction of the antibody with C1q on the C1 complex, antibody-dependent cell-mediated cytotoxicity (ADCC), mediated by the binding of the Fc region of an antibody to an Fc receptor on an effector cell, and phagocytosis. Effector function can be assessed using various assays, such as Fc receptor binding assays, C1q binding assays, and cytolysis assays.

[0067]

[0099] An "isolated" material is altered by the hand of man from its natural state. If an "isolated" composition or material occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or polypeptide that naturally occurs in a living animal is not "isolated," but the same polynucleotide or polypeptide is "isolated" if it is sufficiently separated from the coexisting materials in its natural state and exists in a substantially pure state. An "isolated nucleic acid sequence" refers to a sequence of an isolated nucleic acid molecule. In certain embodiments, an "isolated antibody or antigen-binding fragment thereof" refers to an antibody or antigen-binding fragment thereof that has a purity of at least 60%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% as determined by electrophoretic methods (such as SDS-PAGE, isoelectric focusing, capillary electrophoresis) or chromatographic methods (ion exchange chromatography or reverse phase HPLC).

[0068]

[0100] As used herein, the term "vector" refers to a vehicle into which a genetic element is operably inserted and expression of the genetic element occurs, thereby producing a protein, RNA, or DNA encoded by the genetic element, or replicating the genetic element. A vector may be used to transform, transduce, or transfect a host cell to cause expression of the genetic element it carries into the host cell. Examples of vectors include plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses. A vector may contain various elements for controlling expression, such as promoter sequences, transcription initiation sequences, enhancer sequences, selectable elements, and reporter genes. Additionally, a vector may contain an origin of replication. A vector may contain substances that aid in the entry of the vector into a cell, including, but not limited to, viral particles, liposomes, or protein coatings. A vector may be an expression vector or a cloning vector. The present disclosure provides a vector (e.g., an expression vector) comprising a nucleic acid sequence provided herein encoding an antibody or antigen-binding fragment thereof, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selectable marker.

[0069]

[0101] As used herein, the phrase "host cell" means a cell into which an exogenous polynucleotide and / or vector can be introduced or has been introduced.

[0102] The term "subject" includes human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals such as non-human primates, mice, rats, cats, rabbits, sheep, dogs, cows, chickens, amphibians, and reptiles. Except where noted, the terms "patient" or "subject" are used interchangeably herein.

[0070]

[0103] As used herein, "treating" a disease, disorder, or condition or "treatment" thereof includes preventing or alleviating the disease, disorder, or condition, slowing the onset or rate of occurrence of the disease, disorder, or condition, reducing the risk of developing the disease, disorder, or condition, preventing or delaying the onset of symptoms associated with the disease, disorder, or condition, reducing or terminating symptoms associated with the disease, disorder, or condition, completely or partially ameliorating the disease, disorder, or condition, curing the disease, disorder, or condition, or some combination thereof.

[0071]

[0104] The terms "diagnosis", "diagnose" or "diagnosing" refer to the identification of a pathological condition, disease or condition, such as the identification of a TSLP-associated disease, or the identification of a subject having a TSLP-associated disease that may benefit from a particular treatment regimen. Here, a TSLP-associated disease refers to a disease or disorder that is responsive to TSLP inhibition. In some embodiments, diagnosis includes the identification of an abnormal amount or activity of TSLP. In some embodiments, diagnosis refers to the identification of an inflammatory disease, an autoimmune disease, or a cancer in a subject.

[0072]

[0105] As used herein, the term "biological sample" or "sample" refers to a biological composition comprising cells and / or other molecular entities obtained or derived from a subject of interest and characterized and / or identified based on, for example, physical, biochemical, chemical, and / or physiological characteristics. Biological samples include, but are not limited to, cells, tissues, organs, and / or biological fluids of a subject obtained by any method known to those of skill in the art. In some embodiments, the biological sample is a fluid sample. In some embodiments, the fluid sample is whole blood, plasma, serum, mucus (including nasal drip and sputum), peritoneal fluid, pleural fluid, saliva, urine, synovial fluid, cerebrospinal fluid (CSF), thoracentesis, abdominal fluid, peritoneal fluid, or pericardial fluid. In some embodiments, the biological sample is tissue or cells obtained from the heart, liver, spleen, lungs, kidneys, skin, or blood vessels of a subject.

[0073]

[0106] As used herein, "TSLP" refers to thymic stromal lymphopoietin, a protein that belongs to the cytokine family and is known to be involved in type 2 inflammation through activation of dendritic cells, mast cells, and ILC2. TSLP signals through a heterodimeric receptor complex composed of the thymic stromal lymphopoietin receptor CRLF2 and the IL-7R alpha chain. Following binding of TSLP to the heterodimeric receptor complex, STAT5 phosphorylation is induced, resulting in the expression of upstream transcription factors.

[0074]

[0107] To exert its biological activity on a wide range of cell types, TSLP binds to a heterodimeric receptor consisting of the IL-7 receptor alpha chain (IL-7Rα) and the TSLPR chain, which is closely related to the common receptor gamma chain (γc). TSLPR alone has low affinity for TSLP, but the binding of TSLPR to IL-7Rα creates a high affinity binding site for TSLP and triggers signaling. In some embodiments, a fusion protein of human IL7Ra and human thymic stromal lymphopoietin receptor (TSLPR) can be used to trigger TSLP signaling. In some embodiments, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:70.

[0075]

[0108] Amino acid sequence of the huIL7Ra-huTSLPR fusion protein (SEQ ID NO:70):

[0109] MGWSCIILFLVATATGVHSESGYAQNGDLEDAELDDYSFSCYSQLEVNGSQHSLTCAFEDPDVNITNLEFEICGALVEVKCLNFRKLQEIYFIETKKFLLIGKSNICVKVGEKSLTCKKIDLTTIV KPEAPFDLSVVYREGANDFVVTFNTSHLQKKYVKVLMHDVAYRQEKDENKWTHVNLSSTKLTLLQRKLQPAAMYEIKVRSIPDHYFKGFWSEWSPSYYFRTPEINNSSGEMDGGSGGSGGSGGSGGS GGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSGGSQGGAAEGVQIQIIYFNLETVQVTWNASKYSRTNLTFHYRFNGDEAYDQCTNYLLQEGHTSGCLLDAEQRDDILYFSIRNGTH PVFTASRWMVYYLKPSSPKHVRFSWHQDAVTVTCSDLSYGDLLYEVQYRSPFDTEWQSKQENTCNVTIEGLDAEKCYSFWVRVKAMEDVYGPDTYPSDWSEVTCWQRGEIRDACAETPTPPKPKLSK

[0110] Human IL-7Rα has the amino acid sequence set forth in SEQ ID NO:71.

[0076]

[0111] Amino acid sequence of human IL-7Rα protein (SEQ ID NO:71):

[0112] MTILGTTFGMVFSLLQVVSGESGYAQNGDLEDAELDDYSFSCYSQLEVNGSQHSLTCAFEDPDVNITNLEFEICGALVEVKCLNFRKLQEIYFIETKKFLLIGKSNICVKVGEK SLTCKKIDLTTIVKPEAPFDLSVVYREGANDFVVTFNTSHLQKKYVKVLMHDVAYRQEKDENKWTHVNLSSTKLTLLQRKLQPAAMYEIKVRSIPDHYFKGFWSEWSPSYYFRTP EINNSSGEMDPILLTISILSFFSVALLVILACVLWKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDV QSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ

[0113] Human TSLPR comprises the amino acid sequence set forth in SEQ ID NO:72.

[0077]

[0114] Amino acid sequence of human TSLPR (SEQ ID NO:72):

[0115] MGRLVLLWGAAVFLLGGWMALGQGGAAEGVQIQIIYFNLETVQVTWNASKYSRTNLTFHYRFNGDEAYDQCTNYLLQEGHTSGCLLDAEQRDDILYFSIRNGTHPVFTASRWMVYYLKPSSPKHVRFSWHQDAVTVTCSDLSYGDLLYEVQYRSPFDTEWQSKQENTCNVTIEGLDAEKCYSFWV RVKAMEDVYGPDTYPSDWSEVTCWQRGEIRDACAETPTPPKLSKFILISSLAILLMVSLLLLSLWKLWRVKKFLIPSVPDPKSIFPGLFEIHQGNFQEWITDTQNVAHLHKMAGAEQESGPEEPLVVQLAKTEAESPRMLDPQTEEKEASGGSLQLPHQPLQGGDVVTIGGFTFVMNDRSYVAL

[0116] Two isoforms of TSLP have been described in mice, a short and a long isoform, but the functional consequences of this variation are unclear. In humans, the major isoform expressed during steady-state conditions is the short form of TSLP, whereas the long form of TSLP is upregulated under inflammatory conditions.

[0078]

[0117] In some embodiments, the antibodies or antigen-binding fragments disclosed herein bind to long TSLP but not short TSLP. In some embodiments, the TSLP to which the antibodies or antigen-binding fragments disclosed herein specifically bind is a digested form of TSLP. In some embodiments, the antibodies or antigen-binding fragments disclosed herein bind more strongly to the digested form of TSLP compared to existing antibodies (e.g., AMG 157).

[0079]

[0118] In some embodiments, long-form TSLP comprises amino acid residues 29 to 159 of full-length TSLP, while short-form TSLP comprises amino acid residues 97 to 159 of full-length TSLP. In some embodiments, the amino acid sequence of full-length TSLP is set forth in the sequence having the NCBI accession number of NP_149024.1 (SD.hTSLP, i.e., SEQ ID NO: 85).

[0080]

[0119] MFPFALLYVLSVSFRKIFILQLVGLVLTYDFTNCDFEKIKAAYLSTISKDLITYMSGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEMFAMKTKAALAIWCPGYSETQINATQAMKKRRKRKVTTNKCLEQVSQLQGLWRRFNRPLLKQQ (SEQ ID NO: 85)

[0120] Native full-length or long-chain human TSLP protein is susceptible to enzymatic cleavage, for example, by the PCSK3 enzyme. The PCSK3 enzyme can specifically target positions 127-130 of the long-chain TSLP protein and specifically cleave the protein at a site between positions 130 and 130, where the amino acid numbering follows the sequence of the full-length human TSLP protein. To prevent such enzymatic cleavage and stabilize TSLP in its long-chain form, certain mutations can be introduced at the enzymatic cleavage site (e.g., R127 and R130). Thus, in the present disclosure, reference to long-chain TSLP should be understood to encompass both native long-chain TSLP as well as mutant and stabilized long-chain TSLP, for example, mutant TSLP with R127A and R130A mutations (e.g., SEQ ID NO: 73) or mutant TSLP with R127A and R130S mutations (e.g., the TSLP sequence in SEQ ID NO: 80). In some embodiments, long-form TSLP comprises the amino acid sequence set forth in SEQ ID NO: 73. In some embodiments, short-form TSLP comprises the amino acid sequence set forth in SEQ ID NO: 74.

[0081]

[0121] Amino acid sequence of long form of TSLP (SEQ ID NO:73):

[0122] YDFTNCDFEKIKAAYLSTISKDLITYMSGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEMFAMKTKAALAIWCPGYSETQINATQAMKKARKAKVTTNKCLEQVSQLQGLWRRFNRPLLKQQ

[0123] Amino acid sequence of short-form TSLP (SEQ ID NO:74):

[0124] MFAMKTKAALAIWCPGYSETQINATQAMKKARKAKVTTNKCLEQVSQLQGLWRRFNRPLLKQQ

[0125] The term "digested form of TSLP" as used herein refers to an enzymatic digestion product of long-chain TSLP. In some embodiments, the digested form of TSLP can be obtained by digestion of wild-type long-chain human TSLP protein with PCSK3 enzyme. In some embodiments, the PCSK3 enzyme specifically targets positions 127-130 of the long-chain TSLP protein and specifically cleaves the protein at a site between positions 130 and 130, where the amino acid numbering is according to the sequence of the full-length human TSLP protein. In some embodiments, the digested form of TSLP includes a TSLP fragment comprising amino acid residues 29-124 of full-length TSLP and / or a TSLP fragment comprising amino acid residues 131-159 of full-length TSLP. In some embodiments, the TSLP fragment further comprises a His tag at the C-terminal region. In some embodiments, the digested form of TSLP is presented as a heterodimerized form in which two heterologous monomers are linked via a disulfide bond. It is known in the art that expression levels of digested forms of TSLP, particularly those presented as heterodimerized forms, are significantly higher under inflammatory physiological conditions compared to normal physiological conditions.

[0082]

[0126] The term "anti-TSLP antibody" refers to an antibody that can specifically bind to long form of TSLP (e.g., human long form TSLP). In certain embodiments, an anti-TSLP antibody is further capable of specifically binding to a digestion product of TSLP. The term "anti-human TSLP antibody" refers to an antibody that can specifically bind to human TSLP or a digested form of human TSLP.

[0083]

[0127] A "TSLP-associated" disease, disorder, or condition refers to any disease or condition caused by, exacerbated by, or otherwise associated with increased or decreased expression or activity of long form of TSLP. In some embodiments, a TSLP-associated disease or disorder is a disease or disorder that is responsive to inhibition of long form of TSLP.

[0084]

[0128] In some embodiments, the TSLP-related disease, disorder, or condition is associated with dysregulation of long-form TSLP-mediated signaling compared to a control level. In some embodiments, the dysregulation of long-form TSLP-mediated signaling comprises dysregulation of STAT5 activation by phosphorylation. In some embodiments, the dysregulation of long-form TSLP-mediated signaling comprises dysregulation of TSLPR-expressing cell proliferation. In some embodiments, the control level is the level in a healthy individual.

[0085]

[0129] In some embodiments, the TSLP-related disease, disorder, or condition is an immune-related disorder, such as, for example, an autoimmune disease. In some embodiments, the TSLP-related disease, disorder, or condition is a disorder associated with excessive cell proliferation, such as, for example, cancer. In certain embodiments, the TSLP-related disease or condition is characterized by expression or overexpression of the TSLP gene. In certain embodiments, the TSLP-related disease or condition is characterized by overexpression of TSLP and / or dysregulation of TSLP-mediated signaling.

[0086]

[0130] The term "pharmaceutical acceptable" indicates that the specified carrier, vehicle, diluent, excipient, and / or salt is generally chemically and / or physically compatible with the other ingredients that make up the formulation and physiologically compatible with the recipient thereof.

[0087]

[0131] The term "TSLP-positive cells" as used herein refers to cells that exhibit abnormal TSLP expression levels compared to control cells. The abnormal expression levels can be up-regulated or down-regulated compared to the levels of control cells, and can be associated with dysregulation of TSLP-mediated signal transduction. The control cells can be normal or healthy counterpart cells, which may or may not express TSLP. If the control cells express TSLP, the abnormal expression levels of TSLP-positive cells can be up-regulated or down-regulated. If the control cells do not express TSLP, the abnormal expression levels of TSLP-positive cells can be up-regulated.

[0088]

[0132] Anti-TSLP Antibody

[0133] The present disclosure provides anti-TSLP antibodies and antigen-binding fragments thereof. The anti-TSLP antibodies and antigen-binding fragments provided herein are capable of specifically binding to the long form of TSLP.

[0089]

[0134] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein exhibit a 10 -7 M or less, 8x10 -8 M or less, 5×10 -8 Under M, 2x10 -8 M or less, 8x10 -9 M or less, 5×10 -9 Under M, 2x10 -9 M or less, 10 -9 M or less, 8x10 -10 M or less, 7x10 -10 M or less, or 6 x 10 -10 K below M D It specifically binds to human TSLP (i.e., long-chain TSLP) at high antibody titers (see, e.g., Murphy, M. et al., Current protocols in protein science, Chapter 19, unit 19.14, 2006).

[0090]

[0135] The binding of the antibodies or antigen-binding fragments thereof provided herein to human TSLP is measured using a "50% effective concentration" (EC 50 ) value. 50 The values ​​represent the concentration of antibody at which 50% of its maximal binding is observed. EC 50 Values ​​can be measured by binding assays known in the art, for example, direct or indirect binding assays such as enzyme-linked immunosorbent assays (ELISA), flow cytometry assays, and other binding assays.

[0091]

[0136] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein exhibit no detectable binding to cynomolgus monkey TSLP, rat TSLP, or mouse TSLP, or exhibit binding to cynomolgus monkey, rat, or mouse TSLP at a level comparable to that of a negative control antibody under comparable assay conditions.In addition, the negative control antibody can be any antibody known not to bind to cynomolgus monkey TSLP, rat TSLP, or mouse TSLP.

[0092]

[0137] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein can block TSLP signaling induced by a TSLP agonist (e.g., a fusion protein comprising TSLP and IL-7Rα) as measured by a TSLP reporter assay.

[0093]

[0138] In certain embodiments, TSLP reporter assay is the assay for measuring the level of STAT5 activation.In certain embodiments, TSLP reporter assay is the assay for measuring the secretion of TARC.In certain embodiments, TSLP reporter assay is the assay for measuring TSLP-dependent BaF3 proliferation.

[0094]

[0139] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein specifically bind to a digested form of TSLP with a binding affinity that is higher than that of a control antibody (e.g., AMG157). In some embodiments, the binding affinity of the antibodies or antigen-binding fragments thereof provided herein to a digested form of TSLP is at least 100% higher (e.g., at least 150% higher, 200% higher, 300% higher, 400% higher, 500% higher, 600% higher, 700% higher, 800% higher, 900% higher, etc.) than that of AMG157. Binding affinity can be measured by conventional means well known to those skilled in the art, such as Western blotting, ELISA binding assays, fluorescence-activated cell sorting (FACS) assays, biolayer interferometry (BLI) assays, and surface plasmon resonance (SPR) assays.

[0095]

[0140] Exemplary Anti-TSLP Antibodies

[0141] In certain embodiments, the disclosure provides anti-TSLP antibodies (e.g., anti-human TSLP antibodies) and antigen-binding fragments thereof, comprising the group consisting of SEQ ID NO:11, IFPGDGX1T (SEQ ID NO:59), and ARX2GX3X4X5X6X7X8YAMDY (SEQ ID NO:60) (wherein X1 is E or D, X2 is G or S, X3 is Y or F, X4 is V or D, X5 is N or Y, X6 is absent or G, X7 is F or Y, and X8 is L or F), or SYWX9N (SEQ ID NO:61), QIFPGDGX 10 TX 11 YNGX 12 FKG (SEQ ID NO: 62), and X 13 GX 14 X 15 X 16 X 17 X 18 X 19 The group consisting of YAMDY (SEQ ID NO: 63) (wherein X9 is M or I, 10 is E or D, and X 11 is N or T, and X 12 is K or N, and X 13is G or S, and X 14 is Y or F, and X 15 is V or D, and X 16 is N or Y, and X 17 does not exist or is G, and X 18 is F or Y, and X 19 is L or F. In certain embodiments, the disclosure further encompasses antibodies and antigen-binding fragments thereof having no more than one, no more than two, or no more than three amino acid residue substitutions relative to any of the sequences herein.

[0096]

[0142] In certain embodiments, the disclosure provides anti-TSLP antibodies (e.g., anti-human TSLP antibodies) and antigen-binding fragments thereof, comprising the group consisting of SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, or SEQ ID NO:53, GTSX 20 LAS (SEQ ID NO: 64), and the group consisting of SEQ ID NO: 16 (X 20 is T or N). In certain embodiments, the disclosure further encompasses antibodies and antigen-binding fragments thereof having no more than one, no more than two, or no more than three amino acid residue substitutions relative to any of the sequences herein.

[0097]

[0143] As used herein, the antibody "39H11" refers to a monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:1 and a light chain variable region having the sequence of SEQ ID NO:2.

