Anti-TSLP antibody constructs and their use

Abstract

The present invention relates to isolated antibody constructs that specifically recognize TSLP. Compositions, kits, methods for preparation, and methods of use (e.g., for treating inflammatory diseases) are also provided.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority to U.S. Provisional Patent Application No. 63 / 578,947, filed on 25 August 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] (Refer to the electronic sequence list) The contents of the electronic sequence listing (253272000240seqlist.xml, size: 121,903 bytes, and creation date: August 19, 2024) are incorporated in their entirety herein by reference.

[0003] (Field of Invention) The present invention relates to an isolated antibody construct (e.g., an anti-TSLP antibody construct) that specifically recognizes thymic stromal lymphopoietin (TSLP), a pharmaceutical composition comprising the isolated anti-TSLP antibody construct, a method for treating a disease using the isolated anti-TSLP antibody construct, and a kit comprising the isolated anti-TSLP antibody construct. Methods for producing the same are also provided. [Background technology]

[0004] Many abnormal cells and tissues, as well as many diseases, including autoimmune disorders or inflammatory diseases such as asthma, exhibit unique antigens that can be utilized for immune cell-mediated clearance. Each of these inflammatory diseases may exhibit one or more different target antigens or one or more different epitopes of the same target antigen. For example, some antigens are overexpressed, mutagenic, or selectively mutagenic in inflammatory tissues. Therefore, antibodies targeting specific antigens present in diseases with systemic or local inflammation can be used as therapeutic agents.

[0005] Thymic stromal lymphopoietin (TSLP) is a pleiotropic cytokine characterized as a lymphocyte growth factor. TSLP binds to a high-affinity heteromeric complex composed of the thymic stromal lymphopoietin receptor (TSLPR) chain and interleukin-7 receptor-α (IL-7Rα). TSLP is mainly expressed by intestinal and pulmonary epithelial cells, dermal keratinocytes, and dendritic cells (DC), but can be produced by, for example, airway smooth muscle cells, mast cells, monocytes, macrophages, granulocytes, synovial fibroblasts, and the like. Depending on the immune cells targeted by TSLP, it has been reported to not only promote T helper 2 cell (Th2) responses, but also be associated with autoimmune disorders.

[0006] Tezepelumab (Tezspire™) is a fully human monoclonal antibody that binds to TSLP and blocks its interaction with the TSLPR chain. Subsequently, the IL-7Rα chain is not recruited, and no functional TSLP receptor complex is formed. See H. K. Lehman and C. M. Sabella, "Allergic and Immunologic Diseases, A Practical Guide to the Evaluation, Diagnosis and Management of Allergic and Immunologic Diseases," 2022, Pages 1111-1145, "Chapter 38 - New biologics in allergy." It is approved by the US FDA as an add-on maintenance therapy for severe asthma. Summary of the Invention

[0007] In one aspect of the present invention, an isolated antibody construct ("anti-TSLP antibody construct") is provided, comprising an antibody moiety that specifically recognizes thymic interstitial lymphocyte necrosis factor (TSLP) ("anti-TSLP antibody moiety"), wherein the anti-TSLP antibody moiety comprises a heavy chain variable region (VH) and a light chain variable region (VH). (a) VH includes (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 2 or a variant thereof containing up to 3 amino acid mutations, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 3 or a variant thereof containing up to 3 amino acid mutations, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO: 4 or a variant thereof containing up to 3 amino acid mutations, and VL includes (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 6 or a variant thereof containing up to 3 amino acid mutations, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 7 or a variant thereof containing up to 3 amino acid mutations, and (iii) CDR-L3 containing the amino acid sequence of SEQ ID NO: 8 or a variant thereof containing up to 3 amino acid mutations, (b) VH includes (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 12 or a variant thereof containing up to 3 amino acid mutations, ( ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 13 or a variant thereof containing up to three amino acid mutations, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO: 4 or a variant thereof containing up to three amino acid mutations, and VL contains (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 6 or a variant thereof containing up to three amino acid mutations, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 7 or a variant thereof containing up to three amino acid mutations, and (iii) CDR-L3 containing the amino acid sequence of SEQ ID NO: 8 or a variant thereof containing up to three amino acid mutations, (c) VH contains (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 20 or a variant thereof containing up to three amino acid mutations, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 21 or a variant thereof containing up to three amino acid mutations, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO: 22,Alternatively, CDR-H3 may contain a variant of it containing up to three amino acid mutations, and VL may contain (i) the amino acid sequence of SEQ ID NO: 24 or a variant of it containing up to three amino acid mutations, (ii) the amino acid sequence of SEQ ID NO: 25 or a variant of it containing up to three amino acid mutations, and (iii) the amino acid sequence of SEQ ID NO: 26 or a variant of it containing up to three amino acid mutations, and (d) VH may contain (i) the amino acid sequence of SEQ ID NO: 28 or a variant of it containing up to three amino acid mutations (ii) CDR-H1 containing the variant of (ii) the amino acid sequence of SEQ ID NO: 29 or a variant thereof containing up to three amino acid mutations, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO: 30 or a variant thereof containing up to three amino acid mutations, wherein VL is (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 32 or a variant thereof containing up to three amino acid mutations, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 33 or a variant thereof containing up to three amino acid mutations, and (iii) SEQ ID NO: 34 (e) VH includes (i) CDR-H1 including the amino acid sequence of SEQ ID NO: 36 or a variant thereof containing up to three amino acid mutations, (ii) CDR-H2 including the amino acid sequence of SEQ ID NO: 37 or a variant thereof containing up to three amino acid mutations, and (iii) CDR-H3 including the amino acid sequence of SEQ ID NO: 38 or a variant thereof containing up to three amino acid mutations, and VL includes (i) the amino acid sequence of SEQ ID NO: 40 or up to three amino acid mutations (ii) CDR-L1 containing a variant including an amino acid mutation, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO. 41 or a variant containing up to three amino acid mutations, and (iii) CDR-L3 containing the amino acid sequence of SEQ ID NO. 42 or a variant containing up to three amino acid mutations, or (f) VH containing (i) CDR-H1 containing the amino acid sequence of SEQ ID NO. 44 or a variant containing up to three amino acid mutations, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO. 45 or a variant containing up to three amino acid mutations,and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 46, or a variant thereof comprising up to 3 amino acid mutations, wherein the VL comprises (i) a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 48, or a variant thereof comprising up to 3 amino acid mutations, (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 49, or a variant thereof comprising up to 3 amino acid mutations, and (iii) a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 50, or a variant thereof comprising up to 3 amino acid mutations. ,