[0098]

[0144] As used herein, the antibody "35G12" refers to a monoclonal antibody comprising a heavy chain variable region having the sequence of SEQ ID NO:3 and a light chain variable region having the sequence of SEQ ID NO:4.

[0099]

[0145] In one respect, the disclosure provides an antibody or antigen-binding fragment thereof capable of specifically binding to human TSLP, the antibody or antigen-binding fragment thereof comprising a heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3 contained within any one of the heavy chain variable region sequences of SEQ ID NO:1 or 3, and / or a light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3 contained within any one of the heavy chain variable region sequences of SEQ ID NO:2 or 4.

[0100]

[0146] In certain embodiments, the disclosure provides anti-TSLP antibodies and antigen-binding fragments thereof comprising an HCDR1 comprising the sequence of SEQ ID NO:11, an HCDR2 comprising the sequence of SEQ ID NO:12 or 25, and an HCDR3 comprising the sequence of SEQ ID NO:13 or 26, and / or an LCDR1 comprising the sequence of SEQ ID NO:14, an LCDR2 comprising the sequence of SEQ ID NO:15, and an LCDR3 comprising the sequence of SEQ ID NO:16.

[0101]

[0147] In certain embodiments, the disclosure provides anti-TSLP antibodies and antigen-binding fragments thereof comprising an HCDR1 comprising the sequence of SEQ ID NO:11, an HCDR2 comprising the sequence of SEQ ID NO:12, an HCDR3 comprising the sequence of SEQ ID NO:13, and / or an LCDR1 comprising the sequence of SEQ ID NO:14, an LCDR2 comprising the sequence of SEQ ID NO:15, and an LCDR3 comprising the sequence of SEQ ID NO:16.

[0102]

[0148] In certain embodiments, the disclosure provides anti-TSLP antibodies and antigen-binding fragments thereof comprising an HCDR1 comprising the sequence of SEQ ID NO:11, an HCDR2 comprising the sequence of SEQ ID NO:25, an HCDR3 comprising the sequence of SEQ ID NO:26, and / or an LCDR1 comprising the sequence of SEQ ID NO:14, an LCDR2 comprising the sequence of SEQ ID NO:15, and an LCDR3 comprising the sequence of SEQ ID NO:16.

[0103]

[0149] Tables 1 and 2 below show the CDR amino acid sequences of antibodies 39H11 and 35G12, where Table 1 shows the CDR boundaries defined or identified by the IMGT rules and Table 2 shows the CDR boundaries defined or identified by the Kabat rules. Table 3 below shows the heavy and light chain variable region amino acid sequences of antibodies 39H11 and 35G12.

[0104]

[0150]

[0105] [Table 2]

[0106]

[0151]

[0107] [Table 3]

[0108]

[0152]

[0109] [Table 4]

[0110]

[0153] Given that both antibodies 39H11 and 35G12 can bind TSLP and that antigen-binding specificity is provided primarily by the CDR1, CDR2, and CDR3 regions, the HCDR1, HCDR2, and HCDR3 sequences, and the LCDR1, LCDR2, and LCDR3 sequences of antibodies 39H11 and 35G12 can be "mixed and matched" (i.e., CDRs of different antibodies can be mixed and matched, but each antibody must contain HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3) to create anti-TSLP binding molecules of the present disclosure. TSLP binding of such "mixed and matched" antibodies can be tested using the binding assays described above and in the Examples. Preferably, when VH CDR sequences are mixed and matched, the HCDR1, HCDR2, and / or HCDR3 sequences from a particular VH sequence are replaced with structurally similar CDR sequences. Similarly, when VL CDR sequences are mixed and matched, the LCDR1, LCDR2 and / or LCDR3 sequence from a particular VL sequence is preferably replaced with a structurally similar CDR sequence(s). It will be readily apparent to one of ordinary skill in the art that novel VH and VL sequences can be created by replacing one or more VH and / or VL CDR region sequences for monoclonal antibodies 39H11, and 35G12 with structurally similar sequences from the CDR sequences disclosed herein.

[0111]

[0154] It is known that CDR is involved in antigen binding.However, it has been found that not all six CDRs are essential or invariant.In other words, it is possible to replace, change or modify one or more CDRs in anti-TSLP antibody 39H11 and 35G12 while substantially retaining specific binding affinity to TSLP.

[0112]

[0155] In certain embodiments, the antibodies and antigen-binding fragments thereof provided herein comprise suitable framework region (FR) sequences, so long as the antibodies and antigen-binding fragments thereof are capable of specifically binding to TSLP. Although the CDR sequences provided in Table 1 above are derived from mouse antibodies, they can be grafted into any suitable FR sequence of any suitable species, such as mouse, human, rat, rabbit, among others, using suitable methods known in the art, such as recombinant techniques.

[0113]

[0156] In certain embodiments, the antibody and antigen-binding fragment provided herein are humanized. Humanized antibody or antigen-binding fragment is desirable for its reduced immunogenicity in humans. Humanized antibody is chimeric in its variable region because non-human CDR sequence is grafted to human or substantially human FR sequence. Humanization of antibody or antigen-binding fragment can be essentially performed by replacing non-human (such as mouse) CDR gene with corresponding human CDR gene in human immunoglobulin gene (see, for example, Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536).

[0114]

[0157] To this end, suitable human heavy and light chain variable domains can be selected using methods known in the art. In an illustrative example, a "best-fit" approach can be used, in which the variable domain sequence of a non-human (e.g., rodent) antibody is screened or BLASTed against known human variable domain sequences, and the human sequence closest to the non-human query sequence is identified and used as a human scaffold for grafting the non-human CDR sequences (see, e.g., Sims et al., (1993) J. Immunol. 151:2296; Chothia et al. (1987) J. Mot. Biol. 196:901). Alternatively, frameworks derived from the consensus sequence of all human antibodies may be used for grafting of non-human CDRs (see, e.g., Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al. (1993) J. Immunol., 151:2623).

[0115]

[0158] Table 4 below shows the heavy and light chain variable region amino acid sequences of humanized antibodies 39H11 or 35G12, designated as 39H11Z07, 39H11Z11, and mAb39H11, 35G12Z01, 35G12Z02, 35G12Z03, and mAb35G12. Table 4 below shows the heavy and light chain variable region amino acid sequences of humanized antibodies 39H11 or 35G12, designated as 39H11Z07, 39H11Z11, and mAb39H11, 35G12Z01, 35G12Z02, 35G12Z03, and mAb35G12.

[0116]

[0159]

[0117] [Table 5]

[0118]

[0160]

[0119] [Table 6-1]

[0120] [Table 6-2]

[0121]

[0161] In certain embodiments, the humanized antibody or antigen-binding fragment thereof provided herein consists of substantially all human sequences, except for the CDR sequences, which are non-human. In some embodiments, the variable region FR and, if present, the constant region are entirely or substantially from human immunoglobulin sequences. The human FR sequences and the human constant region sequences can be from different human immunoglobulin genes, e.g., the FR sequences are from one human antibody and the constant region is from another human antibody. In some embodiments, the humanized antibody or antigen-binding fragment thereof comprises human heavy chain HFR1-4 and / or light chain LFR1-4.

[0122]

[0162] In some embodiments, the FR region derived from a human may comprise the same amino acid sequence as the original human immunoglobulin from which it is derived. In some embodiments, one or more amino acid residues of the human FR are replaced with the corresponding residue from the parent non-human antibody. This is desirable in certain embodiments to allow the humanized antibody or fragment thereof to closely approximate the structure of the non-human parent antibody, thereby optimizing the binding properties (e.g., increasing binding affinity). In certain embodiments, the humanized antibody or antigen-binding fragment thereof provided herein comprises the substitution of no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues in each of the human FR sequences, or the substitution of no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues in all of the FR sequences of the heavy or light chain variable domains. In some embodiments, such changes in amino acid residues may be present only in the heavy chain FR regions, only in the light chain FR regions, or in both chains. In certain embodiments, one or more amino acids of the human FR sequences are randomly mutated to increase binding affinity. In certain embodiments, one or more amino acids of the human FR sequences are backmutated to the corresponding amino acid of the parent non-human antibody to increase binding affinity.

[0123]

[0163] In certain embodiments, the present disclosure also provides humanized anti-TSLP antibodies and antigen-binding fragments thereof, comprising: 21 VQLVQSGAEVKKPGX 22 SX 23 KX 24 SCKX 25 Heavy chain HFR1, WVRQX, comprising the sequence of S (SEQ ID NO: 65) or a homologous sequence having at least 80% sequence identity thereto 27 PGX 28 Heavy chain HFR2, X comprising the sequence GLEWMG (SEQ ID NO: 66) or a homologous sequence having at least 80% sequence identity thereto 31 VTX 32 X 33 X 34 DX 35 SX 36 STX 37 YX 38 X 39 X40 SSLX 41 X 42 X 43 DTAX 44 and a heavy chain HFR3 having a sequence of SEQ ID NO: 35 or a homologous sequence having at least 80% sequence identity thereto, and a heavy chain HFR4 having a sequence of SEQ ID NO: 35 or a homologous sequence having at least 80% sequence identity thereto, 21 is E or Q, and X 22 is E, S, or A, and X 23 is L or V, and X 24 is I or V, and X 25 is S or A, and X 27 is M or A, and X 28 is K or Q, and X 31 is Q or R and X 32 is I or M, and X 33 is S or T, and X 34 is A or R, and X 35 is K or T, and X 36 is I or T, and X 37 is A or V, and X 38 is L or M, and X 39 is Q or E and X 40 is W or L, and X 41 is K or R, and X 42 is A or S, and X 43 is S or E, and X 44 is M or V,

[0124]

[0164] In certain embodiments, the present disclosure provides humanized anti-TSLP antibodies and antigen-binding fragments thereof, comprising a light chain LFR1, WYQQKPGQSPRPWIX, comprising the sequence of SEQ ID NO: 36 or a homologous sequence having at least 80% sequence identity thereto. 45(SEQ ID NO: 68) or a homologous sequence having at least 80% sequence identity thereto, a light chain LFR3 comprising the sequence of SEQ ID NO: 38 or a homologous sequence having at least 80% sequence identity thereto, and a light chain LFR4 comprising the sequence of SEQ ID NO: 39 or a homologous sequence having at least 80% sequence identity thereto, 45 is Y or F.

[0125]

[0165] In certain embodiments, the disclosure also provides humanized TSLP antibodies and antigen-binding fragments thereof, comprising a heavy chain HFR1 comprising a sequence selected from the group consisting of SEQ ID NOs: 32, 40, 42, 47, and 87, a heavy chain HFR2 comprising a sequence selected from the group consisting of SEQ ID NOs: 33, 43, and 48, a heavy chain HFR3 comprising a sequence selected from the group consisting of SEQ ID NOs: 34, 41, 44, and 49, and a heavy chain HFR4 comprising the sequence of SEQ ID NO: 35, and / or a light chain LFR1 comprising a sequence from the group consisting of SEQ ID NO: 36, a light chain LFR2 comprising a sequence selected from the group consisting of SEQ ID NOs: 37 and 45, a light chain LFR3 comprising the sequence of SEQ ID NO: 38, and a light chain LFR4 comprising the sequence of SEQ ID NO: 39.

[0126]

[0166] In certain embodiments, the disclosure also provides humanized anti-TSLP antibodies and antigen-binding fragments thereof that include HFR1, HFR2, HFR3 and / or HFR4 sequences contained in a heavy chain variable region selected from the group consisting of 39H11Z07-VH (SEQ ID NO:5), 39H11Z11-VH (SEQ ID NO:7), 35G12Z01-VH (SEQ ID NO:86), 35G12Z02-VH (SEQ ID NO:8), and 35G12Z03-VH (SEQ ID NO:10).

[0127]

[0167] In certain embodiments, the disclosure also provides humanized anti-TSLP antibodies and antigen-binding fragments thereof that include an LFR1, LFR2, LFR3 and / or LFR4 sequence contained in a light chain variable region selected from the group consisting of 39H11Z07-VL / 39H11Z11-VL (SEQ ID NO: 6), 35G12Z01-VL / 35G12Z03-VL (SEQ ID NO: 9).

[0128]

[0168] In certain embodiments, the humanized anti-TSLP antibodies and antigen-binding fragments thereof provided herein comprise a heavy chain variable domain sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:86, and SEQ ID NO:10, and / or a light chain variable domain sequence selected from the group consisting of SEQ ID NO:6 and SEQ ID NO:9.

[0129]

[0169] The present disclosure also provides 1) "39H11Z07" comprising the heavy chain variable region of 39H11Z07-VH (SEQ ID NO: 5) and the light chain variable region of 39H11Z07-VL (SEQ ID NO: 6); 2) "mAb39H11", which comprises the heavy chain variable region of 39H11Z07-VH (SEQ ID NO: 5) and the light chain variable region of 39H11Z07-VL (SEQ ID NO: 6), and further comprises an IgG4 Fc region (SEQ ID NO: 80) with the PAA-YTE mutation; 3) "39H11Z11", comprising the heavy chain variable region of 39H11Z11-VH (SEQ ID NO: 7) and the light chain variable region of 39H11Z11-VL (SEQ ID NO: 6); 4) "35G12Z01" comprising the heavy chain variable region of 35G12Z01-VH (SEQ ID NO: 86) and the light chain variable region of 35G12Z01-VL (SEQ ID NO: 9); 5) "35G12Z02" comprising the heavy chain variable region of 35G12Z02-VH (SEQ ID NO: 8) and the light chain variable region of 35G12Z02-VL (SEQ ID NO: 9); 6) "35G12Z03" comprising the heavy chain variable region of 35G12Z03-VH (SEQ ID NO: 10) and the light chain variable region of 35G12Z03-VL (SEQ ID NO: 9); 7) "mAb35G12", which comprises the heavy chain variable region of 35G12Z03-VH (SEQ ID NO: 10) and the light chain variable region of 35G12Z03-VL (SEQ ID NO: 9), and further comprises an IgG4 Fc region (SEQ ID NO: 75) having a PAA-YTE mutation; 8) "mAb35G12_IgG1" containing the heavy chain variable region of 35G12Z03-VH (SEQ ID NO: 10) and the light chain variable region of 35G12Z03-VL (SEQ ID NO: 9) and further containing the Fc region of IgG1 (SEQ ID NO: 88) Exemplary humanized antibodies of 39H11 or 35G12 are also provided, including:

[0130]

[0170] These exemplary humanized anti-TSLP antibodies retained specific binding ability or affinity for TSLP and were at least as good or better in that respect than the parental murine antibodies 39H11 or 35G12. Data are provided, for example, in Example 7.

[0131]

[0171] In some embodiments, the anti-TSLP antibodies and antigen-binding fragments provided herein comprise all or a portion of the heavy chain variable domain and / or all or a portion of the light chain variable domain. In one embodiment, the anti-TSLP antibodies or antigen-binding fragments provided herein are single domain antibodies consisting of all or a portion of the heavy chain variable domain provided herein. Further information on such single domain antibodies is available in the art (see, for example, U.S. Patent No. 6,248,516).

[0132]

[0172] In certain embodiments, the anti-TSLP antibodies or antigen-binding fragments thereof provided herein further comprise an immunoglobulin (Ig) constant region, optionally including heavy and / or light chain constant regions. In certain embodiments, the heavy chain constant region comprises a CH1, hinge, and / or CH2-CH3 region (or optionally a CH2-CH3-CH4 region). In certain embodiments, the anti-TSLP antibodies or antigen-binding fragments thereof provided herein comprise a human IgG1, IgG2, IgG3, or IgG4 heavy chain constant region. In certain embodiments, the light chain constant region comprises a Cκ or Cλ. The constant region of the anti-TSLP antibodies or antigen-binding fragments thereof provided herein may be identical to the sequence of the wild-type constant region or may differ in one or more mutations.

[0133]

[0173] In certain embodiments, the heavy chain constant region comprises an Fc region. The Fc region is known to mediate effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of antibodies. The Fc regions of different Ig isotypes have different abilities to induce effector functions. For example, it is recognized that the Fc regions of IgG1 and IgG3 induce both ADCC and CDC more effectively than the Fc regions of IgG2 and IgG4. In certain embodiments, the anti-TSLP antibodies and antigen-binding fragments thereof provided herein comprise an Fc region of an IgG1 or IgG3 isotype capable of inducing ADCC or CDC, or comprise a constant region of an IgG4 or IgG2 isotype with reduced or depleted effector function. In certain embodiments, the anti-TSLP antibodies and antigen-binding fragments thereof provided herein comprise a wild-type human IgG4 Fc region or other wild-type human IgG4 alleles. In certain embodiments, the anti-TSLP antibody or antigen-binding fragment thereof provided herein comprises a human IgG4 Fc region comprising one or more mutations selected from the group consisting of S228P, F234A, L235A, M252Y, S254T, T256E, and K447del. In certain embodiments, the anti-TSLP antibody or antigen-binding fragment thereof provided herein comprises a human IgG4 Fc region comprising S228P, F234A, L235A, M252Y, S254T, T256E, and K447del. In certain embodiments, the anti-TSLP antibody or antigen-binding fragment thereof provided herein comprises a heavy chain constant region having the amino acid sequence set forth in SEQ ID NO: 75. In certain embodiments, the anti-TSLP antibody or antigen-binding fragment thereof provided herein comprises a human IgG1 Fc region comprising one or more mutations selected from the group consisting of L234A, L235A, M252Y, S254T, and T256E. In certain embodiments, the anti-TSLP antibodies or antigen-binding fragments thereof provided herein comprise a human IgG1 Fc region that includes the following mutations: L234A, L235A, M252Y, S254T, and T256E.In certain embodiments, an anti-TSLP antibody or antigen-binding fragment thereof provided herein comprises a heavy chain constant region having the amino acid sequence set forth in SEQ ID NO:88.

[0134]

[0174] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein further comprise a signal peptide at the N-terminus of the heavy chain variable region and / or a signal peptide at the N-terminus of the light chain variable region. In certain embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 76 (MGWSCIILFLVATATGVHS).

[0135]

[0175] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein have sufficient specific binding affinity for TSLP to provide for diagnostic and / or therapeutic uses.

[0136]

[0176] The antibodies or antigen-binding fragments thereof provided herein can be monoclonal, polyclonal, humanized, chimeric, recombinant, bispecific, multispecific, labeled, bivalent, anti-idiotypic, or fusion proteins. Recombinant antibodies are antibodies prepared using recombinant methods in vitro, rather than in an animal.

[0137]

[0177] In certain embodiments, the disclosure provides an anti-TSLP antibody or antigen-binding fragment thereof that does not compete for binding to human TSLP with an antibody comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 77 and a light chain variable region comprising the sequence of SEQ ID NO: 78, and wherein the antibody or antigen-binding fragment thereof is not AMG157.

[0138]

[0178] As used herein, "AMG157" means an antibody or antigen-binding fragment thereof comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO:77 and a light chain variable region having the amino acid sequence of SEQ ID NO:78.

[0139]

[0179] Antibody variants

[0180] The antibodies and antigen-binding fragments thereof provided herein also encompass various variants of the antibody sequences provided herein.

[0140]

[0181] In certain embodiments, antibody variants comprise one or more of the CDR sequences provided in Tables 1 and 2 above, one or more of the non-CDR sequences of the heavy or light chain variable regions provided in Tables 3 and 4 above, and / or one or more modifications or substitutions in the constant region (e.g., Fc region). Such variants retain the binding specificity of their parent antibodies for TSLP, but have one or more desirable properties imparted by the modifications or substitutions. For example, antibody variants may have improved antigen binding affinity, improved glycosylation patterns, reduced risk of glycosylation, reduced deamination, reduced or eliminated effector function, improved FcRn receptor binding, increased pharmacokinetic half-life, pH sensitivity, and / or suitability for conjugation (e.g., one or more introduced cysteine ​​residues).