[0008] In some embodiments of the isolated anti-TSLP antibody constructs described above, the anti-TSLP antibody moiety comprises (a) VH comprising (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 2, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 3, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO: 4, and (b) VL comprising (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 6, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 7, and (iii) CDR-L3 containing the amino acid sequence of SEQ ID NO: 8, and (b) CDR-H comprising (i) CDR-H 1. VH containing (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 13, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO: 4, and VL containing CDR-L1 containing the amino acid sequence of SEQ ID NO: 6, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 7, and (iii) CDR-L3 containing the amino acid sequence of SEQ ID NO: 8, (c) VH containing (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 20, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 21, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO: 22, and SEQ ID NO: 24 (ii) VL containing the amino acid sequence of (i) 25, CDR-L2, and (iii) CDR-L3 containing the amino acid sequence of (iii) 26, (d) VH containing the amino acid sequence of (i) 28, CDR-H2, and (iii) CDR-H3 containing the amino acid sequence of (iii) 30, and CDR-L1, (ii) CDR-L2, and (iii) CDR-H3 containing the amino acid sequence of (iii) 32, CDR-L1, (ii) CDR-L2, and (iii) CDR-L3 containing the amino acid sequence of (iii) 34. VL containing DR-L3, (e)(i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 36, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 37, and (iii) VH containing CDR-H3 containing the amino acid sequence of SEQ ID NO: 38, and VL containing CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and (iii) CDR-L3 containing the amino acid sequence of SEQ ID NO: 42, or (f)(i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 44, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 45,(iii) VH containing CDR-H3 containing the amino acid sequence of SEQ ID NO: 46, and CDR-L1 containing the amino acid sequence of SEQ ID NO: 48, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 49, and (iii) VL containing CDR-L3 containing the amino acid sequence of SEQ ID NO: 50.

[0009] In some embodiments of any of the isolated anti-TSLP antibody constructs described above, (a) VH comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 1, and VL comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 5, (b) VH comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 9, and VL comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 10, (c) VH comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 11, and VL comprises (d) VH contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 15, and VL contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 19, and VL contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 23, (e) VH contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 27, and VL contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 31, (f) VH contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 35, and VL contains (g)VH contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 39, VL contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 43, (h)VH contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 63, VL contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 53, (i)VH contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 63, VL contains (j)VH contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 54, VL contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 56, (k)VH contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 55, VL contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 51, (l)VH contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 56, VL contains(m)VH contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 51, VL contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 63, (n)VH contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 64, VL contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 51, (o)VH contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 55, VL contains (p)VH contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 52, VL contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 58, (q)VH contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 60, VL contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 52, (r)VH contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 64, VL contains (s)VH contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 52, VL contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 55, (t)VH contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 53, VL contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 57, (u)VH contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 53, VL contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 58, (v)VH contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 53, VL contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 62, (w)VH contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 64, VL contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 53, (x)VH contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 55, VL contains(y)VH contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 54, VL contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 61, (z)VH contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 62, VL contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 54, (aa)VH contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 64, VL contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 54 (bb)VH contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 63, and VL contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 52, (cc)VH contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 188, and VL contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 189, or (dd)VH contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 188, and VL contains an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 190.

[0010] In some embodiments of any of the isolated anti-TSLP antibody constructs described above, (a) VH contains the amino acid sequence of SEQ ID NO: 1 and VL contains the amino acid sequence of SEQ ID NO: 5, (b) VH contains the amino acid sequence of SEQ ID NO: 9 and VL contains the amino acid sequence of SEQ ID NO: 10, (c) VH contains the amino acid sequence of SEQ ID NO: 11 and VL contains the amino acid sequence of SEQ ID NO: 15, (d) VH contains the amino acid sequence of SEQ ID NO: 19 and VL contains the amino acid sequence of SEQ ID NO: 23, (e) VH contains the amino acid sequence of SEQ ID NO: 27 and VL contains (f) VH contains the amino acid sequence of sequence number 31, VL contains the amino acid sequence of sequence number 35, (g) VH contains the amino acid sequence of sequence number 43, VL contains the amino acid sequence of sequence number 47, (h) VH contains the amino acid sequence of sequence number 63, VL contains the amino acid sequence of sequence number 53, (i) VH contains the amino acid sequence of sequence number 63, VL contains the amino acid sequence of sequence number 54, (j) VH contains the amino acid sequence of sequence number 56, VL contains the amino acid sequence of sequence number 52, (k) VH contains, (l)VH contains the amino acid sequence of SEQ ID NO: 55, and VL contains the amino acid sequence of SEQ ID NO: 51, (m)VH contains the amino acid sequence of SEQ ID NO: 56, and VL contains the amino acid sequence of SEQ ID NO: 51, (n)VH contains the amino acid sequence of SEQ ID NO: 63, and VL contains the amino acid sequence of SEQ ID NO: 51, (o)VH contains the amino acid sequence of SEQ ID NO: 55, and VL contains the amino acid sequence of SEQ ID NO: 52, (p)VH contains the amino acid sequence of SEQ ID NO: 58, and VL contains (q)VH contains the amino acid sequence of sequence number 52, VL contains the amino acid sequence of sequence number 60, (r)VH contains the amino acid sequence of sequence number 64, VL contains the amino acid sequence of sequence number 52, (s)VH contains the amino acid sequence of sequence number 55, VL contains the amino acid sequence of sequence number 53, (t)VH contains the amino acid sequence of sequence number 57, VL contains the amino acid sequence of sequence number 53, (u)VH contains the amino acid sequence of sequence number 58, VL contains the amino acid sequence of sequence number 53, (v)VH is(w)VH contains the amino acid sequence of SEQ ID NO: 62, and VL contains the amino acid sequence of SEQ ID NO: 53; (x)VH contains the amino acid sequence of SEQ ID NO: 55, and VL contains the amino acid sequence of SEQ ID NO: 54; (y)VH contains the amino acid sequence of SEQ ID NO: 61, and VL contains the amino acid sequence of SEQ ID NO: 54; (z)VH contains the amino acid sequence of SEQ ID NO: 62, and VL contains the amino acid sequence of SEQ ID NO: 54; (aa)VH contains the amino acid sequence of SEQ ID NO: 64, and VL contains the amino acid sequence of SEQ ID NO: 54; (bb)VH contains the amino acid sequence of SEQ ID NO: 63, and VL contains the amino acid sequence of SEQ ID NO: 52; (cc)VH contains the amino acid sequence of SEQ ID NO: 188, and VL contains the amino acid sequence of SEQ ID NO: 189; or (dd)VH contains the amino acid sequence of SEQ ID NO: 188, and VL contains the amino acid sequence of SEQ ID NO: 190.

[0011] In some embodiments of any of the isolated anti-TSLP antibody constructs described above, the anti-TSLP antibody portion is selected from the group consisting of full-length antibody, Fab, Fab', F(ab')2, diabody, and scFv.

[0012] In some embodiments of any of the isolated anti-TSLP antibody constructs described above, the anti-TSLP antibody portion is a full-length antibody ("anti-TSLP full-length antibody"). In some embodiments, the anti-TSLP full-length antibody contains a CL comprising an amino acid sequence selected from the group consisting of SEQ ID NOs.73-75. In some embodiments, the anti-TSLP full-length antibody contains an Fc domain derived from human IgG (e.g., human IgG1, IgG2, or IgG4). In some embodiments, the Fc domain is derived from human IgG1, and i) the first and second subunits of the Fc domain each contain one amino acid sequence from sequence numbers 76 to 79; ii) the first subunit of the Fc domain contains the amino acid sequence of sequence number 80 and the second subunit of the Fc domain contains the amino acid sequence of sequence number 81; iii) the first subunit of the Fc domain contains the amino acid sequence of sequence number 81 and the second subunit of the Fc domain contains the amino acid sequence of sequence number 80; iv) the first subunit of the Fc domain contains the amino acid sequence of sequence number 95 and the second subunit of the Fc domain contains the amino acid sequence of sequence number 96; or v) the first subunit of the Fc domain contains the amino acid sequence of sequence number 96 and the second subunit of the Fc domain contains the amino acid sequence of sequence number 95. In some embodiments, the first and second subunits of the Fc domain each contain the amino acid sequence of sequence number 77 or 79. In some embodiments, the full-length anti-TSLP antibody comprises i) two heavy chains, each containing the amino acid sequence of SEQ ID NO: 104, and two light chains, each containing the amino acid sequence of SEQ ID NO: 103; ii) two heavy chains, each containing the amino acid sequence of SEQ ID NO: 105, and two light chains, each containing the amino acid sequence of SEQ ID NO: 103; or iii) a first heavy chain containing the amino acid sequence of SEQ ID NO: 136, a second heavy chain containing the amino acid sequence of SEQ ID NO: 137, and two light chains, each containing the amino acid sequence of SEQ ID NO: 103.

[0013] In some embodiments of any of the isolated anti-TSLP antibody constructs described above, the anti-TSLP antibody moiety is scFv ("anti-TSLP scFv"). In some embodiments, anti-TSLP scFv comprises one of the amino acid sequences of SEQ ID NOs: 106, 107, 191, and 192.

[0014] In some embodiments of any of the isolated anti-TSLP antibody constructs described above, the anti-TSLP antibody portion is Fab ("anti-TSLP Fab"). In some embodiments, anti-TSLP Fab comprises a first polypeptide having the amino acid sequence of SEQ ID NO: 109 and a second polypeptide having the amino acid sequence of SEQ ID NO: 103.

[0015] In some embodiments using any of the isolated anti-TSLP antibody constructs described above, the isolated anti-TSLP antibody construct is monospecific.

[0016] In some embodiments using any of the isolated anti-TSLP antibody constructs described above, the isolated anti-TSLP antibody construct is a multispecific anti-TSLP antibody construct comprising a second antibody moiety that specifically recognizes a second target antigen (e.g., IL-13). In some embodiments, the second antibody moiety is selected from the group consisting of full-length antibody, Fab, Fab', F(ab')2, sdAb, diabody, and scFv. In some embodiments, the second antibody moiety is scFv. In some embodiments, the second antibody moiety is Fab. In some embodiments, the second antibody moiety is a full-length antibody. In some embodiments, the anti-TSLP antibody moiety and the second antibody moiety are fused to each other via a linker such as a linker containing GG and one of the amino acid sequences of SEQ ID NOs. 14, 16-18, 97-99, and 163-172, for example, GG and one of the amino acid sequences of SEQ ID NOs. 98-99.

[0017] Also provided are pharmaceutical compositions comprising one of the isolated anti-TSLP antibody constructs described herein and a pharmaceutically acceptable carrier.

[0018] Another aspect of the present invention provides a method for treating an inflammatory disease in an individual (e.g., a human), comprising administering to the individual an effective amount of any of the isolated anti-TSLP antibody constructs described herein or a pharmaceutical composition thereof (e.g., any of the pharmaceutical compositions described herein). In some embodiments, the inflammatory disease is asthma, atopic dermatitis, or chronic obstructive pulmonary disease (COPD).

[0019] Also provided are isolated nucleic acids encoding any polypeptide portion of the above-mentioned isolated anti-TSLP antibody constructs, vectors containing such isolated nucleic acids, and host cells containing such isolated nucleic acids or vectors.

[0020] Furthermore, a method for producing an anti-TSLP antibody construct is also provided, comprising: i) culturing a host cell containing either an isolated nucleic acid or vector encoding any polypeptide portion of the above-mentioned anti-TSLP antibody construct, or a host cell encoding any of the above-mentioned anti-TSLP antibody constructs, under conditions suitable for the expression of the anti-TSLP antibody construct; and ii) obtaining the expressed anti-TSLP antibody construct, for example, from the host cell or from a cell culture (for example, from a cell culture medium).

[0021] These and other aspects and advantages of the present invention will become apparent from the following detailed description and accompanying drawings. It should be understood that other embodiments of the present invention may be formed by combining one, some, or all of the characteristics of the various embodiments described herein.