[0141]

[0182] Methods known in the art, such as "alanine scanning mutagenesis," may be used to screen parent antibody sequences to identify suitable or preferred residues for modification or substitution (see, e.g., Cunningham and Wells (1989) Science, 244:1081-1085). Briefly, target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) can be identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine), and modified antibodies are produced and screened for the desired properties. If substitution at a particular amino acid position demonstrates a desired change in function, that position can be identified as a potential residue for modification or substitution. Potential residues may be further evaluated by substituting different types of residues (e.g., cysteine ​​residues, positively charged residues, etc.).

[0142]

[0183] Affinity variants

[0184] The affinity variants of the antibodies may include modifications or substitutions in one or more of the CDR sequences provided in Tables 1 and 2 above, one or more of the FR sequences provided in Table 5 above, or the heavy or light chain variable region sequences provided in Tables 3 and 4 above. Since it is known in the art that a CDR region is adjacent to two FR regions in the variable region, the FR sequences can be easily identified by one of skill in the art based on the CDR sequences in Tables 1 and 2 above and the variable region sequences in Tables 3 and 4 above. The affinity variants retain the specific binding affinity of the parent antibody to TSLP, or even have improved specific binding affinity for TSLP over the parent antibody. In certain embodiments, at least one (or all) of the substitutions in the CDR sequences, FR sequences, or variable region sequences include conservative substitutions.

[0143]

[0185] Those skilled in the art will understand that one or more amino acid residues may be substituted in the CDR sequences provided in Tables 1 and 2 above, and the variable region sequences provided in Tables 3 and 4 above, and the resulting antibody or antigen-binding fragment still retains or even has improved binding affinity or ability to TSLP. To this end, various methods known in the art can be used. For example, phage display technology can be used to generate and express libraries of antibody variants (such as Fab or scFv variants), which are then screened for binding affinity to human TSLP. As another example, computer software can be used to virtually mimic the binding of an antibody to human TSLP, and amino acid residues on the antibody that form the binding interface can be identified. Such residues may be avoided in substitutions to prevent a reduction in binding affinity, or may be targeted for substitution to provide stronger binding.

[0144]

[0186] In certain embodiments, the humanized antibody or antigen-binding fragment thereof provided herein comprises one or more amino acid residue substitutions in one or more of the CDR sequences and / or in one or more of the FR sequences. In certain embodiments, the affinity variant comprises a total of 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or less substitutions in the CDR sequences and / or in the FR sequences.

[0145]

[0187] In certain embodiments, an anti-TSLP antibody or antigen-binding fragment thereof comprises one, two, or three CDR sequences that have at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to the CDRs listed in Tables 1 and 2 above, yet retains specific binding affinity for TSLP at similar or even higher levels than its parent antibody.

[0146]

[0188] In certain embodiments, an anti-TSLP antibody or antigen-binding fragment thereof comprises one or more variable region sequences having at least 80% (e.g., at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) sequence identity to a variable region sequence listed in Tables 3 and 4 above, yet retains specific binding affinity for TSLP at a similar or even higher level than its parent antibody. In some embodiments, a total of 1-10 amino acids are substituted, inserted, or deleted in the variable region sequences listed in Tables 3 and 4 above. In some embodiments, the substitutions, insertions, or deletions occur in regions outside of the CDRs (e.g., within the FRs).

[0147]

[0189] Glycosylation variants

[0190] The anti-TSLP antibodies or antigen-binding fragments thereof provided herein also encompass glycosylation variants obtained by increasing or decreasing the degree of glycosylation of the antibodies or antigen-binding fragments thereof.

[0148]

[0191] The antibody or antigen-binding fragment thereof may contain one or more modifications that introduce or remove glycosylation sites. A glycosylation site is an amino acid residue having a side chain to which a carbohydrate moiety (e.g., an oligosaccharide structure) can be attached. Glycosylation of an antibody is typically N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue, e.g., an asparagine residue in a tripeptide sequence, such as asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline. O-linked glycosylation refers to the attachment of one of the sugars, N-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid, most commonly serine or threonine. Removal of a native glycosylation site can be conveniently accomplished, for example, by modifying the amino acid sequence such that one of the above tripeptide sequences (for N-linked glycosylation sites) or a serine or threonine residue (for O-linked glycosylation sites) present in the sequence is substituted. New glycosylation sites can likewise be created by introducing such a tripeptide sequence or a serine or threonine residue.

[0149]

[0192] In certain embodiments, the anti-TSLP antibodies and antigen-binding fragments provided herein contain a mutation at N297 (e.g., N297A, N297Q, or N297G) to remove a glycosylation site.

[0150]

[0193] Cysteine ​​engineered variants

[0194] The anti-TSLP antibodies or antigen-binding fragments thereof provided herein also include cysteine ​​engineered variants that contain one or more introduced free cysteine ​​amino acid residues.

[0151]

[0195] A free cysteine ​​residue is a residue that is not part of a disulfide bridge. Cysteine ​​engineered variants are useful for conjugating, for example, with cytotoxic and / or imaging compounds, labels, or especially radioisotopes, at the site of engineered cysteine, for example, by maleimide or haloacetyl. Methods for engineering antibodies or antigen-binding fragments thereof to introduce free cysteine ​​residues are known in the art, see, for example, WO2006 / 034488.

[0152]

[0196] Fc variants

[0197] The anti-TSLP antibodies or antigen-binding fragments thereof provided herein also include Fc variants that contain modifications or substitutions of one or more amino acid residues in the Fc region and / or hinge region to provide altered effector functions, such as, for example, ADCC and CDC. Methods for modifying ADCC activity by antibody engineering have been described in the art, see, for example, Shields RL.et al., J Biol Chem.2001.276(9):6591-604; Idusogie EE.et al., J Immunol.2000.164(8):4178-84; Steurer W.et al., J Immunol.1995,155(3):1165- 74; Idusogie EE.et al., J Immunol.2001,166(4):2571-5; Lazar GA.et al., PNAS,2006,103(11):4005-4010; Ryan MC.et al., Mol.Cancer Ther.,2007,6:3009-3018; Richards JO,.et al., Mol Cancer See Ther. 2008, 7(8):2517-27; Shields R Let al., J. Biol. Chem, 2002, 277:26733-26740; Shinkawa T. et al., J. Biol. Chem, 2003, 278:3466-3473.

[0153]

[0198] The CDC activity of the antibodies or antigen-binding fragments provided herein can also be modified, for example, by improving or diminishing C1q binding and / or CDC (see, e.g., WO99 / 51642; Duncan & Winter Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO94 / 29351 for other examples of Fc region variants). One or more amino acids selected from amino acid residues 329, 331, and 322 of the Fc region can be replaced with different amino acid residues to modify C1q binding and / or reduce or eliminate complement-dependent cytotoxicity (CDC) (see, e.g., U.S. Patent No. 6,194,551 by Idusogie et al.). One or more amino acid substitutions can also be introduced to modify the ability of the antibody to fix complement (see, PCT Publication WO 94 / 29351 by Bodmer et al.).

[0154]

[0199] In certain embodiments, an anti-TSLP antibody or antigen-binding fragment thereof provided herein has reduced effector function and comprises one or more amino acid substitutions in IgG1 at positions selected from the group consisting of 234, 235, 237, and 238, 268, 297, 309, 330, and 331. In certain embodiments, an anti-TSLP antibody or antigen-binding fragment thereof provided herein is of the IgG1 isotype and comprises one or more amino acid substitutions selected from the group consisting of N297A, N297Q, N297G, L235E, L234A, L235A, L234F, L235E, P331S, and any combination thereof. In certain embodiments, the anti-TSLP antibodies or antigen-binding fragments thereof provided herein are of the IgG2 isotype and contain one or more amino acid substitutions selected from the group consisting of H268Q, V309L, A330S, P331S, V234A, G237A, P238S, H268A, or any combination thereof (e.g., H268Q / V309L / A330S / P331S, V234A / G237A / P238S / H268A / V309L / A330S / P331S). In certain embodiments, the anti-TSLP antibodies or antigen-binding fragments thereof provided herein are of the IgG4 isotype and contain one or more amino acid substitutions selected from the group consisting of N297A, N297Q, N297G, L235E, L234A, L235A, M252Y / S254T / T256E, T307Q / N434, and any combination thereof. In certain embodiments, the anti-TSLP antibodies or antigen-binding fragments thereof provided herein are of the IgG2 / IgG4 cross-isotype. Examples of IgG2 / IgG4 cross-isotypes are described in Rother RP et al., Nat Biotechnol 25:1256-1264 (2007).

[0155]

[0200] In certain embodiments, the anti-TSLP antibodies and antigen-binding fragments provided herein are of the IgG4 isotype and contain the following amino acid substitutions: S228P / F234A / L235A (PAA). In certain embodiments, the anti-TSLP antibodies and antigen-binding fragments provided herein are of the IgG4 isotype and contain the following amino acid substitutions: M252Y / S254T / T256 (YTE). In certain embodiments, the anti-TSLP antibodies and antigen-binding fragments provided herein are of the IgG4 isotype and contain the following amino acid deletions: K447del.

[0156]

[0201] In certain embodiments, the anti-TSLP antibodies and antigen-binding fragments provided herein are of the IgG4 isotype and contain one or more amino acid substitutions, for example at position 228. In certain embodiments, the anti-TSLP antibodies and antigen-binding fragments provided herein are of the IgG4 isotype and contain one or more mutations in the Fc region selected from the group consisting of S228P, F234A, L235A, M252Y, S254T, T256E, and K447del, or any combination thereof. In certain embodiments, the anti-TSLP antibodies or antigen-binding fragments provided herein comprise a heavy chain constant region having the amino acid sequence set forth in SEQ ID NO:75.

[0157]

[0202] In certain embodiments, the anti-TSLP antibodies and antigen-binding fragments provided herein are of the IgG1 isotype and contain the following amino acid substitutions: L234A / L235A (LALA). In certain embodiments, the anti-TSLP antibodies and antigen-binding fragments provided herein are of the IgG1 isotype and contain the following amino acid substitutions: M252Y / S254T / T256 (YTE). In certain embodiments, the anti-TSLP antibodies and antigen-binding fragments provided herein are of the IgG1 isotype and contain one or more mutations in the Fc region selected from the group consisting of L234A, L235A, M252Y, S254T, and T256E, or any combination thereof. In certain embodiments, the anti-TSLP antibodies or antigen-binding fragments provided herein contain a heavy chain constant region having the amino acid sequence set forth in SEQ ID NO:88.

[0158]

[0203] In certain embodiments, the anti-TSLP antibody or antigen-binding fragment thereof contains one or more amino acid substitutions that improve pH-dependent binding to the neonatal Fc receptor (FcRn). Such variants can have an extended pharmacokinetic half-life because they bind to FcRn at acidic pH, allowing it to escape degradation in lysosomes and then be translocated and released extracellularly. Methods for engineering antibodies or antigen-binding fragments thereof to improve binding affinity to FcRn are known in the art, see, e.g., Vaughn, D. et al., Structure, 6(1):63-73, 1998; Kontermann, R. et al., Antibody Engineering, Volume 1, Chapter 27: Engineering of the Fc region for improved PK, published by Springer, 2010; Yeung, Y. et al., Cancer Research, 70:3269-3277(2010); and Hinton, P. et al., J. Immunology, 176:346-356(2006).

[0159]

[0204] In certain embodiments, the anti-TSLP antibody or antigen-binding fragment thereof comprises one or more amino acid substitutions that facilitate and / or promote heterodimerization at the interface of the Fc region. These modifications include the introduction of a protuberance into a first Fc polypeptide and a cavity into a second Fc polypeptide, where the protuberance is located within the cavity to facilitate interaction of the first and second Fc polypeptides to form a heterodimer or complex. Methods for generating antibodies with these modifications are known in the art, for example, as described in U.S. Patent No. 5,731,168.

[0160]

[0205] Antigen-binding fragments

[0206] Anti-TSLP antigen-binding fragments are also provided herein. Various types of antigen-binding fragments are known in the art and can be developed based on the anti-TSLP antibodies provided herein, including, for example, the exemplary antibodies whose CDRs are shown in Tables 1 and 2 above and whose variable sequences are shown in Tables 3 and 4 above, as well as various variants thereof (affinity variants, glycosylation variants, Fc variants, cysteine ​​engineered variants, etc.).

[0161]

[0207] In certain embodiments, the anti-TSLP antigen-binding fragments provided herein are diabodies, Fab, Fab', F(ab')2, Fd, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), multispecific antibodies, camelized single-chain domain antibodies, nanobodies, domain antibodies, and bivalent domain antibodies.

[0162]

[0208] A variety of techniques can be used to produce such antigen-binding fragments. Illustrative methods include enzymatic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)), recombinant expression by host cells such as E. coli (e.g., for Fab, Fv, and ScFv antibody fragments), screening from phage display libraries as discussed above (e.g., for ScFv), and chemical coupling of two Fab'-SH fragments to form F(ab')2 fragments (Carter et al., Bio / Technology 10:163-167 (1992)). Other techniques for the production of antibody fragments will be apparent to those skilled in the art.

[0163]

[0209] In certain embodiments, the antigen-binding fragment is an scFv. The generation of scFvs is described, for example, in WO 93 / 16185; U.S. Patent Nos. 5,571,894 and 5,587,458. ScFvs can be fused to effector proteins at the amino or carboxyl terminus to provide fusion proteins (see, for example, Antibody Engineering, edited by Borrebaeck).

[0164]

[0210] In certain embodiments, the anti-TSLP antibodies or antigen-binding fragments thereof provided herein are bivalent, tetravalent, hexavalent, or multivalent. Any molecule with more than two valencies is considered multivalent, including, for example, trivalent, tetravalent, hexavalent, etc.

[0165]

[0211] A bivalent molecule is monospecific if both binding sites are specific for binding to the same antigen or epitope. In certain embodiments, this provides stronger binding to antigen or epitope than its monovalent counterpart. Similarly, multivalent molecules can also be monospecific. In certain embodiments, in a bivalent or multivalent antigen-binding moiety, the first valency of the binding site and the second valency of the binding site are structurally identical (i.e., have the same sequence) or structurally different (i.e., have the same specificity but different sequence).

[0166]

[0212] Bivalent molecules can also be bispecific if the two binding sites are specific for different antigens or epitopes. This also applies to multivalent molecules. For example, a trivalent molecule can be bispecific if the two binding sites are monospecific for a first antigen (or epitope) and the third binding site is specific for a second antigen (or epitope).

[0167]

[0213] Bispecific antibodies

[0214] In certain embodiments, the anti-TSLP antibody or antigen-binding fragment thereof is bispecific, hi certain embodiments, the antibody or antigen-binding fragment thereof is further linked to a second functional moiety having a different binding specificity than the TSLP antibody or antigen-binding fragment thereof.

[0168]

[0215] In certain embodiments, the bispecific antibodies or antigen-binding fragments thereof provided herein are capable of specifically binding to a second antigen other than TSLP, or a second epitope on TSLP. In some embodiments, the second antigen other than TSLP is selected from the group consisting of IL-33, IL-25, IL-4, IL-5, IL-4R, IL-13, etc.

[0169]

[0216] Conjugates

[0217] In some embodiments, the anti-TSLP antibody or antigen-binding fragment thereof further comprises one or more conjugate moieties. The conjugate moiety can be linked to the antibody or antigen-binding fragment thereof. The conjugate moiety is a moiety that can bind to the antibody or antigen-binding fragment thereof. It is contemplated that various conjugate moieties can be linked to the antibody or antigen-binding fragment thereof provided herein (see, for example, "Conjugate Vaccines", Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr. (eds.), Carger Press, New York, (1989)). These conjugate moieties can be linked to the antibody or antigen-binding fragment thereof by covalent binding, affinity binding, intercalation, coordinate binding, complexing, association, blending, or addition, among others. In some embodiments, the antibody or antigen-binding fragment thereof can be linked to one or more conjugates via a linker.

[0170]

[0218] In certain embodiments, the antibodies or antigen-binding fragments thereof provided herein may be engineered to contain specific sites in addition to the epitope-binding moiety that are available for attachment to one or more conjugate moieties. For example, such sites may contain one or more reactive amino acid residues, such as cysteine ​​or histidine residues, that facilitate covalent linkage to the conjugate moiety.

[0171]

[0219] In some embodiments, the antibody moiety is linked to the conjugate moiety through a chemical bond or linker, hi some embodiments, the antibody moiety and the conjugate moiety are linked using a variety of well-known bifunctional reagents and chemistries suitable for conjugation to proteins. Such reagents include, but are not limited to, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HQ), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds bis-(p-azidobenzoyl)-hexane-diamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene-2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene).

[0172]

[0220] In certain embodiments, the antibody or antigen-binding fragment thereof may be indirectly linked to the conjugate moiety or through another conjugate moiety.For example, the antibody or antigen-binding fragment thereof provided herein may be conjugated to biotin and then indirectly conjugated to a second conjugate conjugated to avidin.In some embodiments, the conjugate moiety comprises a clearance modifier (e.g., a polymer such as PEG that extends half-life), a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a detectable label (e.g., a luminescent label, a fluorescent label, an enzyme substrate label), a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binder, a purification moiety, or other therapeutic agent or drug.

[0173]

[0221] Therapeutic agents or drugs useful as conjugate moieties can be those useful for treating asthma, polypoid rhinosinusitis, chronic obstructive pulmonary disease (COPD), urticaria, eosinophilic esophagitis (EoE), atopic dermatitis, and the like.

[0174]

[0222] In some embodiments, the conjugate moiety comprises a therapeutic agent or drug for treating asthma. In some embodiments, the conjugate moiety comprises a beta2-adrenergic receptor agonist (SABA, e.g., salbutamol, salmeterol, formoterol, etc.), an adrenergic agonist (e.g., inhaled epinephrine), a corticosteroid (preferably in an inhaled form, e.g., beclomethasone), a leukotriene receptor antagonist (e.g., montelukast and zafirlukast), a mast cell stabilizer (e.g., cromolyn sodium), a macrolide antibiotic, or a combination thereof.

[0175]

[0223] In some embodiments, the conjugate moiety includes a therapeutic agent or drug for treating polypoid rhinosinusitis, hi some embodiments, the conjugate moiety includes antibiotics and small molecules such as kinase inhibitors, steroids, and the like.

[0176]

[0224] In some embodiments, the conjugate moiety comprises a therapeutic agent or drug for treating COPD. In some embodiments, the conjugate moiety comprises an antibiotic (including amoxicillin, doxycycline, and azithromycin), a beta2-adrenergic agonist, an anticholinergic (e.g., ipratropium and tiotropium), a long-acting muscarinic antagonist (LAMA, e.g., tiotropium), aclidinium, the LAMA umeclidinium bromide, a corticosteroid, a phosphodiesterase-4 inhibitor (PDE4 inhibitor, e.g., roflumilast), and the like.

[0177]

[0225] In some embodiments, the conjugate moiety comprises a therapeutic agent or drug for treating urticaria. In some embodiments, the conjugate moiety comprises an antihistamine, a systemic steroid, a leukotriene-receptor antagonist, an anti-inflammatory drug, omalizumab, an immunosuppressant, etc. Non-limiting exemplary antihistamines include diphenhydramine, hydroxyzine, loratadine, cetirizine, desloratadine, fexofenadine, etc.

[0178]

[0226] In some embodiments, the conjugate moiety comprises a therapeutic agent or drug for treating EoE, hi some embodiments, the conjugate moiety comprises a proton pump inhibitor (PPI), a corticosteroid, or the like.