[0022] All publications, patents, patent applications, and published patent application disclosures referenced herein are incorporated herein by reference in their entirety. [Brief explanation of the drawing]

[0023] [Figure 1] This report shows the percentage of inhibition of TSLP binding to the TSLP receptor complex expressed on STAT5-HEK293 reporter cells when treated with exemplary anti-TSLP antibodies (i.e., 51B2, 51A3, 51A4, 54A2, 54B3, and 54C4). STAT5-HEK293 reporter cells were incubated with various concentrations of each antibody and with 10 ng / mL of TSLP. TSLP-inducible luciferase production by reporter cells served as a readout of TSLP-binding activity, and the IC90 values ​​for each antibody were calculated based on antibody dose-dependent binding inhibition curves. Data were analyzed and presented using GraphPad. Ab, antibody; Conc., concentration. [Figure 2] This shows 51B2 and the US-FDA approved anti-TSLP Ab (reference antibody #1 or "Ref.Ab.#1") that bind to overlapping but different epitopes on TSLP in ELISA competitive testing. Ab, antibody; OD, optical density. [Figure 3A] Compared to the US-FDA-approved anti-TSLP Ab (Ref. Ab. #1), 51B2 shows complete inhibition of TSLP-induced CCL17 secretion from PBMCs (Figure 3A). Figure 3A shows inhibition of TSLP-induced CCL17 secretion from human PBMCs seeded in 96-well plates and incubated with 50 ng / ml TSLP and multiple concentrations of 51B2 or Ref. Ab. #1. Cell culture supernatants collected after 48 hours were analyzed by ELISA for the levels of secreted CCL17. Ab, antibody; PBMC, peripheral blood mononuclear cells. [Figure 3B]Compared to the US-FDA-approved anti-TSLP Ab (Ref. Ab. #1), 51B2 shows complete inhibition of TSLP-induced CCL17 secretion from isolated dendritic cells (Figure 3B). Figure 3B shows inhibition of TSLP-induced CCL17 secretion from isolated CD1c+ dendritic cells seeded in a 96-well plate and incubated with 5 ng / ml TSLP and multiple concentrations of 51B2 or Ref. Ab. #1. Cell culture supernatants collected after 24 hours were analyzed by ELISA for the levels of secreted CCL17. Data were analyzed and presented using GraphPad Prism. Ab, antibody; PBMC, peripheral blood mononuclear cells. [Figure 4-1] The IC50(nM) and IC90(nM) values ​​obtained for each humanized 51B2 antibody produced in human and cynomolgus monkey ("cyno") TSLP inhibition assays using HEK293 reporter cells transfected with the TSLP receptor complex are shown, compared to the parental Ch51B2 clone. Humanized antibody clones separated by boxes indicate clones selected for further analysis. [Figure 4-2] The IC50(nM) and IC90(nM) values ​​obtained for each humanized 51B2 antibody produced in human and cynomolgus monkey ("cyno") TSLP inhibition assays using HEK293 reporter cells transfected with the TSLP receptor complex are shown, compared to the parental Ch51B2 clone. Humanized antibody clones separated by boxes indicate clones selected for further analysis. [Figure 5A] This report shows the percentage of inhibition of TSLP binding to the TSLP receptor complex expressed on STAT5-HEK293 reporter cells when treated with selected humanized anti-TSLP antibodies (ch51B2 parental chimeric clone, hz51B2 L2H2, hz51B2 L2H9, and hz51B2 L3H9). STAT5-HEK293 reporter cells were incubated with 10 ng / mL of TSLP and with multiple concentrations of each antibody, and the IC90 value for each antibody was calculated based on antibody dose-dependent binding inhibition curves. Data were analyzed and presented using GraphPad. Ab, antibody; Conc., concentration. [Figure 5B] This shows the equilibrium binding analysis of hz51B2 L3H9 binding to human TSLP in solution. [Modes for carrying out the invention]

[0024] This application provides novel anti-TSLP antibody constructs, including both monospecific and multispecific anti-TSLP antibody constructs. These novel anti-TSLP antibody constructs offer several advantages. Firstly, they demonstrate improved potency and ability to completely block TSLP signaling compared to US-FDA approved reference anti-TSLP antibodies that could only partially block TSLP signaling. For example, the anti-TSLP antibody constructs described herein demonstrated activity to completely block CCL17 secretion by immune cells such as peripheral blood mononuclear cells (PBMCs) and dendritic cells (DCs), demonstrating complete suppression of TSLP signaling. While not bound by theory, the anti-TSLP antibody constructs described herein are thought to bind to different epitopes than the reference anti-TSLP antibodies. Unlike reference anti-TSLP antibodies that block only TSLP / TSLPR interactions, the anti-TSLP antibody constructs described herein are thought to block interactions between TSLP and both TSLPR and IL-7Rα, and therefore may provide a more potent and more ideal TSLP blocking profile. Secondly, the anti-TSLP antibody constructs described herein exhibit very strong binding affinity to human TSLP, comparable to, and in some cases even stronger than, US-FDA-approved reference anti-TSLP antibodies. Thirdly, the anti-TSLP antibody constructs described herein exhibit cross-reactivity and strong binding affinity to cynomolgus monkey TSLP, which facilitates the extrapolation of toxicity and efficacy study results from cynomolgus monkeys to human clinical studies.

[0025] Accordingly, in one embodiment, the present invention provides an isolated antibody construct ("anti-TSLP antibody construct") comprising an antibody moiety that specifically recognizes TSLP ("anti-TSLP antibody moiety"). In some embodiments, the anti-TSLP antibody moiety is a full-length antibody ("anti-TSLP full-length antibody"). In some embodiments, the anti-TSLP antibody moiety is an scFv ("anti-TSLP scFv"). In some embodiments, the anti-TSLP antibody moiety is a Fab ("anti-TSLP Fab"). In some embodiments, the isolated anti-TSLP antibody construct is monospecific. In some embodiments, the isolated anti-TSLP antibody construct is a multispecific anti-TSLP antibody construct further comprising a second antibody moiety that specifically recognizes a second target antigen (e.g., IL-13). Also provided are isolated anti-TSLP antibody constructs that can compete with any of the isolated anti-TSLP antibody constructs and / or anti-TSLP antibody moieties described herein with respect to binding to TSLP.

[0026] Furthermore, pharmaceutical compositions and kits comprising any of the isolated anti-TSLP antibody constructs described herein, as well as methods of using any of the isolated anti-TSLP antibody constructs described herein or the pharmaceutical compositions thereof, such as for treating inflammatory diseases, such as asthma, atopic dermatitis, or COPD.

[0027] I. Definition As used herein, the term “treatment” refers to a clinical intervention designed to alter the natural course of an individual or cell being treated in the course of clinicopathology. Desired effects of treatment include a decrease in the rate of disease progression, improvement or mitigation of the disease state, and remission or improved prognosis. For example, if one or more symptoms associated with an inflammatory disease are alleviated or eliminated, the individual is successfully “treated,” which includes, but is not limited to, reducing local or systemic inflammation, decreasing symptoms resulting from the disease, increasing the quality of life of the affected individual, or reducing the dosage of other drugs required to treat the disease.

[0028] As used herein, “effective dose” refers to the amount of an agent or drug that is effective in treating a disease or disorder in a subject (an individual, e.g., a human). In the case of inflammatory diseases, an effective dose of an agent may reduce the number of active immune cells, reduce the amount of pro-inflammatory cytokines, inhibit (i.e., slow, preferably stop) inflammatory immune cell activity locally and / or systemically, and / or alleviate to some extent one or more of the symptoms associated with the inflammatory disease. As understood in clinical contexts, an effective dose of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Thus, “effective dose” may be considered in a situation in which one or more therapeutic agents are administered, and a single agent may be considered to be given in an effective dose if, in combination with one or more other agents, the desired outcome can or would be achieved.

[0029] As used herein, “individual” or “subject” refers to mammals, including but not limited to humans, cattle, horses, cats, dogs, rodents, or non-human primates. In some embodiments, the individual is a human.

[0030] The term "antibody" is used broadly and specifically includes monoclonal antibodies (including full-length monoclonal antibodies), multispecific antibodies (e.g., bispecific antibodies), and antibody fragments insofar as they exhibit the desired biological activity. As used herein, the terms "immunoglobulin" (Ig) and "antibody" are interchangeable.

[0031] The term "full-length antibody" is used herein to refer to an antibody in its substantially intact form, rather than an antibody fragment as defined below. This term specifically refers to an antibody having a heavy chain containing an Fc region. A full-length antibody is typically a heterotetrameric glycoprotein with approximately 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains.

[0032] The term "constant domain" refers to the portion of the immunoglobulin molecule that contains the antigen-binding site and has a more conserved amino acid sequence compared to the variable domain. The constant domain is the C of the heavy chain. H 1, C H 2, and C H 3 domains (collectively, CH) and the light chain C H It contains an L (or CL) domain.

[0033] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain. The variable domain of the heavy chain may be referred to as "VH," and the variable domain of the light chain may be referred to as "VL." These domains are generally the most variable parts of the antibody and contain the antigen-binding site.

[0034] The term "variable" refers to the fact that certain portions of the variable domain have widely different sequences across antibodies, and these are used in the binding and specificity of each particular antibody to a particular antigen. However, variability is not evenly distributed throughout the variable domain of an antibody. It is concentrated in three segments called hypervariable regions (also known as HVRs or CDRs) in both the light and heavy chain variable domains. A more highly conserved portion of the variable domain is called the framework region (FR). The variable domains of the naive heavy and light chains each contain four FRs, most of which take the form of a beta-sheet structure, connected by three HVRs, forming loops that connect the beta-sheet structures, and in some cases forming parts of the beta-sheet structures. The HVRs in each chain are held together in close proximity by the FR regions and, using HVRs from other chains, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domain does not directly participate in antibody binding to antigens, but exhibits various effector functions, such as the antibody's involvement in antibody-dependent cytotoxicity. Unless otherwise indicated, residues of the constant domain are described herein based on EU numbering.

[0035] As used herein, the terms “hypervariable region,” “HVR,” or “HV” refer to regions of antibody variable domains that are hypervariable in sequence and / or form structurally defined loops. Generally, antibodies contain six HVRs: three VH (HVR-H1, HVR-H2, HVR-H3) and three VL (HVR-L1, HVR-L2, HVR-L3). In natural antibodies, HVR-H3 and HVR-L3 exhibit the highest diversity among the six HVRs, and HVR-H3 in particular is thought to play a unique role in conferring superior specificity to antibodies. See, for example, Xu et al., Immunity 13:37-45 (2000) and Johnson and Wu, in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, NJ, 2003). In fact, naturally occurring camel antibodies consisting solely of heavy chains are functional and stable in the absence of light chains. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993) and Sheriff et al., Nature Struct. Biol. 3:733-736 (1996). HVR is also referred to as "CDR" or "complementarity-determining region".

[0036] The structure and location of immunoglobulin variable regions can be determined by referring to Kabat, EA et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, 1987, and its latest edition (currently available on the internet (immuno.bme.nwu.edu)). Any immunoglobulin variable region definition scheme, including but not limited to Kabat, Chothia, AbM, Contact, or IMGT, may be used.

[0037] As used herein, the terms “CDR” or “complementarity-determining region” are intended to mean discontinuous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. These specific regions have been described in Kabat et al., J. Biol. al., J.Mol.Biol.,273:927-948(1997), MacCallum et al.,J.Mol.Biol.262:732-745(1996), Abhinandan and Martin,Mol.Immunol.,45:3832-3839(2008), Lefranc MPet The definitions are described in al., Dev. Comp. Immunol., 27:55-77 (2003), and Honegger and Pluckthun, J. Mol. Biol., 309:657-670 (2001), and include overlaps or subsets of amino acid residues when compared to one another. Nevertheless, the application of any definition to refer to a CDR of an antibody or grafted antibody or its variant is intended to be within the scope of the terms defined and used herein. For comparison, the amino acid residues encompassing a CDR as defined by each of the above cited references are shown in Table A below. CDR prediction algorithms and interfaces are known in the art, including, for example, Abhinandan and Martin, Mol.Immunol., 45:3832-3839 (2008), Ehrenmann F. et al., Nucleic Acids Res., 38:D301-D307 (2010), and Adolf-Bryfogle J. et al., Nucleic Acids Res., 43:D432-D438 (2015).The contents of the references cited in this paragraph are incorporated herein by reference in their entireties for use in the present application and to enable inclusion in one or more claims of the present specification. Unless otherwise indicated, amino acid residues of CDRs provided herein are set forth herein based on Kabat.

[0038]

Table 1

[0039] "Framework" or "FR" residues are variable domain residues other than the HVR or CDR residues as defined herein. Unless otherwise indicated, FR amino acid residues are set forth herein based on Kabat numbering.

[0040] The "light chains" of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two distinct types, namely kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0041] As used herein, the term IgG "isotype" or "subclass" refers to any of the subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions.