[0179]

[0227] In some embodiments, the conjugate moiety comprises a therapeutic agent or drug for treating atopic dermatitis. In some embodiments, the conjugate moiety comprises a corticosteroid (e.g., hydrocortisone), a calcineurin inhibitor (e.g., tacrolimus or pimecrolimus), tacrolimus, pimecrolimus, a PDE-4 inhibitor (e.g., crisaborole), a systemic immunosuppressant (e.g., cyclosporine, methotrexate, interferon gamma-1b, mycophenolate mofetil, and azathioprine), an additional antibody (e.g., dupilumab, tralokinumab), and the like.

[0180]

[0228] In some embodiments, the conjugate moiety comprises an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordi protein, dianthin protein, Phytolacca americana protein, Momordica chalantia inhibitor, curcin, crotin, Sapoonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and the trichothecenes.

[0181]

[0229] A "toxin" can be any agent that is detrimental to or damages or kills a cell. Examples of toxins include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, MMAE, MMAF, DM1, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin and analogs thereof, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethate, 5-fluorouracil decarbazine), 5-fluorouracil decarbazine, ... These include, without limitation, cisplatin, thioepachlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP) cisplatin, anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), antimitotic agents (e.g., vincristine and vinblastine), topoisomerase inhibitors, and tubulin binders.

[0182]

[0230] Examples of detectable labels include fluorescent labels (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or Texas Red), enzyme substrate labels (e.g., horseradish peroxidase, alkaline phosphatase, luciferase, glucoamylase, lysozyme, saccharide oxidase, or β-D-galactosidase), radioisotopes, luminescent labels, chromogenic moieties, digoxigenin, biotin / avidin, DNA molecules, or gold for detection. A variety of radioisotopes are available for the production of such radioconjugates. Examples include: 211 At,131 I, 125 I, 90 Y, 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P, 212 Radioisotopes include Pb, and Lu. In some embodiments, the conjugate moiety may include a radioisotope for scintigraphic detection, or a spin label for NMR detection or MRI. Suitable radioisotopes or spin labels include: 123 I, 131 I, 111 In, 13 C. 19 F, 15 N, 17 Examples include various isotopes of O, Gd, Mn, and Fe.

[0183]

[0231] In certain embodiments, the conjugate moiety may be a clearance modifier that helps to extend the half-life of the antibody. Illustrative examples include water-soluble polymers, such as PEG, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, ethylene glycol / propylene glycol copolymers, and others. The polymers may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody varies, and when two or more polymers are attached, they may be the same or different molecules.

[0184]

[0232] In certain embodiments, the conjugated moiety can be a purification moiety, such as a magnetic bead.

[0233] In certain embodiments, an antibody or antigen-binding fragment thereof provided herein is used as the basis for a conjugate.

[0185]

[0234] Polynucleotides and Recombinant Methods

[0235] The present disclosure provides an isolated polynucleotide encoding an anti-TSLP antibody or antigen-binding fragment thereof provided herein. As used herein, the term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single- or double-stranded form. Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed base and / or deoxyinosine residues (see Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0186]

[0236] DNA encoding a monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of the antibody). The encoding DNA may also be obtained by synthetic methods.

[0187]

[0237] The isolated polynucleotide encoding an anti-TSLP antibody or antigen-binding fragment thereof can be inserted into a vector for further cloning (amplification of the DNA) or expression using recombinant techniques known in the art. Many vectors are available. The vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter (e.g., SV40, CMV, EF-1α), and a transcription termination sequence.

[0188]

[0238] The present disclosure provides a vector comprising the isolated polynucleotide provided herein. In certain embodiments, the polynucleotide provided herein encodes an antibody or antigen-binding fragment thereof, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selectable marker. Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papilloma viruses, papova viruses (e.g., SV40), lambda phage, and M13 phage, plasmids pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, p GEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, p Includes LexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos, etc.

[0189]

[0239] A vector containing a polynucleotide sequence encoding an antibody or an antigen-binding fragment thereof can be introduced into a host cell for cloning or gene expression. Suitable host cells for cloning or DNA expression in a vector herein are prokaryotic cells, yeast, or higher eukaryotic cells as described above. Suitable prokaryotic cells for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, such as Escherichia, Enterobacteriaceae, such as E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, such as Serratia marcescans, and Shigella, as well as Bacillus, such as B. subtilis and B. licheniformis, Pseudomonas, such as P. aeruginosa, and Streptomyces.

[0190]

[0240] In addition to prokaryotes, eukaryotic microbes, such as filamentous fungi or yeast, are suitable cloning or expression hosts for anti-TSLP antibody-encoding vectors. Among lower eukaryotic host microorganisms, Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used. However, Schizosaccharomyces pombe is capable of inhibiting Kluyveromyces hosts such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus, Yarrowia (EP 402,226), Pichia pastoris (EP 183,070), Candida, Trichoderma reesia (EP 244,234), Neurospora crassa, Schwanniomyces Many other genera, species, and strains are commonly available and useful herein, such as Schwanniomyces, e.g., A. occidentalis, and filamentous fungi, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, e.g., A. nidulans and A. niger.

[0191]

[0241] Suitable host cells for the expression of the glycosylated antibodies or antigen-binding fragments thereof provided herein are derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains and variants have been identified, as well as corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori. Various virus strains for transfection, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, are publicly available, and such viruses may be used as viruses herein in accordance with the present invention, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco may also be used as hosts.

[0192]

[0242] However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become routine procedure. Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651), human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK, ATCC CCL 10), Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human hepatocytes (Hep G2, HB 8065), mouse mammary tumor (MMT 060562, ATCC CCL51), TRI cells (Mather et al., Annals NYAcad. Sci. 383:44-68 (1982)), MRC5 cells, FS4 cells, and human hepatocellular carcinoma line (Hep G2). In some embodiments, the host cell is a mammalian cultured cell line, such as CHO, BHK, NS0, 293, and their derivatives.

[0193]

[0243] The host cell is transformed with the above expression or cloning vector for the production of anti-TSLP antibody and cultured in a conventional nutrient medium modified as appropriate for inducing promoters, selecting transformants, or amplifying genes encoding desired sequences. In another embodiment, the antibody may be produced by homologous recombination as known in the art. In certain embodiments, the host cell can produce the antibody or antigen-binding fragment thereof provided herein.

[0194]

[0244] The present disclosure also provides a method for expressing the antibody or antigen-binding fragment thereof provided herein, comprising culturing a host cell provided herein under conditions in which the vector of the present disclosure is expressed. The host cells used to produce the antibody or antigen-binding fragment thereof provided herein may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM) (Sigma) are suitable for culturing the host cells. Additionally, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, or 5,122,469, WO 90 / 03430, WO 87 / 00195, or U.S. Pat. Re. 30,985 may be used as a culture medium for the host cells. Any of these media may be supplemented as necessary with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as the GENTAMYCIN™ drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements known to those of skill in the art may also be included at appropriate concentrations. Temperature, pH, and other culture conditions will be those previously used with the host cell selected for expression and will be apparent to those of skill in the art.

[0195]

[0245] When recombinant techniques are used, antibodies can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, particulate debris of host cells or lysed fragments are removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) describes a procedure for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes. Cell debris can be removed by centrifugation. If the antibody is secreted into the medium, the supernatant from such expression systems is generally first concentrated using a commercially available protein concentration filter, for example, a Pellicon ultrafiltration unit from Amicon or Millipore. A protease inhibitor such as PMSF may be included in any of the above steps to inhibit proteolysis, and an antibiotic may be included to prevent the growth of adventitious contaminants.

[0196]

[0246] Anti-TSLP antibodies or antigen-binding fragments thereof prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, DEAE-cellulose ion exchange chromatography, ammonium sulfate precipitation, salting out, and affinity chromatography, with affinity chromatography being the preferred purification technique.

[0197]

[0247] In certain embodiments, Protein A immobilized on a solid phase is used for immunoaffinity purification of antibodies and their antigen-binding fragments. The suitability of Protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human gamma 1, gamma 2, or gamma 4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human gamma 3 (Guss et al., EMBO J. 5:1567 1575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, although other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. If the antibody contains a CH3 domain, Bakerbond ABX™ resin (JT Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification, such as fractionation on ion exchange columns, ethanol precipitation, reversed-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, are also available depending on the antibody to be recovered.

[0198]

[0248] Following any preliminary purification steps, the mixture containing the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH of about 2.5 to 4.5, preferably performed at a low salt concentration (e.g., about 0 to 0.25 M salt).

[0199]

[0249] Pharmaceutical Compositions

[0250] The disclosure further provides pharmaceutical compositions comprising an anti-TSLP antibody, or antigen-binding fragment thereof, and one or more pharma- ceutically acceptable carriers.

[0200]

[0251] The present disclosure further provides a pharmaceutical composition comprising a polynucleotide encoding an anti-TSLP antibody or an antigen-binding fragment thereof, and one or more pharma- ceutically acceptable carriers. The antibodies provided herein can also be produced in vivo by delivery of a polynucleotide encoding an antibody or an antigen-binding fragment thereof, such as an in-vitro transcribed mRNA or an expression vector. Methods for delivery of polynucleotides for in vivo antibody expression are known in the art, see, for example, Rybakova, Y. et al, Molecular Therapy, vol. 27 (8), pp. 1415-1423 (2019); Deal, CE et al, Vaccines, 2021, 9, 108.

[0201]

[0252] The disclosure further provides a pharmaceutical composition comprising an expression vector comprising a polynucleotide encoding an anti-TSLP antibody, or antigen-binding fragment thereof, and one or more pharma- ceutically acceptable carriers.

[0202]

[0253] In certain embodiments, the expression vector comprises a viral vector or a non-viral vector. Examples of viral vectors include, but are not limited to, adeno-associated viral (AAV) vectors, lentiviral vectors, retroviral vectors, and adenoviral vectors. Examples of non-viral vectors include naked DNA, plasmids, exosomes, mRNA, and the like. In certain embodiments, the expression vector is suitable for gene therapy in humans. Vectors suitable for gene therapy include, for example, adeno-associated viral (AAV) or adenoviral vectors. In certain embodiments, the expression vector comprises a DNA vector or an RNA vector. In certain embodiments, the pharmaceutically acceptable carrier is a polymer excipient, for example, but not limited to, a microsphere, a microcapsule, a polymeric micelle, and a dendrimer. The polynucleotides or polynucleotide vectors of the present disclosure may be encapsulated in, attached to, or coated with polymer-based moieties by methods known in the art (see, e.g., W. Heiser, Nonviral gene transfer techniques, published by Humana Press, 2004; U.S. Pat. No. 6,025,337; Advanced Drug Delivery Reviews, 57(15):2177-2202 (2005)).

[0203]

[0254] Pharmaceutically acceptable carriers for use in the pharmaceutical compositions disclosed herein may include, for example, pharma- ceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, sequestrants or chelating agents, diluents, adjuvants, excipients, or other non-toxic auxiliary substances, ingredients known in the art, or various combinations thereof.

[0204]

[0255] Suitable ingredients may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickening agents, coloring agents, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylated hydroxyanisole, butylated hydroxytoluene, and / or propyl gallate. As disclosed herein, the inclusion of one or more antioxidants, such as methionine, in compositions comprising the antibodies or antigen-binding fragments thereof and conjugates provided herein reduces oxidation of the antibodies or antigen-binding fragments thereof. This reduction in oxidation prevents or reduces loss of binding affinity, thereby improving the stability of the antibodies and maximizing shelf life. Thus, in certain embodiments, pharmaceutical compositions are provided that include one or more antibodies or antigen-binding fragments thereof disclosed herein and one or more antioxidants, such as methionine. Further provided are methods of preventing oxidation, extending shelf life, and / or improving efficacy of the antibodies or antigen-binding fragments provided herein by combining the antibodies or antigen-binding fragments with one or more antioxidants, such as methionine.

[0205]

[0256] To further illustrate, pharma- ceutically acceptable carriers may include, for example, aqueous vehicles such as sodium chloride for injection, Ringer's injection, isotonic dextrose injection, sterile water for injection, or dextrose and lactated Ringer's injection; non-aqueous vehicles such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil, or peanut oil; antimicrobial agents in bacteriostatic or fungistatic concentrations; isotonic agents such as sodium chloride or dextrose; buffers such as phosphates or citrates; antioxidants such as sodium bisulfate; local anesthetics such as procaine hydrochloride; suspending and dispersing agents such as sodium carboxymethylcellulose, hydroxypropylmethylcellulose, or polyvinylpyrrolidone; emulsifying agents such as Polysorbate 80 (TWEEN-80); sequestrants or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid); ethyl alcohol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid, or lactic acid. Pharmaceutical compositions in multi-dose containers may contain antimicrobial agents utilized as carriers, including phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride, and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol, or ethanol. Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffers, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrins.

[0206]

[0257] The pharmaceutical compositions can be liquid solutions, suspensions, emulsions, pills, capsules, tablets, sustained release formulations, or powders. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, etc.

[0207]

[0258] In certain embodiments, the pharmaceutical composition is formulated into an injectable composition. The injectable pharmaceutical composition may be prepared in any conventional form, such as liquid solution, suspension, emulsion, or solid form suitable for producing liquid solution, suspension, emulsion. Preparations for injection may include sterile and / or non-pyrogenic solutions ready for injection, sterile dry soluble products such as lyophilized powders ready for mixing with a solvent immediately before use, including tablets for subcutaneous injection, sterile suspensions ready for injection, sterile dry insoluble products ready for mixing with a vehicle immediately before use, and sterile and / or non-pyrogenic emulsions. The solutions may be aqueous or non-aqueous.

[0208]

[0259] In certain embodiments, unit dose preparations are packaged in ampoules, vials, or syringes with needles. All preparations for parenteral administration should be sterile and nonpyrogenic, as known and practiced in the art.

[0209]

[0260] In certain embodiments, a sterile lyophilized powder is prepared by dissolving the antibody or antigen-binding fragment disclosed herein in a suitable solvent. The solvent may contain excipients that improve the stability or other pharmacological factors of the powder or a reconstituted solution prepared from the powder. Excipients that may be used include, but are not limited to, water, dextrose, sorbital, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, or other suitable agents. The solvent may contain a buffer, such as citrate, sodium or potassium phosphate, or other such buffers known to those of skill in the art, at about neutral pH in one embodiment. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides the desired formulation. In one embodiment, the resulting solution is dispensed into vials for lyophilization. Each vial may contain a single dose or multiple doses of an anti-TSLP antibody or antigen-binding fragment thereof, or a composition thereof. Overfilling the vial by a small amount (e.g., about 10%) more than needed for a dose or set of doses is permissible to facilitate accurate sample removal and accurate dosing. The lyophilized powder can be stored under appropriate conditions, such as at about 4° C. to room temperature.

[0210]

[0261] The lyophilized powder is reconstituted with water for injection to provide a formulation for use in parenteral administration.In one embodiment, for reconstitution, sterile and / or non-pyrogenic water or other suitable liquid carrier is added to the lyophilized powder.The exact amount depends on the selected therapy given and can be determined empirically.

[0211]

[0262] kit

[0263] In certain embodiments, the present disclosure provides a kit comprising an antibody or antigen-binding fragment thereof provided herein. In certain embodiments, the present disclosure provides a kit comprising an antibody or antigen-binding fragment thereof provided herein and a second therapeutic agent.

[0212]

[0264] Therapeutic agents or drugs useful as conjugate moieties can be those useful for treating asthma, polypoid rhinosinusitis, chronic obstructive pulmonary disease (COPD), urticaria, eosinophilic esophagitis (EoE), atopic dermatitis, and the like.

[0213]

[0265] In some embodiments, the second therapeutic agent comprises a therapeutic agent or drug for treating asthma. In some embodiments, the conjugate moiety comprises a beta2-adrenergic receptor agonist (SABA, e.g., salbutamol, salmeterol, formoterol, etc.), an adrenergic agonist (e.g., inhaled epinephrine), a corticosteroid (preferably in an inhaled form, e.g., beclomethasone), a leukotriene receptor antagonist (e.g., montelukast and zafirlukast), a mast cell stabilizer (e.g., cromolyn sodium), a macrolide antibiotic, or a combination thereof.

[0214]

[0266] In some embodiments, the second therapeutic agent comprises a therapeutic agent or drug for treating polypoid rhinosinusitis. In some embodiments, the conjugate moiety comprises an antibiotic, and a small molecule, such as a kinase inhibitor, a steroid, and the like.

[0215]

[0267] In some embodiments, the second therapeutic agent comprises a therapeutic agent or drug for treating COPD. In some embodiments, the conjugate moiety comprises an antibiotic (including amoxicillin, doxycycline, and azithromycin), a beta2-adrenergic agonist, an anticholinergic agent (e.g., ipratropium and tiotropium), a long-acting muscarinic antagonist (LAMA, e.g., tiotropium), aclidinium, LAMA umeclidinium bromide, a corticosteroid, a phosphodiesterase-4 inhibitor (PDE4 inhibitor, e.g., roflumilast), and the like. In some embodiments, the second therapeutic agent is a monoclonal antibody, e.g., dupilumab or itepekimab.

[0216]

[0268] In some embodiments, the second therapeutic agent comprises a therapeutic agent or drug for treating urticaria. In some embodiments, the conjugate moiety comprises an antihistamine, a systemic steroid, a leukotriene-receptor antagonist, an anti-inflammatory drug, omalizumab, an immunosuppressant, and the like. Non-limiting exemplary antihistamines include diphenhydramine, hydroxyzine, loratadine, cetirizine, desloratadine, fexofenadine, and the like. In some embodiments, the second therapeutic agent is a monoclonal antibody, for example, dupilumab or itepekimab.

[0217]

[0269] In some embodiments, the second therapeutic agent comprises a therapeutic agent or drug for treating EoE. In some embodiments, the conjugate moiety comprises a proton pump inhibitor (PPI), a corticosteroid, etc. In some embodiments, the second therapeutic agent is a monoclonal antibody, e.g., dupilumab or itepekimab.

[0218]

[0270] In some embodiments, the second therapeutic agent comprises a therapeutic agent or drug for treating atopic dermatitis. In some embodiments, the conjugate moiety comprises a corticosteroid (e.g., hydrocortisone), a calcineurin inhibitor (e.g., tacrolimus or pimecrolimus), tacrolimus, pimecrolimus, a PDE-4 inhibitor (e.g., crisaborole), a systemic immunosuppressant (e.g., cyclosporine, methotrexate, interferon gamma-1b, mycophenolate mofetil, and azathioprine), an additional antibody (e.g., dupilumab, tralokinumab), and the like. In some embodiments, the second therapeutic agent is a monoclonal antibody, e.g., dupilumab or itepekimab.

[0219]

[0271] In some embodiments, the second therapeutic agent is a drug that targets IL-33, IL-25, IL-4, IL-5, IL-4R, IL-13, etc.

[0272] Such kits may further include, if desired, for example, a container containing one or more pharma- ceutically acceptable carriers, additional containers, and one or more of a variety of other conventional pharmaceutical kit components, as will be readily apparent to one of skill in the art. Instructions as an insert or label indicating the amounts of the components to be administered, guidelines for administration, and / or guidelines for mixing the components may also be included in the kit.