[0042] Depending on the amino acid sequence of the constant domains of their heavy chains, antibodies (immunoglobulins) can be assigned to different "classes." There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further classified into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known, and generally, see, for example, Abbas et al., Cellular and Mol. Immunology, 4. th This is described in ed. (WBSaunders, Co., 2000). The antibody may be part of a larger fusion molecule formed by covalent or noncovalent bonding between the antibody and one or more other proteins or peptides.

[0043] An "antibody fragment" comprises a portion of an intact antibody, preferably including its antigen-binding region. In some embodiments, the antibody fragment described herein is an antigen-binding fragment. Examples of antibody fragments or antigen-binding fragments include Fab, Fab', F(ab')2, and Fv fragments (single-chain variable fragments, such as scFv), diabodies; linear antibodies; single-chain antibody (sdAb) molecules; and multispecific antibodies formed from antibody fragments.

[0044] Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab" fragments, each with a single antigen-binding site, while the remaining "Fc" fragment, whose name reflects its ability to easily crystallize, is also produced. Pepsin treatment yields an F(ab')2 fragment, which has two antigen-binding sites and can still crosslink antigens.

[0045] "Fv" is the smallest antibody fragment containing a complete antigen-binding site. In one embodiment, a double-stranded Fv species consists of a dimer of one heavy-chain variable domain and one light-chain variable domain that are rigidly non-covalently associated. In a single-stranded Fv (scFv) species, the one heavy-chain and one light-chain variable domain can be covalently linked by a flexible peptide linker so that the light and heavy chains can associate in a "dimer" structure similar to that in a double-stranded Fv species. In this configuration, the three HVRs in each variable domain interact to define the antigen-binding site on the surface of the VH-VL dimer. Collectively, the six HVRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific HVRs) has the ability to recognize and bind to an antigen, albeit with lower affinity than a complete binding site.

[0046] The Fab fragment contains heavy chain and light chain variable domains (VH, VL), as well as a constant domain (CL) of the light chain and a first constant domain (C) of the heavy chain. H 1) It has two polypeptide chains, which also contain. The Fab' fragment is a heavy chain C containing one or more cysteines from the antibody hinge region. H It differs from the Fab fragment by having several residues added to the carboxyl terminus of one domain. Fab'-SH is the heretical designation for Fab' having a free thiol group on the cysteine ​​residue of the constant domain. The F(ab')2 antibody fragment was originally produced as a pair with Fab' fragments having a hinged cysteine ​​between them. Other chemical couplings of antibody fragments are also known.

[0047] A "single-stranded Fv" or "scFv" antibody fragment contains the VH and VL domains of the antibody, and these domains are present within a single polypeptide chain. Generally, scFv polypeptides further include a polypeptide linker between the VH and VL domains, which allows the scFv to form a desired structure for antigen binding. For an overview of scFv, see, for example, Pluckthun, The Pharmacology of Monoclonal Antibodies. Springer Berlin Heidelberg, 1994. 269-315.

[0048] The "Fc" fragment contains the carboxyl terminal portions of both heavy chains, held together by a disulfide. The effector function of the antibody is determined by the sequence of the Fc region, which is also the region recognized by the Fc receptor (FcR) found on cells of certain species.

[0049] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, where, for example, the individual antibodies constituting the population are identical except for any possible mutations that may be present in trace amounts, such as spontaneously occurring mutations. Thus, the modifier “monoclonal” indicates a characteristic of the antibody that it is not a mixture of distinct antibodies. In some embodiments, such a monoclonal antibody typically comprises an antibody containing a target-binding polypeptide sequence, and the target-binding polypeptide sequence is obtained by a process comprising the selection of a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process may be the selection of a unique clone from a plurality of clones, such as a hybridoma clone, a phage clone, or a pool of recombinant DNA clones. It should be understood that the selected target-binding sequence may be further modified, for example, to improve affinity for a target, to humanize the target-binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to produce a multispecific antibody, and that an antibody containing a modified target-binding sequence is also a monoclonal antibody of the present invention. In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is against a single determinant on an antigen. In addition to their specificity, monoclonal antibody preparations have the advantage of being typically free from contamination by other immunoglobulins.

[0050] The modifier "monoclonal" indicates the characteristic of antibodies obtained from a substantially homogeneous population of antibodies and should not be interpreted as requiring antibody production by any particular method. For example, monoclonal antibodies used in accordance with the present invention are, for example, produced by hybridoma (e.g., Kohler and Milstein, Nature 256:495-97 (1975), Hongo et al., Hybridoma 14(3):253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2 nd(ed. 1988), Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981), Recombinant DNA method (see, e.g., U.S. Patent No. 4,816,567), Phage display technology (e.g., Clackson et al., Nature 352:624-628 (1991), Marks et al., J.Mol.Biol.222:581-597 (1992), Sidhu et al., J.Mol.Biol.338(2):299-310 (2004), Lee et al., J.Mol.Biol.340(5):1073-1093 (2004), Fellouse, Proc. Natl. Acad. Sci. USA See 101(34):12467-12472(2004) and Lee et al., J.Immunol.Methods 284(1-2):119-132(2004), as well as techniques for producing human or human-like antibodies in animals having some or all of the human immunoglobulin locus or genes encoding human immunoglobulin sequences (e.g., International Publication No. 1998 / 24893, International Publication No. 1996 / 34096, International Publication No. 1996 / 33735, International Publication No. 1991 / 10741, Jakobovits et al., Proc.Natl.Acad.Sci.USA 90:2551(1993), Jakobovits et al., Nature 362:255-258(1993), Bruggemann et al., Year in Immunol.7:33 (1993), U.S. Patent Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016, Marks et al.,Bio / Technology 10:779-783(1992), Lonberg et al., Nature 368:856-859(1994), Morrison, Nature 368:812-813(1994), Fishwild et al., Nature Biotechnol.It can be prepared by various techniques, including (see 14:845-851 (1996), Neuberger, Nature Biotechnol. 14:826 (1996), and Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995)).

[0051] The term "monoclonal antibody" as used herein specifically includes "chimeric" antibodies in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence of an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the rest of the chain is identical or homologous to a corresponding sequence of an antibody derived from another species or belonging to another antibody class or subclass, and also includes fragments of such antibodies insofar as they exhibit the desired biological activity (see, for example, U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies include PRIMATIZED® antibodies in which the antigen-binding region of the antibody is derived, for example, from an antibody produced by immunizing macaque monkeys with the antigen of interest.