[0220]

[0273] Chimeric Antigen Receptor (CAR) Compositions

[0274] The present disclosure also provides a chimeric antigen receptor (CAR) comprising the anti-TSLP antigen-binding domain and a T cell activation domain provided herein. A chimeric antigen receptor (CAR) is an engineered chimeric receptor that combines the antigen-binding domain of an antibody with one or more signaling domains for T cell activation. Immune cells, e.g., T cells and natural killer (NK) cells, can be engineered to express a CAR. T cells expressing a CAR are referred to as CAR-T cells. CARs can mediate antigen-specific cellular immune activity in T cells, allowing the CAR-T cells to eliminate cells (e.g., tumor cells) that express the targeted antigen. In one embodiment, binding of a CAR-T cell provided herein to TSLP expressed on a cell, e.g., a cancer cell, results in the proliferation and / or activation of the CAR-T cell, where the activated CAR-T cell can release cytotoxic factors, e.g., perforin, granzymes, and granulysin, to initiate cytolysis and / or apoptosis of the cancer cell.

[0221]

[0275] In some embodiments, the T cell activation domain of a CAR comprises a costimulatory signaling domain and a TCR signaling domain, which can be linked to each other in a random or specified order, optionally using a short peptide linker (e.g., a glycine-serine double linker) having, for example, a length of between 2 and 10 amino acids.

[0222]

[0276] In some embodiments, the CAR further comprises a transmembrane domain. When expressed in a cell, the anti-TSLP antigen-binding domain is extracellular and the T cell activation domain is intracellular.

[0223]

[0277] In certain embodiments, the CAR comprises an anti-TSLP antigen-binding domain, a transmembrane domain, a costimulatory signaling region, and a TCR signaling domain, where the antigen-binding domain specifically binds to TSLP and comprises an antigen-binding fragment of an antibody provided herein. 1. Antigen-binding domain

[0278] In some embodiments, the anti-TSLP antigen-binding domain of the CAR comprises one or more CDR sequences provided herein, one or more heavy chain variable domains or light chain variable domains provided herein, or one or more antigen-binding fragments derived from any of the anti-TSLP antibodies provided herein.

[0224]

[0279] In some embodiments, it is beneficial for the antigen-binding domain to be derived from the same species in which the CAR will ultimately be used. For example, for use in humans, it may be beneficial for the antigen-binding domain used in the CAR to be derived from a human antibody or a humanized antibody. In some embodiments, the antigen-binding domain comprises a single chain variable fragment (scFv). In some embodiments, the antigen-binding domain may exist in a variety of other forms, including, for example, Fv, Fab, and (Fab')2, as well as bifunctional (i.e., bispecific) hybrid antibody fragments (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)). In certain embodiments, the antigen-binding domain comprises a Fab or scFv. 2. Transmembrane domain

[0280] In certain embodiments, the CAR comprises a transmembrane domain fused to the extracellular antigen-binding domain of the CAR.In one embodiment, the transmembrane domain can be selected to naturally associate with one of the domains in the CAR.In some examples, the transmembrane domain can be selected or modified to avoid binding to the transmembrane domain of other members of the T cell receptor complex.

[0225]

[0281] The transmembrane domain of the CARs provided herein can be derived from the transmembrane domain of any naturally occurring membrane-bound or transmembrane protein, such as the alpha, beta, or zeta chains of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In some embodiments, the transmembrane domain of the CAR can also use various human hinges, such as human Ig (immunoglobulin) hinges.

[0226]

[0282] Alternatively, the transmembrane domain of the CAR provided herein may be synthetic, for example, containing mainly hydrophobic residues, such as leucine and valine. In one embodiment, a triplet of phenylalanine, tryptophan, and valine is included at each end of the synthetic transmembrane domain. Optionally, a short oligopeptide or polypeptide linker between 2 and 10 amino acids in length may form the link between the transmembrane domain of the CAR and the intracellular signaling domain. A glycine-serine doublet provides a particularly preferred linker. 3. TCR signaling domain

[0283] The T cell activation domain of the CAR provided herein comprises a TCR signaling domain.The TCR signaling domain can activate the T cell expressing the CAR to perform at least one of the normal TCR effector functions of T cell, such as cytolytic activity or helper activity, including secretion of cytokines.The TCR signaling domain can be either the full length of the natural intracellular signaling domain or a fragment thereof sufficient to transmit TCR effector function signals.

[0227]

[0284] Exemplary intracellular signaling domains useful in the CARs provided herein include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor binding, as well as any derivative or variant of these sequences and any synthetic sequence having the same functional capability.

[0228]

[0285] The TCR signaling domain that acts in a stimulatory manner can comprise signaling motifs known as immune receptor tyrosine-based activation motifs or ITAMs.The TCR signaling domains that comprise ITAMs that are useful in the CARs provided herein include TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.In certain embodiments, the TCR signaling domain comprises the cytoplasmic signaling sequence derived from CD3-zeta. 4.Costimulatory signaling regions

[0286] In certain embodiments, the T cell activation domain of the CAR provided herein may further comprise a costimulatory signaling region. The costimulatory signaling region may be derived from a costimulatory molecule that acts to mediate TCR activation in an antigen-independent manner and is required for the efficient response of lymphocytes to antigen. Exemplary costimulatory molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and the ligand that specifically binds to CD83. 5. Polynucleotide sequence encoding CAR

[0287] In one aspect, the present disclosure further provides a nucleic acid sequence encoding a CAR provided herein, comprising a first polynucleotide sequence encoding an antigen-binding domain of the CAR provided herein, and optionally a second polynucleotide sequence encoding a transmembrane domain and a T cell activation domain provided herein. In some embodiments, the sequence encoding the antigen-binding domain is operably linked to the sequence encoding the transmembrane domain and the T cell activation domain. The nucleic acid sequence encoding the desired molecule can be obtained using recombinant methods known in the art, for example, by screening a library from a cell expressing the gene, by inducing the gene from a vector known to contain it, or by directly isolating it from a cell or tissue containing it, using standard techniques. Alternatively, the gene of interest can be produced synthetically rather than cloned.

[0229]

[0288] In one aspect, the present disclosure provides a vector comprising a nucleic acid sequence encoding a CAR provided herein.In some embodiments, the vector is a retroviral and lentiviral vector construct expressing the CAR of the present disclosure that can be directly transduced into cells, or an RNA construct that can be directly transfected into cells.

[0230]

[0289] In one aspect, the disclosure provides an isolated cell that comprises a nucleic acid sequence encoding a CAR provided herein and / or expresses a CAR provided herein.

[0231]

[0290] In certain embodiments, the cells that contain the nucleic acid encoding the CAR or express the CAR are selected from the group consisting of T cells, NK cells, cytotoxic T lymphocytes (CTLs), and regulatory T cells. In one embodiment, the cells that contain the nucleic acid encoding the CAR or express the CAR exhibit anti-tumor immunity when the antigen-binding domain of the CAR binds to its corresponding antigen. The cytotoxic lymphocytes are preferably autologous cells, although xenogeneic or allogeneic cells may also be used. As used herein, "autologous" refers to any material derived from the same individual that is subsequently reintroduced into that individual.

[0232]

[0291] In one aspect, the disclosure further provides a method for stimulating a T cell-mediated immune response to a TSLP-rich environment or tissue in a subject, comprising administering to the subject an effective amount of a cell genetically modified to express a CAR provided herein.

[0233]

[0292] In one aspect, the present disclosure further provides a method for treating a mammal having a disease, disorder, or condition associated with increased expression of TSLP, comprising administering to the mammal an effective amount of a cell genetically modified to express the CAR provided herein, thereby treating the mammal.In certain embodiments, the cell is an autologous T cell.In certain embodiments, the mammal has been diagnosed with a disease, disorder, or condition associated with increased expression of TSLP.

[0234]

[0293] How to use

[0294] In another embodiment, a method is provided for treating a disease, disorder, or condition in a subject that would benefit from modulating TSLP activity.In another embodiment, a method is provided for treating a TSLP-related disease or disorder in a subject in need thereof.In another embodiment, a method is provided for treating a disease, disorder, or condition that is responsive to TSLP inhibition in a subject in need thereof.

[0235]

[0295] In some embodiments, the method comprises administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein, or a polynucleotide encoding an antibody or antigen-binding fragment thereof provided herein, and / or a pharmaceutical composition provided herein. In certain embodiments, the subject is a human.

[0236]

[0296] In some embodiments, TSLP-related disease or disorder is TSLP-positive disease or disorder.In some embodiments, the subject to be treated is identified as having TSLP-positive disease or disorder.In some embodiments, TSLP-related disease, disorder or condition is responsive to TSLP inhibition.In some embodiments, TSLP-related disease, disorder or condition is associated with dysregulation of TSLP-mediated signal transduction, more particularly associated with upregulation of TSLP signal transduction.

[0237]

[0297] In some embodiments, the disease or disorder is associated with dysregulation of TSLP-mediated signaling in cells. In some embodiments, the dysregulation of TSLP-mediated signaling comprises dysregulation of STAT5 activation by phosphorylation. In some embodiments, the dysregulation of TSLP-mediated signaling comprises dysregulation of proliferation of TSLPR-expressing cells compared to control levels (e.g., levels in healthy subjects).

[0238]

[0298] In some embodiments, the disease or disorder is an inflammatory disease, an autoimmune disease, or cancer.

[0299] In some embodiments, the disease or disorder is asthma (including severe asthma), idiopathic pulmonary fibrosis, atopic dermatitis (AD), allergic conjunctivitis, allergic rhinitis (AR), Netherton syndrome (NS), eosinophilic esophagitis (EoE), food allergy, allergic diarrhea, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis (ABPA), allergic fungal sinusitis, chronic pruritus, cancer, rheumatoid arthritis, chronic obstructive pulmonary disease (COPD), systemic sclerosis, and / or chronic pulmonary fibrosis. In one embodiment, the present invention relates to a method for treating eosinophilic bowel disease, aortic ulcers, multiple sclerosis, keloids, ulcerative colitis, chronic rhinosinusitis (CRS), polyposis, chronic eosinophilic pneumonia, eosinophilic bronchitis, allergic bronchopulmonary aspergillosis, celiac disease, eosinophilic gastroenteritis, Churg-Strauss disease, eosinophilic myalgia syndrome, hypereosinophilic syndrome, eosinophilic granulomatosis with polyangiitis, eosinophilic esophagitis, inflammatory bowel disease, fibrotic disorders, inflammatory bowel disease, Hodgkin's lymphoma, and systemic lupus erythematosus.

[0239]

[0300] In some embodiments, the cancer is selected from breast cancer, pancreatic cancer, colon cancer, lung cancer, ovarian cancer, prostate cancer, and B-cell acute lymphoblastic leukemia.

[0301] In some embodiments, the fibrotic disorder is selected from systemic and localized scleroderma, keloids and hypertrophic scars, interstitial lung disease (ILD), idiopathic pulmonary fibrosis (IPF), liver fibrosis due to chronic hepatitis B or C infection, radiation-induced fibrosis, and fibrosis resulting from wound healing, atherosclerosis, restenosis, lung inflammation and fibrosis, cirrhosis of the liver, kidney disease, heart disease due to scar tissue, and eye diseases such as macular degeneration, and retinal and vitreous retinopathy, fibrosis due to chemotherapy drugs, and trauma and burns.

[0240]

[0302] In some embodiments, the disease is selected from the group consisting of asthma, polypoid rhinosinusitis, COPD, urticaria, EoE, and atopic dermatitis.

[0303] The presence and / or amount of TSLP in a biological sample of interest may indicate whether the subject from which the biological sample is derived is likely to respond to anti-TSLP antibodies. Various methods can be used to determine the presence and / or amount of TSLP in a test biological sample obtained from a subject. For example, the test biological sample may be exposed to an anti-TSLP antibody or an antigen-binding fragment thereof that binds to and detects the expressed TSLP protein. Alternatively, TSLP can also be detected at the nucleic acid expression level using methods such as qPCR, reverse transcriptase PCR, microarray, serial analysis of gene expression (SAGE), and fluorescent in situ hybridization (FISH). In some embodiments, the test sample is derived from epithelial tissue. In certain embodiments, the presence or upregulated level of TSLP in a test biological sample indicates the likelihood of responsiveness. The term "upregulated" as used herein refers to an overall increase in the expression level of TSLP in a test sample of 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, or more, compared to the expression level of TSLP in a reference sample detected using the same method. The reference sample can be a control sample obtained from a healthy or non-diseased individual, or a healthy or non-diseased sample obtained from the same individual from which the test sample was obtained.

[0241]

[0304] The therapeutically effective amount of an antibody or antigen-binding fragment provided herein will depend on a variety of factors known in the art, such as, for example, weight, age, past medical history, current medications, the subject's health status and potential for cross-reactivity, allergies, sensitivities, and adverse side reactions, as well as the route of administration and the extent of disease occurrence. Dosages may be proportionately reduced or increased by the practitioner (e.g., physician or veterinarian) as dictated by these and other circumstances or requirements.

[0242]

[0305] In some embodiments, the antibodies or antigen-binding fragments provided herein may be administered at a therapeutically effective dose of about 0.01 mg / kg to about 100 mg / kg. In certain embodiments, the administered dose may vary over the course of treatment. For example, in certain embodiments, the initial administered dose may be higher than subsequent administered doses. In certain embodiments, the administered dose may vary over the course of treatment depending on the subject's response.

[0243]

[0306] Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response), and may for example be administered as a single dose or as several divided doses administered over time.

[0244]

[0307] The antibodies or antigen-binding fragments thereof provided herein may be administered by any route known in the art, such as parenteral (e.g., subcutaneous, intraperitoneal, intravenous including intravenous infusion, intramuscular, or intradermal injection) or non-parenteral (e.g., oral, intranasal, intraocular, sublingual, rectal, or topical) routes.

[0245]

[0308] In some embodiments, the antibodies or antigen-binding fragments thereof provided herein may be administered alone or in combination with a therapeutically effective amount of a second therapeutic agent. For example, the antibodies or antigen-binding fragments thereof disclosed herein may be administered in combination with a second therapeutic agent.

[0246]

[0309] Therapeutic agents or drugs useful as conjugate moieties can be those useful for treating asthma, polypoid rhinosinusitis, chronic obstructive pulmonary disease (COPD), urticaria, eosinophilic esophagitis (EoE), atopic dermatitis, and the like.

[0247]

[0310] In some embodiments, the second therapeutic agent comprises a therapeutic agent or drug for treating asthma. In some embodiments, the second therapeutic agent comprises a beta2-adrenergic receptor agonist (SABA, e.g., salbutamol, salmeterol, formoterol, etc.), an adrenergic agonist (e.g., inhaled epinephrine), a corticosteroid (preferably in an inhaled form, e.g., beclomethasone), a leukotriene receptor antagonist (e.g., montelukast and zafirlukast), a mast cell stabilizer (e.g., cromolyn sodium), a macrolide antibiotic, or a combination thereof.

[0248]

[0311] In some embodiments, the second therapeutic agent comprises a therapeutic agent or drug for treating polypoid rhinosinusitis. In some embodiments, the conjugate moiety comprises an antibiotic, a kinase inhibitor, or a steroid.

[0249]

[0312] In some embodiments, the second therapeutic agent comprises a therapeutic agent or drug for treating COPD. In some embodiments, the conjugate moiety comprises an antibiotic (including amoxicillin, doxycycline, and azithromycin), a beta2-adrenergic agonist, an anticholinergic agent (e.g., ipratropium and tiotropium), a long-acting muscarinic antagonist (LAMA, e.g., tiotropium), aclidinium, the LAMA umeclidinium bromide, a corticosteroid, a phosphodiesterase-4 inhibitor (PDE4 inhibitor, e.g., roflumilast), and the like.

[0250]

[0313] In some embodiments, the second therapeutic agent comprises a therapeutic agent or drug for treating urticaria. In some embodiments, the conjugate moiety comprises an antihistamine, a systemic steroid, a leukotriene-receptor antagonist, an anti-inflammatory drug, omalizumab, an immunosuppressant, etc. Non-limiting exemplary antihistamines include diphenhydramine, hydroxyzine, loratadine, cetirizine, desloratadine, fexofenadine, etc.

[0251]

[0314] In some embodiments, the second therapeutic agent comprises a therapeutic agent or drug for treating EoE, hi some embodiments, the second therapeutic agent comprises a proton pump inhibitor (PPI), a corticosteroid, or the like.

[0252]

[0315] In some embodiments, the second therapeutic agent comprises a therapeutic agent or drug for treating atopic dermatitis. In some embodiments, the conjugate moiety comprises a corticosteroid (e.g., hydrocortisone), a calcineurin inhibitor (e.g., tacrolimus or pimecrolimus), tacrolimus, pimecrolimus, a PDE-4 inhibitor (e.g., crisaborole), a systemic immunosuppressant (e.g., cyclosporine, methotrexate, interferon gamma-1b, mycophenolate mofetil, and azathioprine), an additional antibody (e.g., dupilumab, tralokinumab), and the like.

[0253]

[0316] In certain of these embodiments, an antibody or antigen-binding fragment thereof provided herein that is administered in combination with one or more additional therapeutic agents may be administered simultaneously with the one or more additional therapeutic agents, and in certain of these embodiments, the antibody or antigen-binding fragment thereof and the additional therapeutic agent may be administered as part of the same pharmaceutical composition. However, an antibody or antigen-binding fragment thereof that is administered "in combination" with another therapeutic agent may not be administered simultaneously with or in the same composition as the agent. An antibody or antigen-binding fragment thereof that is administered before or after another agent is considered to be administered "in combination" with the agent, as this phrase is used herein, even if the antibody or antigen-binding fragment and the second agent are administered via different routes. When possible, additional therapeutic agents administered in combination with an antibody or antigen-binding fragment thereof disclosed herein are administered according to the schedule listed in the product information sheet for that additional therapeutic agent, or according to protocols known in the art, such as the Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Edition; Medical Economics Company; ISBN: 1563634457; 57th Edition (November 2002)).

[0254]

[0317] In another aspect, the disclosure further provides a method of modulating the activity of TSLP comprising exposing a TSLP- and TSLPR-positive cell to an antibody or antigen-binding fragment thereof provided herein.

[0255]

[0318] In another aspect, the disclosure provides a method of detecting the presence or amount of TSLP in a sample, comprising contacting the sample with an antibody or antigen-binding fragment thereof provided herein and determining the presence or amount of TSLP in the sample.

[0256]

[0319] In another aspect, the disclosure provides a method of diagnosing a TSLP-related disease, disorder, or condition in a subject comprising the steps of: a) contacting a sample obtained from the subject with an antibody or antigen-binding fragment thereof provided herein; b) determining the presence or amount of TSLP in the sample; and c) correlating the presence or amount of TSLP with the presence or status of the TSLP-related disease, disorder, or status in the subject.

[0257]

[0320] In another aspect, the disclosure provides a kit comprising an antibody or antigen-binding fragment thereof provided herein, optionally conjugated to a detectable moiety, useful for detecting a TSLP-related disease, disorder, or condition. The kit may further comprise instructions for use.

[0258]

[0321] In another aspect, the disclosure also provides the use of an antibody or antigen-binding fragment thereof provided herein in the manufacture of a medicament for treating, preventing, or ameliorating a TSLP-related disease, disorder, or condition in a subject, and in the manufacture of a diagnostic reagent for diagnosing a TSLP-related disease, disorder, or condition.

[0259]

[0322] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. All specific compositions, materials, and methods described below are within the scope of the invention, either in whole or in part. These specific compositions, materials, and methods are not intended to limit the invention, but merely illustrate specific embodiments within the scope of the invention. Those skilled in the art can develop equivalent compositions, materials, and methods without exercising inventive ability and without departing from the scope of the invention. It will be understood that many variations can be made in the procedures described herein while remaining within the boundaries of the invention. It is the intention of the inventors that such variations are included within the scope of the invention. EXAMPLES

[0260] Example 1 Expression of TSLPR-hIL7Ra protein

[0323] Human IL7RA was obtained from human IL-7R (NM_002185) cDNA clone (Fenghui biological G120380), and human TSLPR_23-231 gene fragment was obtained from human CRLF2 (NM_022148) cDNA clone (Fenghui biological G157739). The two gene fragments were connected by a synthetic GGS linker and constructed into vector Ori.Vec.E016-pTT5-hIgG1.CH1.His. An IgG light chain signal peptide sequence was added to the N-terminus of the sequence, and a 10xHis peptide was added to the C-terminus. The target vector huIL7Ra-huTSLPR His fusion was used to obtain pure receptor complex. After the constructed vector was verified by sequencing, the correct clone was extracted for plasmid extraction. Transfection level plasmid was prepared and transfected into freestyle 293T cells. The supernatant was collected on the 7th day, then affinity purified by nickel column, and the protein was exchanged into a buffer of 20mM PB and 150mM NaCl by dialysis. SDS-PAGE electrophoresis and ELISA binding assay results showed that the purity of the protein was more than 90% and the binding activity was qualified. The purified TSLPR-hIL7Ra protein can be used for subsequent functional screening.

[0261] Example 2 Establishment of cell lines

[0324] To develop monoclonal antibodies that bind to TSLP and block its function, we constructed the following cell lines: All constructed stable overexpressing cell lines were packaged with lentivirus and used to infect the relevant cell lines.

[0262]

[0325] 2.1 Cells expressing long and short membrane-form human TSLP

[0326] Long (29-159) and short (97-159) human TSLP (huTSLP) were obtained from the synthetic gene fragment SD.hTSLP (NP_149024.1, R127A, R130S) and then constructed into the vector Ori.Vec.E055-pCDH.CMV-Sig.CD3TM-EF1.GFP.T2A.Puro-RE to obtain two overexpression vectors pCDH.CMV-Sig.CD3TM-EF1.GFP.T2A.Puro-RE-hLTSLP and pCDH.CMV-Sig.CD3TM-EF1.GFP.T2A.Puro-RE-hSTSLP, which display the TSLP protein on the cell membrane through the transmembrane domain of CD3. The amino acid sequence of the membrane long huTSLP fusion protein is shown as SEQ ID NO:80. The amino acid sequence of the membrane short huTSLP fusion protein is shown as SEQ ID NO:79. After sequencing the plasmids to verify the correct expression frame, the lentiviruses were packaged and infected into HEK-293 cells to construct overexpressing cell lines of the relevant cellular variants.

[0263]

[0327] Short TSLP was contained in a membrane short TSLP fusion protein, which was expressed on the cell membrane. The membrane short TSLP fusion protein further contains a signal peptide at the N-terminus of the short TSLP sequence and a transmembrane domain at the C-terminus of the short TSLP sequence, and the amino acid sequence is shown in SEQ ID NO:79.

[0264]

[0328] Amino acid sequence of the membrane short TSLP fusion protein (SEQ ID NO:79):

[0329] MALPVTALLLPLALLLHAARPMFAMKTKAALAIWCPGYSETQINATQAMKKARKSKVTTNKCLEQVSQLQGLWRRFNRPLLKQQTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0330] Long TSLP was contained in a membrane long TSLP fusion protein, which was expressed on the cell membrane. The membrane long TSLP fusion protein further contains a signal peptide at the N-terminus of the long TSLP sequence and a transmembrane domain at the C-terminus of the long TSLP sequence, and the amino acid sequence is shown in SEQ ID NO:80.

[0265]

[0331] Amino acid sequence of membrane long TSLP fusion protein (SEQ ID NO:80):

[0332] MALPVTALLLPLALLLHAARPYDFTNCDFEKIKAAYLSTISKDLITYMSGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEMFAMKTKAALAIWCPGYSETQINATQAMKKARKSKVTTNKCLEQVSQLQGLWRRFNRPLLKQQTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV HTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0333] 2.2 Membrane CyTSLP-expressing cells

[0334] According to the amino acid and nucleotide sequence information of cynomolgus monkey TSLP (XM_00557498.2) in NCBI, gene CyTSLP was synthesized and constructed into vector Ori.Vec.E055-pCDH.CMV-Sig.CD3TM-EF1.GFP.T2A.Puro-RE, and the overexpression vector pCDH.CMV-Sig.CD3TM-EF1.GFP.T2A.Puro-RE-CynoTSLP was obtained, which displays CyTSLP protein on cell membrane through the transmembrane region of CD3. The amino acid sequence of membrane CyTSLP fusion protein is shown in SEQ ID NO: 81. After sequencing the plasmid to verify the correct expression frame, the overexpression cell line of the relevant cells was constructed by packaging the lentivirus and infecting HEK-293 and Ba / F3 cells.

[0266]

[0335] Amino acid sequence of membrane CyTSLP fusion protein (SEQ ID NO:81):

[0336] MALPVTALLLPLALLLHAARPYDFTNCDFQKIEADYLRTISKDLITYMSGTKSTDFNNTVSCSNRPHCLTEIQSLTFNPTPRCASLAKEMFARKTKATLALWCPGYSETQINATQAMKKRRKRKVTTNKCLEQVSQLLGLWRRFIRTLLKKQTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV HTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0337] 2.3 Membrane huTSLPR-huIL7Ra expressing cells

[0338] The full length human IL7RA gene fragment and human TSLPR gene fragment were obtained from human IL7R (NM_002185) cDNA clone (Fenghui biological G120380) and human CRLF2 (nm_022148) cDNA clone (Fenghui biological G157739). They were constructed into vector pLVX-IRES-Neo and vector Ori.Vec.E025-pCDH-CMV.MCS-EF1.CopGFP.T2A.Puro, respectively, by homologous recombination to obtain two overexpression vectors, human IL7Ra-pLVX-IRES-Neo and human TSLPR-pCDH-CMV-Puro. The amino acid sequence of human IL7RA protein is shown as SEQ ID NO:71, respectively. The amino acid sequence of human TSLPR protein is shown as SEQ ID NO:72. After sequencing the plasmids to verify the correct expression frame, the lentiviruses were packaged and infected into HEK-293 and Ba / F3 cells to construct overexpressing cell lines of the relevant cells.

[0267]

[0339] 2.4 293T cells expressing hTSLPR-hIL7Ra and STAT5-luciferase

[0340] STAT5 luciferase virus was used to infect 293T-hTSLPR-hIL7Ra stable cells. After 24 hours, new culture medium was replaced and 100ug / ml hygro was added for screening. After 8 days of screening, the positive rate of FACS was detected. The positive rate of expressed cells was above 90%. The expression of luciferase could be detected after stimulation with hTSLP, indicating that the receptor expressed by the cells has the function of activating STAT5 luciferase.

[0268] Example 3 immunization

[0341] To obtain antibodies that specifically bind to long-chain TSLP, healthy SPF Balb / C female mice aged 6 to 8 weeks (purchased from Zhejiang Vital River Experimental Animal Technology Co., Ltd.) were selected. The mice were immunized multiple times with recombinant human long-chain TSLP antigen (purchased from ACRO, TSP-H52Ha, mutant long-chain TSLP with the amino acid sequence of SEQ ID NO: 73 and mutations at R127A and R130A) and an equal amount of Freund's adjuvant, using Freund's complete adjuvant for the first round and Freund's incomplete adjuvant for boosting. From the second booster immunization, orbital blood of the mice was collected within 6 to 8 days after each booster immunization, and the immune serum titers of the mice were detected by indirect ELISA. The results showed that the mice could produce high-titer antibodies specific to human long-chain TSLP after immunization, indicating that this scheme could be met for the preparation of subsequent hybridoma antibodies.

[0269] Example 4 Hybridoma generation and screening

[0342] Mice with stable and qualified serum titers after two booster immunizations were selected for cell fusion. Splenic lymphocytes of these mice were electrofused with SP2 / 0 myeloma cells. Fusion clones were screened by hypoxanthine aminopterin thymidine (HAT) and cultured for 7 days. Cell supernatants were detected by indirect ELISA (the same method as described below in section 4.1 of this example).

[0270]

[0343] Flow cytometry (same as the method described below in section 4.2 of this Example) was used to detect the blocking activity of the supernatants against the binding of human long TSLP (His tag, TSP-H52Ha) and monkey long TSLP (His tag, TSP-C52H8) (all purchased from ACRO) to 293T-hTSLP / hIL7Ra cells.

[0271]

[0344] According to the results of ELISA and flow cytometry, clones with good ELISA binding and FACS blocking activity were selected, and subcloned 2-3 times by limiting dilution. When all the subclone cultures were ELISA positive and at least two monoclonal cultures were flow cytometry positive, two clones from each hybridoma were selected for line identification and expansion.

[0272]

[0345] After screening of the subclones, 98 human TSLP-specific hybridoma cell lines were finally obtained for further development.

[0346] Through the analysis of the heavy and light chain sequences of hybridoma antibodies, 69 hybridoma cells with different VH sequences were cultured in serum-free medium to express hybridoma antibodies. The culture supernatant was collected, protein G affinity purification was performed, and the antibody was replaced by 20 mM PB and 150 mM NaCl buffer by dialysis. Through SDS-PAGE electrophoresis and binding verification, the purity of the antibody was more than 90%, and the binding activity was qualified, which can be used for antibody screening at a later stage.

[0273]

[0347] 4.1 ELISA binding assay

[0348] Human long TSLP protein (Acro, tsp-h5255 or tsp-h52ha) or cynomolgus monkey long TSLP protein (Acro, tsp-c52h8) antigen solution at 1ug / ml was prepared in PBS and coated on 384-well plates at 25ul per well overnight at 4°C. The antigen coating solution was discarded. 3% milk was added at 80ul / well and sealed at room temperature for 1.5 hours. The supernatant was discarded and the plate was washed 3 times with PBST (0.1% T20). 25ul of sample was added to the 384-well plate for 1 hour incubation at room temperature. The supernatant was discarded and the plate was washed 3 times with PBST (0.1% T20). Secondary antibody was added at 25ul per well to the 384-well plate for 1 hour incubation at room temperature. The supernatant was discarded and the plate was washed 7 times with PBST (0.1% T20). HRP substrate TMB was added at 25 ul / well. After 12 minutes of incubation, the reaction was terminated by adding 25 ul of stop solution and 2M HCl. Absorbance was read at 450 nm using a microplate reader. Antibodies that showed specific binding to human long TSLP were identified and selected.

[0274]

[0349] 4.2 FACS blocking test

[0350] Antibodies were diluted in cell culture medium and transferred to a new 96-well plate at 50ul per well. 0.012ug / ml of human long TSLP protein (Acro, tsp-h52ha) or cynomolgus monkey long TSLP protein (Acro, tsp-c52h8) was added at 10ul per well for 0.5h incubation at 4°C. 293T-TSLPR-IL7R stable cells (constructed according to section 2.3 of Example 2) were added to the 96-well plate and the supernatant was discarded after centrifugation. Pre-incubated antibodies and TSLP were transferred to the cell plate at 50ul, mixed and incubated at 4°C for 0.5h. After centrifugation, the supernatant was washed 3 times with PBS and after centrifugation, the supernatant was discarded. 50ul of secondary antibody was added to the 96-well plate for 0.5h incubation at 4°C. After centrifugation, the supernatant was washed three times with PBS, and after centrifugation, the supernatant was discarded. The cells were resuspended in 100ul PBS, and the fluorescence value was read by flow cytometry. Antibodies that showed specific blocking of long human TSLP binding to 293T-TSLPR-IL7R stable cells were identified and selected.

[0275]

[0351] 4.3 Binding assay of membrane CyTSLP-expressing cells

[0352] CyTSLP expressing cells (constructed according to section 2.2 of Example 2) were added to a 96-well plate, and the supernatant was discarded after centrifugation. 50ul of antibody solution diluted in D10 (DMEM+10% FBS) was transferred to the cell plate for 0.5h incubation at 4°C. After centrifugation, the plate was washed 3 times with PBS, and the supernatant was discarded after centrifugation. 50ul of secondary antibody was added to the 96-well plate for 0.5h binding at 4°C. After centrifugation, the supernatant was removed, the plate was washed 3 times with PBS, and the supernatant was discarded after centrifugation. The cells were resuspended in 100ul of PBS, and the fluorescence value was read by flow cytometry.

[0276]

[0353] 4.4 TSLP-dependent BaF3 proliferation assay

[0354] After thawing BaF3-hTSLPR-hIL7R cells, they were washed twice with hybridoma serum-free medium, diluted to 4E5 / ml, and added to 384-well cell culture plates at 30ul per well. Human long TSLP protein (Acro, tsp-h52ha) was prepared at 20ng / ml using hybridoma serum-free medium and added to cell culture plates at 15ul per well. Antibodies were prepared in hybridoma serum-free medium and added to cell culture plates at 15ul per well for 72 hours of incubation in a cell culture box at 37°C and 5% CO2. The 384-well cell culture plates were removed and 15ul of CTG (Promega, g7572) was added. The plates were shaken for 5 minutes after centrifugation, centrifuged again, and luminescence readings were read within 30 minutes. Antibodies that showed specific inhibition of recombinant long TSLP-induced BaF3 proliferation were identified and selected.

[0277]

[0355] Through ELISA, FACS block test and function test described in Examples 4.1, 4.2 and 4.4, clones with good activity were selected for preparation of chimeric antibodies. These candidate sequences were further verified and screened by chimeric expressed antibodies.

[0278] Example 5 Generation and screening of chimeric antibodies

[0356] According to the hybridoma antibody screening results, we constructed recombinant expression vectors and purified 15 selected antibodies. The process was as follows: the heavy chain sequencing plasmid obtained by hybridoma sequencing was used as a template, the heavy chain variable region fragment was amplified by PCR, and cloned into the pTT5-hIgG2.CH vector containing the amino acid sequence of the IgG2 heavy chain constant region by homologous recombination to obtain a chimeric antibody heavy chain expression vector. Similarly, the light chain sequencing plasmid obtained by hybridoma sequencing was used as a template, the light chain variable region fragment was amplified by PCR, and cloned into the pTT5-hKappa.CL vector containing the amino acid sequence of the light chain constant region CL of the kappa light chain to obtain a chimeric antibody light chain expression vector.

[0279]

[0357] The constructed transient expression vector was used to prepare 50-100ug of endotoxin-free plasmid, which was transfected into freestyle 293T cells, affinity purified with protein A, and the antibodies were dialyzed into 20mM PB and 150mM NaCl buffer. Among them, two clones were not obtained due to low expression. After SDS-PAGE electrophoresis and binding verification, the other 13 antibodies were found to be more than 90% pure and qualified in binding activity, which could be used for antibody screening at a later stage.

[0280]

[0358] Then, two clones, 39H11 and 35G12, with good activity were screened for humanization through FACS blocking test and functional test according to the same method as described in Example 4.

[0281]

[0359] The 39H11 chimeric antibody was named Chi39H11, and the 35G12 chimeric antibody was named Chi35G12. The results of binding assays (FIGS. 1, 2, and 7), blocking assays (FIG. 3), and functional assays (FIG. 8) are shown.

[0282] Example 6 Humanization and lead generation

[0360] 6.1 39H11

[0361] Candidate antibodies were humanized according to commonly known CDR grafting methods. IMGT / V-QUEST tool (http: / / www.imgt.org / IMGT_vquest / input) was used to analyze the sequences of antibody heavy chain variable region (VH) and light chain variable region (VL) to determine the CDR region sequences of antibody light chain and heavy chain. IgBlast tool (https: / / www.ncbi.nlm.nih.gov / igblast / ) was used to analyze the amino acid sequence of antibody to obtain the similar VH and VL sequences of human. The CDR region of the antibody was grafted into the frame region of the selected VH and VL sequences, which were humanized antibody sequences.

[0283]

[0362] Then, back mutations were performed for individual amino acids in the antibody sequence. Homo sapiens codon optimization and gene synthesis were performed, and the heavy chain gene was constructed in the pTT5-hIgG2.CH vector containing the amino acid sequence of the IgG2 heavy chain constant region, and the light chain gene was constructed in the pTT5-hKappa.CL vector containing the amino acid CL sequence of the antibody kappa light chain constant region. The constructed transient expression vectors were paired with the light and heavy chains and transfected into freestyle 293T cells. The antibodies were expressed and purified, and then quantified, bound, blocked, and functionally evaluated. Two humanized antibody clones, 39H11Z07 and 39H11Z11, were selected for further evaluation.

[0284]

[0363] Back mutations to the mouse sequence were introduced into the corresponding sites of the humanized sequence. The mutant vectors were obtained, mated, and transfected into freestyle 293T cells. The antibodies were expressed, purified, and tested for binding, blocking, and functional evaluation.

[0285]

[0364] The results are shown in Figures 4a, 4b, 5a, 5b, and 6. The results show that the blocking activity of 39H11Z07 and 39H11Z11 is comparable to Chi39H11 (Figures 5a, 5b), and functions better than BMK (Figure 6).

[0286]

[0365] 6.2 35G12

[0366] Candidate antibodies were humanized according to commonly known CDR grafting methods. IMGT / V-QUEST tool (http: / / www.imgt.org / IMGT_vquest / input) was used to analyze the sequences of antibody heavy chain variable region (VH) and light chain variable region (VL) to determine the CDR region sequences of antibody light chain and heavy chain. IgBlast tool (https: / / www.ncbi.nlm.nih.gov / igblast / ) was used to analyze the amino acid sequence of antibody to obtain the similar VH and VL sequences of human. The CDR region of the antibody was grafted into the frame region of the selected VH and VL sequences, which were humanized antibody sequences.

[0287]

[0367] Then, back mutations were performed for individual amino acids in the antibody sequence. Codon optimization and gene synthesis of Homo sapiens were performed, and the heavy chain gene was constructed in the pTT5-hIgG2.CH vector containing the amino acid sequence of the IgG2 heavy chain constant region, and the light chain gene was constructed in the pTT5-hKappa.CL vector containing the amino acid CL sequence of the antibody kappa light chain constant region. The constructed transient expression vectors were paired with the light and heavy chains and transfected into freestyle 293T cells. The antibodies were expressed and purified, and then quantified, bound, blocked, and functionally evaluated.

[0288]

[0368] Back mutations to the mouse sequence were introduced into the corresponding sites of the humanized sequence. The mutant vectors were obtained, mated, and transfected into freestyle 293T cells. The antibodies were expressed, purified, and tested for binding, blocking, and functional evaluation. Three humanized antibody clones, 35G12Z01, 35G12Z02, and 35G12Z03, were selected for further evaluation.

[0289]

[0369] The expression and purification of the antibodies were then carried out according to the preparation process of the chimeric antibody. The obtained antibodies were tested for binding activity, blocking activity, and function. The results are shown in Figures 9 to 11. The results show that the inhibitory effects of 35G12Z01, 35G12Z02, and 35G12Z03 on human long-type TSLP-dependent proliferation of BaF3 cells are better than those of BMK and Chi35G12 (Figure 11).

[0290] Example 7 In vitro characterization

[0370] Two of the screened antibodies, 39H11Z07 and 35G12Z03, were modified with an Fc that had reduced ADCC and extended half-life.

[0291]

[0371] The following mutations were introduced to obtain the IgG4-PAA-YTE heavy chain constant region expression vector: S228P\F234A\L235A\M252Y\S254T\T256E\K447del. The amino acid sequence of the IgG4-PAA-YTE heavy chain constant region is shown as SEQ ID NO:75.