[0052] The "humanized" form of a non-human (e.g., mouse) antibody is a chimeric antibody containing a minimal sequence derived from a non-human immunoglobulin. In one embodiment, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues derived from the recipient's HVR are replaced by residues derived from the HVR of a non-human species (donor antibody) (e.g., mouse, rat, rabbit, or non-human primate) having the desired specificity, affinity, and / or capabilities. In some examples, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may contain residues not found in either the recipient antibody or the donor antibody. These modifications may be made to further improve the performance of the antibody. Generally, the humanized antibody will contain substantially all of at least one, typically two, variable domains, in which all or substantially all hypervariable loops correspond to the hypervariable loops of the non-human immunoglobulin, and all or substantially all FRs are FRs of the human immunoglobulin sequence. Humanized antibodies also optionally include the constant region (Fc) of immunoglobulins, typically at least a portion of the Fc of human immunoglobulins. For further details, see, for example, Jones et al., Nature 321:522-525 (1986), Riechmann et al., Nature 332:323-329 (1988), and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See, for example, Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998), Harris, Biochem. Soc. Transactions 23:1035-1038 (1995), Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994), and U.S. Patent Nos. 6,982,321 and 7,087,409.

[0053] A “human antibody” is defined as one having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human, and / or one prepared using any of the techniques for producing human antibodies disclosed herein. Specifically, this definition of a human antibody excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. (Hoogenboom and Winter, J.Mol.Biol.227:381(1991), Marks et al., J.Mol.Biol.222:581(1991)). Methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, 77(1985), and Boerner et al., J.Immunol.147(1):86-95(1991) are also available for the preparation of human monoclonal antibodies. See also van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001). Human antibodies can be prepared by administering an antigen to transgenic animals, such as immunized xenomouses, which are modified to produce such antibodies in response to antigen challenge but whose endogenous gene loci have been deactivated (see, for example, U.S. Patents 6,075,181 and 6,150,584, relating to XENOMOUSE® technology). See also, for example, Li, et al., Proc. Natl. Acad. Sci. USA 103:3557-3562 (2006) (referring to human antibodies produced via human B-cell hybridoma technology).

[0054] As used herein, the term “covalent” refers to a direct bond via one or more chemical bonds, or an indirect bond via one or more linkers. Direct bonds can be made using any suitable chemical bond, including, but not limited to, covalent bonds such as peptide bonds and disulfide bonds, or non-covalent bonds such as hydrogen bonds, hydrophobic bonds, ionic bonds, or van der Waals bonds.

[0055] As used herein, “covalent bond” refers to a stable bond between two atoms that share one or more electrons. Examples of covalent bonds include, but are not limited to, peptide bonds and disulfide bonds. As used herein, “peptide bond” refers to a covalent bond formed between the carboxyl group of an amino acid and the amine group of an adjacent amino acid. As used herein, “disulfide bond” refers to a heavy chain fragment C bonded by one or more disulfide bonds. H This refers to a covalent bond formed between two sulfur atoms, such as a combination of 1 and the light chain fragment CL. One or more disulfide bonds can be formed between two fragments by linking thiol groups in the two fragments. In some embodiments, one or more disulfide bonds can be formed between one or more cysteine ​​groups of the heavy chain fragment and the light chain fragment, respectively. Disulfide bonds can be formed by the oxidation of two thiol groups. In some embodiments, covalent bonds are directly linked by covalent bonds. In some embodiments, covalent bonds are directly linked by peptide bonds or disulfide bonds.

[0056] As used herein, the terms “binding,” “specifically binding,” “specifically recognizing,” or “specific to” refer to measurable and reproducible interactions, such as binding between a target and an antibody, that determine the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that binds to a target (which may be an epitope) or specifically recognizes a target is an antibody that binds to this target with higher affinity, binding strength, more readily, and / or for a longer duration than an antibody that binds to other targets. In one embodiment, the degree of antibody binding to an unrelated target is less than about 10% of the antibody binding to the target, as measured, for example, by radioimmunoassay (RIA). In some embodiments, an antibody that specifically recognizes a target has a dissociation constant (K) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM. d ) has. In some embodiments, the antibody specifically recognizes an epitope on a protein that is conserved between proteins of different species. In other embodiments, specific binding may include, but is not required, exclusive binding.

[0057] As used herein, “amino acid sequence identity percentage (%)” and “homology” for a peptide, polypeptide, or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a particular peptide or polypeptide sequence after the sequences have been aligned and gaps introduced as necessary, without considering any conservative substitutions as part of the sequence identity, to achieve the maximum sequence identity percentage. Alignment for the purpose of determining amino acid sequence identity percentage can be achieved using various methods within the scope of the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN® (DNASTAR) software. A person skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithm necessary to achieve the maximum alignment over the entire length of the sequences being compared.

[0058] Amino acid substitutions may include, but are not limited to, the replacement of one amino acid in a polypeptide with another. Exemplary substitutions are shown in Table 1. Amino acid substitutions may be introduced into the antibody of interest, and the product may be screened for desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0059] [Table 2]

[0060] Amino acids can be classified according to their common side-chain properties: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues that affect chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe. Non-conservative substitutions involve exchanging a member of one of these classes for a member of another class.

[0061] The term "multispecificity," as used in conjunction with antibodies or antigen-binding proteins, refers to antibodies or antigen-binding proteins that possess polyepitope specificity (i.e., they can specifically recognize two, three, or more different epitopes on one biomolecule, or can specifically recognize two, three, or more different epitopes on two, three, or more different biomolecules). Unless otherwise specified, the order in which the antigens to which the multispecific antibody binds are listed in the multispecific antibody name is arbitrary.

[0062] The term "bispecificity," used in conjunction with antibodies or antigen-binding proteins, refers to an antibody or antigen-binding protein that can specifically recognize two different epitopes on one biomolecule, or that can specifically recognize two different epitopes on two different biomolecules. Unless otherwise specified, the order in which the antigens to which a bispecific antibody binds are listed in the bispecific antibody name is arbitrary.