[0292]

[0372] Amino acid sequence of the constant region of the human IgG4-PAA-YTE heavy chain (SEQ ID NO:75): ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG

[0373] The following mutations were introduced to obtain the IgG1-LALA-YTE heavy chain constant region expression vector: L234A\L235A\M252Y\S254T\T256E. The amino acid sequence of the IgG1-LALA-YTE heavy chain constant region is shown as SEQ ID NO: 88. Amino acid sequence of the heavy chain constant region IgG1-LALA-YTE Fc (SEQ ID NO: 88): ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0374] The 39H11Z07 heavy chain variable region fragment was amplified by PCR and cloned by homologous recombination into the pTT5HE-hIgG4CH-PAA-YTE-RE vector containing the IgG4-PAA-YTE heavy chain constant region amino acid sequence to obtain the 39H11Z07 Fc replacement expression vector. Similarly, the 35G12Z03 heavy chain variable region fragment was amplified by PCR using the h35G12.H3 plasmid as a template and cloned by homologous recombination into the pTT5HE-hIgG4CH-PAA-YTE-RE vector containing the IgG4-PAA-YTE heavy chain constant region amino acid sequence.

[0293]

[0375] The 35G12Z03 Fc replacement expression vector was obtained. The constructed transient expression heavy chain vector and its corresponding light chain vector were used to prepare endotoxin-free plasmids, which were transfected into freestyle 293T cells, affinity purified with protein A, and the antibody was exchanged into 20 mm PB and 150 mM NaCl buffer by dialysis or cation chromatography.

[0294]

[0376] In the examples, the benchmark used (abbreviated as BMK) was AMG 157, having a heavy chain comprising the amino acid sequence of SEQ ID NO:77 and a light chain comprising the amino acid sequence of SEQ ID NO:78.

[0295]

[0377] Amino acid sequence of AMG 157 heavy chain (SEQ ID NO:77):

[0378] QMQLVESGGGVVQPGRSLRLSCAASGFTFRTYGMHWVRQAPGKGLEWVAVIWYDGSNKHYADSVKGRFTITRDNSKNTLNLQMNSLRAEDTAVYYCARAPQWELVHEAFDIW GQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKC CVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKT ISKTKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0379] Amino acid sequence of AMG 157 light chain (SEQ ID NO:78):

[0380] SYVLTQPPSVSVAPGQTARITCGGNNLGSKSVHWYQQKPGQAPVLVVYDDSDRPSWIPERFSGSNSGNTATLTISRGEAGDEADYYCQVWDSSSDHVVFGGGTKLTV LGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0381] The obtained antibodies were characterized for binding, blocking, and functional tests, and the results are shown in Figures 12 to 16. The results show that the different Fc antibodies have little difference in binding activity, blocking activity, and function, and are better than BMK.

[0296]

[0382] 7.1 ELISA Binding

[0383] Human long TSLP protein (Acro, tsp-h5255 or tsp-h52ha) or cynomolgus monkey long TSLP protein (Acro, tsp-c52h8) antigen solution at 1ug / ml was prepared in PBS and coated on 384-well plates at 25ul per well overnight at 4°C. The antigen coating solution was discarded. 3% milk was added at 80ul / well and sealed at room temperature for 1.5 hours. The supernatant was discarded and the plate was washed 3 times with PBST (0.1% T20). 25ul of sample was added to the 384-well plate for 1 hour incubation at room temperature. The supernatant was discarded and the plate was washed 3 times with PBST (0.1% T20). Secondary antibody was added at 25ul per well to the 384-well plate for 1 hour incubation at room temperature. The supernatant was discarded and the plate was washed 7 times with PBST (0.1% T20). HRP substrate TMB was added at 25ul / well. After 12 minutes of incubation, the reaction was terminated by adding 25ul of stop solution and 2M HCl. Absorbance was read at 450nm using a microplate reader.

[0297]

[0384] The results are shown in FIG. 12, which demonstrated that the antibodies mAb35G12 and mAb39H11 in this disclosure were capable of binding to the TSLP protein.

[0385] 7.2 Determining affinity

[0386] The affinity of the antibodies was determined using Gator and HFC (Gator, pl168-160003) probes. Five steps were employed, including equilibration, loading, equilibration, association, and dissociation.

[0298]

[0387] Different kinds of antibodies were diluted to a concentration of 10ug / ml for loading, and then the sensor was equilibrated with the same concentration of human long-chain TSLP protein (Acro, tsp-h5255 or tsp-h52ha) and the same concentration of references, 50nm, 25nm, 12.5nm, 6.25nm, 3.12nm, 1.56nm and 0, respectively. The Kon value was tested. After binding to equilibrium, the dissociation curve of the antibody-antigen complex was determined in dissociation buffer, and the koff value was calculated.

[0299]

[0388] The Kd values ​​of the candidate molecules are as follows: The results show that the Kd values ​​of the candidate clones mAb35G12 and mAb39H11 in different Fc formats in combination with antigen are comparable to that of the AMG157 antibody.

[0300] [Table 7]

[0301]

[0389] 7.3 Binding of different species to TSLP

[0390] To detect the binding of antibodies to TSLP of different species, 1ug / ml of human long TSLP protein (ACRO, tsp-h5255 or tsp-h52ha), cynomolgus monkey long TSLP protein (ACRO, tsp-c52h8), mouse long TSLP protein (ACRO, tsp-m52h8), and rat long TSLP protein (ACRO, tsp-r52h3) antigen solutions were prepared in PBS. Each well of a 384-well plate was coated with 25ul and coated overnight at 4°C. After that, the supernatant was discarded, 80ul / well of 3% milk was added, and the plate was sealed at room temperature for 1.5 hours. The supernatant was discarded, and the plate was washed three times with PBST (0.1% T20). 25ul of the sample to be tested was added to the 384-well plate for 1 hour incubation at room temperature. The supernatant was discarded and the plate was washed 3 times with PBST (0.1% T20). Secondary antibody, 25ul per well, was added to the 384-well plate for 1 hour incubation at room temperature. The supernatant was discarded and the plate was washed 7 times with PBST (0.1% T20). HRP substrate TMB was added at 25ul / well. After 12 minutes, the reaction was terminated by adding 25ul of stop solution and 2m HCl. Absorbance was read at 450nm using a microplate reader.

[0302]

[0391] The results are shown in Figures 13a to 13d. The ELISA results show that all of the mAb35G12 and mAb39H11 molecules can bind to human long TSLP, and none of the molecules can bind to monkey, rat, or mouse long TSLP, except for BMK, which can bind to monkey long TSLP with low potency.

[0303]

[0392] 7.4 Epitope Analysis

[0393] The epitope of the antibody was determined using Gator and His probes (Gator, pl168-160009).

[0304]

[0394] After the probe was equilibrated, it was loaded with human long chain TSLP protein (Acro, tsp-h52ha) antigen at a concentration of 5ug / ml. After equilibration, it was loaded with 10ug / ml of the first antibody, and after re-equilibration, it was loaded with 10ug / ml of the second antibody. Finally, the binding thickness (nm) of the second antibody was obtained, and this parameter was used to analyze whether the epitopes were the same.

[0305]

[0395] The results are shown in FIG. 14, which indicate that the binding epitopes of Chi39H11 and Chi35G12 are distinct from that of BMK.

[0396] 7.5 Receptor Blocking

[0397] The protocol for the receptor blocking test of the humanized antibodies was the same as that described in Example 4.1.

[0306]

[0398] The results are shown in FIG. 15, which demonstrates that antibodies mAb35G12 and mAb39H11 in this disclosure can block TSLP binding to the cell surface of 293T-hTSLPR-hIL7R.

[0307]

[0399] 7.6 Binding of short-chain TSLP

[0400] Short huTSLP(97-159) expressing cells were added to a 96-well plate, and the supernatant was discarded after centrifugation. 50ul of antibody solution diluted in D10 was transferred to the cell plate for 0.5h incubation at 4℃. After centrifugation, the plate was washed 3 times with PBS, and the supernatant was discarded after centrifugation. 50ul of secondary antibody was added to the 96-well plate for 0.5h binding at 4℃. After centrifugation, the supernatant was removed, the plate was washed 3 times with PBS, and the supernatant was discarded after centrifugation. The cells were resuspended in 100ul of PBS, and the fluorescence value was read by flow cytometry.

[0308]

[0401] The results are shown in FIG. 16, which demonstrates that neither of the antibodies mAb35G12 nor mAb39H11 of the present disclosure are able to bind to the cell surface expressing truncated huTSLP.

[0309]

[0402] 7.7 Binding of digested TSLP

[0403] Three different forms of TSLP were used in this study to test the binding of antibodies to digested and non-digested forms of TSLP. H52Ha is a mutant long-chain TSLP with the amino acid sequence of SEQ ID NO: 73 and with the mutations R127A and R130A that confer resistance to enzymatic digestion. H52Hb is a native long-chain TSLP with the amino acid sequence of SEQ ID NO: 82, containing a cleavage site at 130 and susceptible to enzymatic digestion.

[0310]

[0404] Digested TSLP H53Hb (ACRO number tsp-h52hb) comprises the amino acid sequence set forth in SEQ ID NO: 82. Undigested TSLP H53Ha (ACRO number tsp-h52ha) comprises the amino acid sequence set forth in SEQ ID NO: 73.

[0311]

[0405] YDFTNCDFEKIKAAYLSTISKDLITYMSGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEMFAMKTKAALAIWCPGYSETQINATQAMKKRRKRKVTTNKCLEQVSQLQGLWRRFNRPLLKQQ (SEQ ID NO: 82)

[0406] YDFTNCDFEKIKAAYLSTISKDLITYMSGTKSTEFNNTVSCSNRPHCLTEIQSLTFNPTAGCASLAKEMFAMKTKAALAIWCPGYSETQINATQAMKKARKAKVTTNKCLEQVSQLQGLWRRFNRPLLKQQ (SEQ ID NO: 73)

[0407] To obtain the digestion product of native long TSLP H53Hb-d, 100 uL of digestion buffer (25 mm Tris, 1 mM CaCl2, 0.5 (w / V) Brij-35, pH 9.0) was prepared in total, where the working concentration of long native TSLP (ACRO number tsp-h52hb) was 10 ug / ml and the working concentration of PCSK3 was 12 ug / ml for 24 hours of enzymatic digestion at 37° C. The digestion product (referred to as 52Hb-d) was then prepared in Western blot system (5 ug per well) of reduced and non-reduced samples, respectively, and non-digested native TSLP H53Hb (ACRO number tsp-h52hb) and non-digested mutant TSLP H53Ha (ACRO number tsp-h52ha) were also loaded for SDS-PAGE electrophoresis in parallel.

[0312]

[0408] Enzyme digestion was detected by reducing SDS-PAGE.52Ha did not show any digestion fragments due to mutation, while wild-type sequence 52Hb showed significant digestion fragments, which can be attributed to the natural enzymes present during the production process.In addition, 52Hb was more completely digested when PCSK3 was added, as shown in Figure 17.

[0313]

[0409] The results indicated that 52Hb contained a mixture of undigested and digested TSLP, while 52Hb-d was a completely digested TSLP sample, both of which were further tested for anti-TSLP antibody binding activity.

[0314]

[0410] Three forms of TSLP protein (52Ha, 52Hb, and 52Hb-d, respectively) were treated and loaded in SDS-PAGE protein loading buffer, and prepared into reduced (with DTT) and non-reduced (without DTT) samples at a concentration of 0.5ug / 20ul. After SDS-PAGE electrophoresis, samples were transferred to membranes (PVDF membranes) using a Beyotime membrane transfer device, membrane transfer current was set at 300mA and membrane transfer time was set at 60 minutes, and sealed (Western blocking solution was added, shaken slowly on a shaking table, blocked at 37°C for more than 1 hour, and blocked at room temperature for more than 2 hours).

[0315]

[0411] Primary antibodies: BMK, 35G12Z03, and 39H11Z07 were each diluted in antibody diluent to a concentration of 2ug / ml, incubated for 2 hours, and washed three times (10 minutes each) with washing solution, where each of the three antibodies contains an IgG2 heavy chain constant region (SEQ ID NO:83) and an IgG2 light chain constant region (SEQ ID NO:84).

[0316]

[0412] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDISVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 83)

[0413] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 84)

[0414] Secondary antibody: Secondary antibody anti-HuIgG HRP (Sino ssa002) 1:2000 was diluted in antibody diluent, incubated for 60 minutes at room temperature, and washed three times with washing solution (10 minutes each time).

[0317]

[0415] Staining using chemiluminescence method: Two anti-PVDF membranes were placed into the fluorescence and chemiluminescence imaging system. Equal volumes of luminescence solution A and B were mixed and evenly distributed on the PVDF membrane. The software of the gel imaging system was run and the exposure time was set.

[0318]

[0416] 52Ha, 52Hb, and 52Hb-d TSLP proteins were immunoblotted with the primary and secondary antibodies indicated above under reducing and non-reducing conditions, respectively. The results are shown in Figures 18a (non-reduced sample) and 18b (reduced sample). The total chemiluminescence of each single lane was measured in gray values.

[0319]

[0417] The results show that the molecules of the present disclosure can strongly bind to the digested form of TSLP (52Hb-d), and such binding activity was much stronger (e.g., at least 2-fold) than that observed for the BMK molecule (see Figures 18a and 18b).

[0320]

[0418] 7.8 Blocking STAT5 activation

[0419] To determine the ability of the antibodies to block long-TSLP stimulated STAT5 activation, 293T-hTSLPR-hIL7Ra and STAT5-luciferase stable cells (constructed according to section 2.4 of Example 2) were used under favorable growth conditions. After trypsin digestion and cell counting, cells were diluted to 2 × 10 5The antibody solutions were diluted to 100 / ml and 15ul / well of cells were added to a 384-well plate. The antibody solutions were diluted in DMEM cell culture medium and 15ul of antibody solutions at different concentrations were added to a 384-well plate for 30 minutes incubation in an incubator. 30ng / ml of human TSLP (ACRO, tsp-h52ha) was prepared in DMEM cell culture medium and 15ul / well of cells were added to a 384-well plate and cultured at 37℃ and 5% CO2 for 4-6 hours. Finally, 25ul of bright glo™ luciferase assay system (Promega, e2620) was added. After shaking for 5 minutes, the luminescence value was read on an enzyme-labeled instrument.

[0321]

[0420] The experimental results are shown in Figure 19. According to the detection results, the presented molecules mAb35G12 and mAb39H11 can successfully block STAT5 activation stimulated by long-chain TSLP. By switching the isotype of mAb35G12, similar blocking potency was maintained. Compared with BMK antibody, the blocking IC50 of candidate clones mAb35G12, mAb35G12_IgG1, and mAb39H11 is significantly better.

[0322]

[0421] 7.9 Blocking native TSLP-induced TARC secretion

[0422] Native TSLP was induced and collected from human lung fibroblasts. Briefly, human lung fibroblasts were harvested at a density of 80%, digested with trypsin, and subcultured into T25 culture flasks according to 1:3. The cell density was monitored, and when it reached a maximum of 80%, the supernatant was discarded. Native TSLP production was induced by cultured human lung fibroblasts in a medium containing 10 ng / ml IL1-β, 20 ng / mL IL-13, and 20 ng / mL TNF-α. After 24 hours of incubation, the TSLP-induced supernatant was collected in a centrifuge tube, then centrifuged at 3000 rpm for 10 minutes, and the supernatant was transferred to a new centrifuge tube.

[0323]

[0423] Antibodies were diluted in cell culture medium. PBMCs were thawed and resuspended in cell culture medium after centrifugation. The cell concentration was 1×10 7 The antibody solutions were adjusted to 50 ul / ml and added to 96-well cell culture plates at 50 ul / well. Native TSLP was prepared as described above and added to 96-well cell culture plates at 25 ul / well to a final concentration of 20 ng / ml. 25 ul of diluted antibody solution was added per well. After 24 hours, TARC expression was detected by ELISA.

[0324]

[0424] 2ug / ml capture antibody (Sino, sek10233) antigen solution was prepared in PBS and coated on 384-well plate at 25ul per well for overnight incubation at 4℃. The supernatant was discarded. 3% milk was added at 80ul / well and sealed at room temperature for 1.5 hours. The supernatant was discarded and the plate was washed 3 times with PBST (0.1% T20). 25ul of the sample to be tested was added to the 384-well plate for 2 hours incubation at room temperature. The supernatant was discarded and the plate was washed 3 times with PBST (0.1% T20). 25ul of secondary antibody solution per well was added to the 384-well plate for 1 hour incubation at room temperature. The supernatant was discarded and the plate was washed 7 times with PBST (0.1% T20). HRP substrate TMB was added at 25ul / well. After 12 min incubation, the reaction was terminated by adding 25 ul of stop solution and 2 m HCl, and the absorbance was read at 450 nm using a microplate reader.

[0325]

[0425] The results are shown in Figure 20. The results showed that the antibodies mAb35G12 and mAb39H11 in the present disclosure were able to block native TSLP-induced TARC secretion better than BMK.

[0326]

[0426] 7.10 Recombinant TSLP-dependent BaF3 growth

[0427] The protocol for recombinant TSLP-dependent BaF3 proliferation assay of humanized antibodies was the same as that described in Example 4.2.

[0327]

[0428] The results are shown in Figure 21, which shows that the antibodies mAb35G12, mAb35G12_IgG1, and mAb39H11 in the present disclosure can block TSLP-induced BaF3-hTSLP-hIL7R cell proliferation better than BMK. By switching the isotype of mAb35G12, relatively similar blocking potency was maintained.

[0328]

[0429] 7.11 Natural TSLP-dependent BaF3 proliferation

[0430] Human lung fibroblasts were harvested at a density of 80%, digested with trypsin, and subcultured into T25 culture flasks according to 1:3. The cell density was monitored, and when it reached a maximum of 80%, the supernatant was discarded. TSLP production was induced by cultured human lung fibroblasts in a medium containing 10 ng / ml IL1-β, 20 ng / mL IL-13, and 20 ng / mL TNF-α. Another T25 bottle containing the above-mentioned medium without cells was obtained. The cells were cultured in a third T25 bottle containing serum-free medium. After 24 hours of incubation, the above-mentioned cell supernatants (TSLP-induced / uninduced / serum-free supernatants) were collected in centrifuge tubes, respectively, and then centrifuged at 3000 rpm for 10 minutes, and the supernatants were transferred to new centrifuge tubes. The antibody solution was diluted in serum-free medium. Baf3-TSLPR-IL7Ra was thawed and washed twice with medium. Resuspend the cells, count them, and confirm the cell density at 4 x 10 5The supernatants were adjusted to 1000 / ml and added to 384-well cell culture plates at 30 μL / well. The TSLP-induced supernatants were spread on 384-well black cell culture plates at 15 ul / well (TSLP-induced supernatants were added to experimental wells, and TSLP-induced, non-induced / cell-free supernatants were added to control wells, respectively) and cultured in a cell culture chamber at 37°C and 5% CO2 for 2 days. The 384-well plates were removed, 15 ul of CTG was added, shaken for 5 minutes, and the luminescence was read within 30 minutes. The proliferation of baf3 was detected.

[0329]

[0431] The results showed that only cytokine-induced fibroblasts stimulated the proliferation of Baf3, indicating that native TSLP expression can be induced by this method.

[0330]

[0432] Then, we tested the inhibitory effect of antibodies on native TSLP-induced Baf3 proliferation by the same method as that of the antibody that inhibits hTSLP protein-stimulated baf3 proliferation (see Example 4.3). The results are shown in Figure 22, which shows that the molecules mAb35G12 and mAb39H11 in the present disclosure can strongly inhibit the proliferation of baf3 cells. The molecules presented have better inhibitory effect than BMK.

Claims

1. An anti-TSLP antibody or antigen-binding fragment thereof, comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, and light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, (a) HCDR1 comprises the amino acid sequence of SEQ ID NO: 11; HCDR2 is IFPGDGX １ T (SEQ ID NO:59), and HCDR3 is ARX ２ GX ３ X ４ X ５ X ６ X ７ X ８ comprising the amino acid sequence of YAMDY (SEQ ID NO: 60), X １ is E or D, and X ２ is G or S, and X ３ is Y or F, and X ４ is V or D, and X ５ is N or Y, and X ６ does not exist or is G, and X ７ is F or Y, and X ８ is L or F, or (b) HCDR1 is SYWX ９ N (SEQ ID NO: 61), HCDR2 is QIFPGDGX １０ TX １１ YNGX １２ comprising the amino acid sequence of FKG (SEQ ID NO: 62), and HCDR3 is X １３ GX １４ X １５ X １６ X １７ X １８ X １９ comprising the amino acid sequence of YAMDY (SEQ ID NO: 63), X ９ is M or I, and X １０ is E or D, and X １１ is N or T, and X １２ is K or N, and X １３ is G or S, and X １４ is Y or F, and X １５ is V or D, and X １６ is N or Y, and X １７ does not exist or is G, and X １８ is F or Y, and X １９ is L or F, and (c) LCDR1 comprises the amino acid sequence of SEQ ID NO: 14; LCDR2 comprises the amino acid sequence of SEQ ID NO: 15, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 16; or (d) LCDR1 comprises the amino acid sequence of SEQ ID NO: 53; LCDR2 comprises the amino acid sequence of GTSX 20 LAS (SEQ ID NO: 64), and LCDR3 comprises the amino acid sequence of SEQ ID NO: 16; X 20 is T or N; An anti-TSLP antibody or an antigen-binding fragment thereof.

2. (a) HCDR1 comprises the amino acid sequence of SEQ ID NO: 11; HCDR2 is IFPGDGX １ T (SEQ ID NO:59), and HCDR3 is ARX ２ GX ３ X ４ X ５ X ６ X ７ X ８ comprising the amino acid sequence of YAMDY (SEQ ID NO: 60), LCDR1 comprises the amino acid sequence of SEQ ID NO: 14; LCDR2 comprises the amino acid sequence of SEQ ID NO: 15, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 16; X １ is E or D, and X ２ is G or S, and X ３ is Y or F, and X ４ is V or D, and X ５ is N or Y, and X ６ is absent or is G, and X ７ is F or Y, and X ８ is L or F, or (b) HCDR1 is SYWX ９ N (SEQ ID NO: 61), HCDR2 is QIFPGDGX １０ TX １１ YNGX １２ comprising the amino acid sequence of FKG (SEQ ID NO: 62), and HCDR3 is X １３ GX １４ X １５ X １６ X １７ X １８ X １９ comprising the amino acid sequence of YAMDY (SEQ ID NO: 63), LCDR1 comprises the amino acid sequence of SEQ ID NO: 53; LCDR2 is GTSX ２０ comprising the amino acid sequence of LAS (SEQ ID NO: 64), and LCDR3 comprises the amino acid sequence of SEQ ID NO: 16; X ９ is M or I, and X １０ is E or D, and X １１ is N or T, and X １２ is K or N, and X １３ is G or S, and X １４ is Y or F, and X １５ is V or D, and X １６ is N or Y, and X １７ is absent or is G, and X １８ is F or Y, and X １９ is L or F, and X ２０ is T or N, where (a) HCDR1 comprises the amino acid sequence of SEQ ID NO: 11; HCDR2 comprises the amino acid sequence of SEQ ID NO: 25 or 12; HCDR3 comprises the amino acid sequence of SEQ ID NO: 26 or 13; LCDR1 comprises the amino acid sequence of SEQ ID NO: 14; LCDR2 comprises the amino acid sequence of SEQ ID NO: 15, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 16; or (b) HCDR1 comprises the amino acid sequence of SEQ ID NO: 55 or 50; HCDR2 comprises the amino acid sequence of SEQ ID NO: 56 or 51; HCDR3 comprises the amino acid sequence of SEQ ID NO: 57 or 52; LCDR1 comprises the amino acid sequence of SEQ ID NO: 53; LCDR2 comprises the amino acid sequence of SEQ ID NO: 58 or 54, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 16; or (c) HCDR1 comprises the amino acid sequence of SEQ ID NO: 11; HCDR2 comprises the amino acid sequence of SEQ ID NO: 25; HCDR3 comprises the amino acid sequence of SEQ ID NO: 26; LCDR1 comprises the amino acid sequence of SEQ ID NO: 14; LCDR2 comprises the amino acid sequence of SEQ ID NO: 15, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 16; or (d) HCDR1 comprises the amino acid sequence of SEQ ID NO: 55; HCDR2 comprises the amino acid sequence of SEQ ID NO:56; HCDR3 comprises the amino acid sequence of SEQ ID NO:57; LCDR1 comprises the amino acid sequence of SEQ ID NO: 53; LCDR2 comprises the amino acid sequence of SEQ ID NO: 58, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 16; or (e) HCDR1 comprises the amino acid sequence of SEQ ID NO: 50; HCDR2 comprises the amino acid sequence of SEQ ID NO:51; HCDR3 comprises the amino acid sequence of SEQ ID NO: 52; LCDR1 comprises the amino acid sequence of SEQ ID NO: 53; LCDR2 comprises the amino acid sequence of SEQ ID NO: 54, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 16; The antibody or antigen-binding fragment thereof according to claim 1.

3. further comprising one or more of heavy chain framework region 1 (HFR1), HFR2, HFR3 and HFR4, and / or one or more of light chain framework region 1 (LFR1), LFR2, LFR3 and LFR4; (a) HFR1 is SEQ ID NO: 17, SEQ ID NO: 27, or X ２１ VQLVQSGAEVKKPGX ２２ SX ２３ KX ２４ SCKX ２５ S (SEQ ID NO: 65) or a homologous sequence having at least 85% sequence identity thereto; HFR2 is SEQ ID NO: 18 or WVRQX ２７ PGX ２８ GLEWMG (SEQ ID NO: 66) or a homologous sequence thereof having at least 85% sequence identity; HFR3 is SEQ ID NO: 19, SEQ ID NO: 28, or X ３１ VTX ３２ X ３３ X ３４ DX ３５ SX ３６ STX ３７ YX ３８ X ３９ X ４０ SSLX ４１ X ４２ X ４３ DTAX ４４ YYC (SEQ ID NO: 67) or a homologous sequence thereof having at least 85% sequence identity; HFR4 comprises the amino acid sequence of SEQ ID NO: 20 or SEQ ID NO: 35, or a homologous sequence having at least 85% sequence identity thereto; LFR1 comprises the amino acid sequence of SEQ ID NO: 21, SEQ ID NO: 29, or SEQ ID NO: 36, or a homologous sequence having at least 85% sequence identity thereto; LFR2 is SEQ ID NO: 22, SEQ ID NO: 30, or WYQQKPGQSPRPWIX ４５ (SEQ ID NO: 68) or a homologous sequence having at least 85% sequence identity thereto; LFR3 comprises the amino acid sequence of SEQ ID NO:23, SEQ ID NO:31, or SEQ ID NO:38, or a homologous sequence having at least 85% sequence identity thereto; and LFR4 comprises the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 39, or a homologous sequence having at least 85% sequence identity thereto; X ２１ is E or Q, and X ２２ is E, S, or A, and X ２３ is L or V, and X ２４ is I or V, and X ２５ is S or A, and X ２７ is M or A, and X ２８ is K or Q, and X ３１ is Q or R, and X ３２ is I or M, and X ３３ is S or T, and X ３４ is A or R, and X ３５ is K or T, and X ３６ is I or T, and X ３７ is A or V, and X ３８ is L or M, and X ３９ is Q or E, and X ４０ is W or L, and X ４１ is K or R, and X ４２ is A or S, and X ４３ is S or E, and X ４４ is M or V, and X ４５ is Y or F, or (b) HFR1 comprises the amino acid sequence of SEQ ID NO: 17, 27, 32, 40, 42, 47, or 87; HFR2 comprises the amino acid sequence of SEQ ID NO: 18, 33, 43, or 48; HFR3 comprises the amino acid sequence of SEQ ID NO: 19, 28, 34, 41, 44, or 49; HFR4 comprises the amino acid sequence of SEQ ID NO: 20 or 35; LFR1 comprises the amino acid sequence of SEQ ID NO: 21, 29, or 36; LFR2 comprises the amino acid sequence of SEQ ID NO: 22, 30, 37, or 45; LFR3 comprises the amino acid sequence of SEQ ID NO: 23, 31, or 38; and LFR4 comprises the amino acid sequence of SEQ ID NO: 24 or 39; or (c) HFR1 comprises the amino acid sequence of SEQ ID NO: 17; HFR2 comprises the amino acid sequence of SEQ ID NO: 18; HFR3 comprises the amino acid sequence of SEQ ID NO: 19; HFR4 comprises the amino acid sequence of SEQ ID NO: 20; LFR1 comprises the amino acid sequence of SEQ ID NO: 21; LFR2 comprises the amino acid sequence of SEQ ID NO: 22; LFR3 comprises the amino acid sequence of SEQ ID NO: 23, and LFR4 comprises the amino acid sequence of SEQ ID NO: 24; or (d) HFR1 comprises the amino acid sequence of SEQ ID NO: 27; HFR2 comprises the amino acid sequence of SEQ ID NO: 18; HFR3 comprises the amino acid sequence of SEQ ID NO: 28; HFR4 comprises the amino acid sequence of SEQ ID NO: 20; LFR1 comprises the amino acid sequence of SEQ ID NO: 29; LFR2 comprises the amino acid sequence of SEQ ID NO: 30; LFR3 comprises the amino acid sequence of SEQ ID NO: 31, and LFR4 comprises the amino acid sequence of SEQ ID NO: 24; or (e) HFR1 comprises the amino acid sequence of SEQ ID NO: 32; HFR2 comprises the amino acid sequence of SEQ ID NO: 33; HFR3 comprises the amino acid sequence of SEQ ID NO: 34; HFR4 comprises the amino acid sequence of SEQ ID NO: 35; LFR1 comprises the amino acid sequence of SEQ ID NO: 36; LFR2 comprises the amino acid sequence of SEQ ID NO: 37; LFR3 comprises the amino acid sequence of SEQ ID NO: 38, and LFR4 comprises the amino acid sequence of SEQ ID NO: 39; or (f) HFR1 comprises the amino acid sequence of SEQ ID NO: 40; HFR2 comprises the amino acid sequence of SEQ ID NO: 33; HFR3 comprises the amino acid sequence of SEQ ID NO: 41; HFR4 comprises the amino acid sequence of SEQ ID NO: 35; LFR1 comprises the amino acid sequence of SEQ ID NO: 36; LFR2 comprises the amino acid sequence of SEQ ID NO: 37; LFR3 comprises the amino acid sequence of SEQ ID NO: 38, and LFR4 comprises the amino acid sequence of SEQ ID NO: 39; or (g) HFR1 comprises the amino acid sequence of SEQ ID NO: 42; HFR2 comprises the amino acid sequence of SEQ ID NO: 43; HFR3 comprises the amino acid sequence of SEQ ID NO: 44; HFR4 comprises the amino acid sequence of SEQ ID NO: 35; LFR1 comprises the amino acid sequence of SEQ ID NO: 36; LFR2 comprises the amino acid sequence of SEQ ID NO: 45; LFR3 comprises the amino acid sequence of SEQ ID NO: 38, and LFR4 comprises the amino acid sequence of SEQ ID NO: 39; or (h) HFR1 comprises the amino acid sequence of SEQ ID NO: 87; HFR2 comprises the amino acid sequence of SEQ ID NO: 33; HFR3 comprises the amino acid sequence of SEQ ID NO: 41; HFR4 comprises the amino acid sequence of SEQ ID NO: 35; LFR1 comprises the amino acid sequence of SEQ ID NO: 36; LFR2 comprises the amino acid sequence of SEQ ID NO: 45; LFR3 comprises the amino acid sequence of SEQ ID NO: 38, and LFR4 comprises the amino acid sequence of SEQ ID NO: 39; or (i) HFR1 comprises the amino acid sequence of SEQ ID NO: 47; HFR2 comprises the amino acid sequence of SEQ ID NO: 48; HFR3 comprises the amino acid sequence of SEQ ID NO:49; HFR4 comprises the amino acid sequence of SEQ ID NO: 35; LFR1 comprises the amino acid sequence of SEQ ID NO: 36; LFR2 comprises the amino acid sequence of SEQ ID NO: 45; LFR3 comprises the amino acid sequence of SEQ ID NO: 38, and LFR4 comprises the amino acid sequence of SEQ ID NO: 39; The antibody or antigen-binding fragment thereof according to claim 1.

4. and / or a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 8, 10 and 86, or a homologous sequence thereof with at least 80% sequence identity. A light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6 and 9, or a homologous sequence thereof having at least 80% sequence identity; The antibody or antigen-binding fragment thereof according to claim 1.

5. at least one of the substitutions or modifications is in one or more of the complementarity determining region (CDR) sequences of the heavy or light chain variable region; and / or at least one of the substitutions or modifications is in one or more of the non-CDR sequences of the heavy or light chain variable region; The antibody or antigen-binding fragment thereof according to claim 1.

6. Fc region, optionally a human immunoglobulin (Ig) Fc region, or optionally a human IgG Fc region, wherein the Fc region is derived from human IgG1 or IgG4, or further optionally the Fc region is derived from human IgG4 and contains one or more mutations selected from the group consisting of S228P, F234A, L235A, M252Y, S254T, T256E, and K447del; The antibody or antigen-binding fragment thereof according to claim 1.

7. The antibody or antigen-binding fragment thereof according to claim 6, wherein the amino acid sequence of the Fc region derived from human IgG4 containing the mutations S228P, F234A, L235A, M252Y, S254T, T256E, and K447del is shown as SEQ ID NO:

75.

8. 8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, which is a monoclonal antibody, a bispecific antibody, a multispecific antibody, a recombinant antibody, a chimeric antibody, a labeled antibody, a bivalent antibody, an anti-idiotypic antibody, a fusion protein, a diabody, Fab, Fab', F(ab')2, Fd, Fv fragment, a disulfide-stabilized Fv fragment (dsFv), (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide-stabilized diabody (dsdiabody), a single-chain antibody molecule (scFv), an scFv dimer (bivalent diabody), a multispecific antibody, a camelized single domain antibody, a nanobody, a domain antibody, or a bivalent domain antibody.

9. further comprising substitutions or modifications of one or more amino acid residues, yet retaining binding specificity for human TSLP, and optionally a) having the ability to specifically bind to human TSLP; b) having the ability to block the binding between TSLP and TSLPR; c) having the ability to block the binding between TSLP and cells expressing TSLPR and IL7R; d) having the ability to inhibit the TSLP-dependent proliferation of BaF3 cells; e) having the ability to inhibit TSLP-dependent TARC secretion from PBMCs; or f) having the ability to inhibit TSLP-dependent STAT5 activation in cells expressing TSLPR and IL7R. having one or more properties selected from the group consisting of: An antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, which binds to an epitope different from that bound by a reference antibody, wherein the amino acid sequence of the heavy chain of the reference antibody is shown as SEQ ID NO: 77 and the amino acid sequence of the light chain of the reference antibody is shown as SEQ ID NO:

78.

11. linked to one or more conjugate moieties, and optionally the conjugate moiety comprises an agent for detection or isolation, such as a clearance modifier, a chemotherapeutic agent, a toxin, a radioisotope, a lanthanide, a luminescent label, a fluorescent label, an enzyme substrate label, a DNA alkylating agent, a topoisomerase inhibitor, a tubulin binder, or other anti-cancer drug; An antibody or antigen-binding fragment thereof according to any one of claims 1 to 7.

12. (i) an antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, or a polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, and (ii) one or more pharmaceutically acceptable carriers, diluents, buffers, or excipients Including, Optionally, further comprising an additional therapeutic agent, further optionally, the additional therapeutic agent is an agent for treating inflammatory diseases, autoimmune diseases, and cancer; Pharmaceutical compositions.

13. A method for expressing an antibody or antigen-binding fragment thereof described in any one of claims 1 to 7, comprising the step of culturing a host cell containing a vector comprising an isolated polynucleotide encoding the antibody or antigen-binding fragment thereof described in any one of claims 1 to 7 under conditions in which the vector is expressed.

14. 10. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 7, or a polynucleotide encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 7, for use in a method for treating, preventing, or alleviating a TSLP-related disease or disorder in a subject, the method comprising the step of administering to a subject a therapeutically effective amount of the antibody or antigen-binding fragment thereof, or the polynucleotide encoding the antibody or antigen-binding fragment thereof.

15. wherein the disease or disorder is associated with dysregulation of TSLPR-mediated signaling compared to control levels, and optionally (a) the dysregulation of TSLP-mediated signaling includes dysregulation of STAT5 activation by phosphorylation, or (b) dysregulation of TSLP-mediated signaling includes dysregulation of proliferation of TSLPR-expressing cells; 15. The method of claim 14.

16. wherein the disease or disorder is selected from the group consisting of inflammatory diseases, autoimmune diseases, and cancer, and optionally the disease or disorder is selected from the group consisting of asthma (including severe asthma), idiopathic pulmonary fibrosis, atopic dermatitis (AD), allergic conjunctivitis, allergic rhinitis (AR), Netherton syndrome (NS), eosinophilic esophagitis (EoE), food allergy, allergic diarrhea, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis (ABPA), allergic fungal sinusitis, chronic pruritus, cancer, rheumatoid arthritis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), systemic sclerosis, multiple sclerosis, keloid, ulcerative colitis, chronic rhinosinusitis (CRS), polyposis, chronic eosinophilic pneumonia, eosinophilic bronchitis, allergic bronchopulmonary aspergillosis, celiac disease, eosinophilic gastroenteritis, Churg-Strauss disease, eosinophilic myalgia syndrome, hypereosinophilic syndrome, eosinophilic granulomatosis with polyangiitis, eosinophilic esophagitis, inflammatory bowel disease, fibrotic disorders, inflammatory bowel disease, Hodgkin's lymphoma, systemic lupus erythematosus, or Further optionally, the fibrotic disorder is selected from systemic and localized scleroderma, keloids and hypertrophic scars, interstitial lung disease (ILD), idiopathic pulmonary fibrosis (IPF), liver fibrosis due to chronic hepatitis B or C infection, radiation-induced fibrosis, and fibrosis resulting from wound healing, atherosclerosis, restenosis, lung inflammation and fibrosis, cirrhosis of the liver, kidney disease, heart disease due to scar tissue, and eye diseases such as macular degeneration, and retinal and vitreoretinopathy, fibrosis due to chemotherapy drugs, and trauma and burns; or Optionally, the cancer is selected from breast cancer, pancreatic cancer, colon cancer, lung cancer, ovarian cancer, prostate cancer, and B-cell acute lymphoblastic leukemia; or Optionally, the disease or disorder is selected from the group consisting of asthma, polypoid rhinosinusitis, COPD, urticaria, EoE, and atopic dermatitis.

16. The pharmaceutical composition according to claim 14 or 15.

17. 10. A method for detecting the presence or amount of TSLP in a sample, comprising contacting the sample with an antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, and determining the presence or amount of TSLP in the sample, and optionally further comprising determining whether TSLP is overexpressed in cells in the sample.

18. 8. A chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, and a TCR signaling domain, wherein the antigen-binding domain specifically binds to TSLP and comprises the antigen-binding fragment of any one of claims 1 to 7, and optionally further comprising a costimulatory domain.

19. 20. A pharmaceutical composition comprising cells genetically modified to express the CAR of claim 18 for use in a method for stimulating a T cell-mediated immune response to a TSLP-rich environment or tissue in a mammal, the method comprising administering to the mammal an effective amount of the cells.

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