[0063] The “knob-in-hole” strategy (see, for example, International Publication No. 2006 / 028936) has its plain, ordinary meaning when read in light of this specification and refers to a strategy that may be used to produce full-length bispecific antibodies. In short, C in human IgG H Selected amino acids that form the interface of the three domains are used to promote heterodimer formation. H Mutations can be induced at positions that affect the three-domain interaction. An amino acid with a small side chain (hole) is introduced into the heavy chain of the antibody that specifically recognizes the first antigen, and an amino acid with a large side chain (knob) is introduced into the heavy chain of the antibody that specifically recognizes the second antigen. After co-expression of the two antibodies, a heterodimer is formed as a result of the preferential interaction between the heavy chain with the "hole" and the heavy chain with the "knob".

[0064] As used herein, the term “vector” means a nucleic acid molecule capable of propagating another ligated nucleic acid. This term includes vectors as self-replicating nucleic acid structures, and vectors incorporated into the genome of a host cell into which they are introduced. Certain vectors can be directed to the expression of nucleic acids into which they are manipulably ligated. Such vectors are referred to herein as “expression vectors.”

[0065] Embodiments of the present invention described herein are understood to include embodiments that "consist" and / or "consisting essentially of".

[0066] In this specification, references to values ​​or parameters "about" include (and describe) variations relating to the value or parameter itself. For example, a statement referring to "about X" includes a statement of "X".

[0067] The terms "approximately" and "about" refer to a percentage of a given value or range that is within 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less than 20% of that value or range.

[0068] Where used herein, references to a value or parameter "not" generally mean and describe something "other than" a value or parameter. For example, "The method is not used to treat inflammatory diseases of type X" means that the method is used to treat inflammatory diseases of types other than X.

[0069] As used herein, the term "approximately X to Y" has the same meaning as "approximately X to approximately Y".

[0070] As used herein and in the appended claims, unless otherwise explicitly indicated by the context, the singular forms "a," "an," and "the" include plural referents.

[0071] II. Isolated anti-TSLP antibody constructs TSLP is a cytokine produced by intestinal and lung epithelial cells, skin keratinocytes, and dendritic cells, but it can also be produced by (e.g.) airway smooth muscle cells, mast cells, monocytes, macrophages, granulocytes, synovial fibroblasts, etc. TSLP is involved in helper 2 T cell (Th2) inflammation and has been identified as a potential therapeutic target in the treatment of asthma. This antigen can be used as a biomarker for the development of certain inflammatory diseases (e.g., allergic or autoimmune diseases), as a prognostic indicator, and as an immunotherapy target for antigen-expressing inflammatory diseases.

[0072] In one embodiment, the application provides an isolated antibody construct (anti-TSLP antibody construct) comprising (or consisting of, or essentially comprising) an anti-TSLP antibody moiety, such as one of the anti-TSLP antibody moieties described herein. In some embodiments, the isolated anti-TSLP antibody construct comprises one or more anti-TSLP antibody moieties. Thus, in some embodiments, the application also provides anti-TSLP antibody moieties, such as one of the anti-TSLP antibody moieties described herein. In some embodiments, the isolated anti-TSLP antibody construct is monospecific. In some embodiments, the isolated anti-TSLP antibody construct is multispecific (e.g., bispecific). In some embodiments, a multispecific anti-TSLP antibody construct is provided, comprising one or more anti-TSLP antibody moieties described herein (e.g., full-length anti-TSLP antibody, anti-TSLP scFv, or anti-TSLP Fab) and one or more additional antibody moieties that specifically recognize one or more additional target antigens or epitopes. Furthermore, isolated anti-TSLP antibody constructs and isolated anti-TSLP antibody moieties are also provided that compete with any of the isolated anti-TSLP antibody constructs or isolated anti-TSLP antibody moieties described herein in terms of binding to TSLP. In some embodiments, isolated anti-TSLP antibody constructs are provided that include means for specifically recognizing TSLP.

[0073] In some embodiments, the isolated anti-TSLP antibody construct comprises (or consists of, or is essentially composed of) a full-length anti-TSLP antibody, Fab, Fab', Fab'-SH, F(ab')2, Fv, scFv, or a combination thereof. In some embodiments, the isolated anti-TSLP antibody construct comprises (or consists of) an anti-TSLP Fab fragment. In some embodiments, the isolated anti-TSLP antibody construct comprises (or consists of) an anti-TSLP scFv. In some embodiments, the isolated anti-TSLP antibody construct comprises (or consists of) a full-length anti-TSLP antibody.

[0074] In some embodiments, the isolated anti-TSLP antibody construct is monovalent. In some embodiments, the isolated anti-TSLP antibody construct is polyvalent. In some embodiments, the isolated anti-TSLP antibody construct is polyvalent and monospecific (i.e., contains two or more antibody moieties that bind to the same TSLP epitope). In some embodiments, the isolated anti-TSLP antibody construct is polyvalent and multispecific.

[0075] In some embodiments, the isolated anti-TSLP antibody construct is selected from the group consisting of monospecific antibody constructs, multispecific antibody constructs (e.g., bispecific antibody constructs), and antibody-detection tag conjugates.

[0076] The present invention further provides a fusion protein comprising any of the anti-TSLP antibody moieties described herein (e.g., full-length antibody, scFv, or Fab) and a conjugate (e.g., a detection tag conjugate) comprising any of the isolated anti-TSLP antibody constructs or isolated anti-TSLP antibody moieties described herein. Also provided are isolated anti-TSLP antibody constructs comprising any of the anti-TSLP antibody moieties described herein (e.g., full-length antibody, scFv, or Fab) and a fusion partner (e.g., another protein, an expression tag, a purification tag, or a moiety for extending the half-life).

[0077] In some embodiments, the N-terminus of one or more polypeptides of the isolated anti-TSLP antibody construct is additionally fused with a signal peptide for better expression.

[0078] In some embodiments, the N-terminus and / or C-terminus of one or more polypeptides of an isolated anti-TSLP antibody construct may include a histidine tag (HIS tag) for protein purification. For example, the C-terminus of one or more polypeptides of a full-length anti-TSLP antibody may further include a histidine tag.

[0079] Anti-TSLP antibody part Any of the anti-TSLP antibody portions described herein may be used in the isolated anti-TSLP antibody construct described herein.

[0080] TSLP antigens can be derived from various species, including humans, non-human primates, mice, rats, rabbits, or other mammals. In some embodiments, the TSLP molecule is human TSLP. In some embodiments, the TSLP molecule is mouse TSLP. In some embodiments, the TSLP molecule is cynomolgus monkey TSLP. In some embodiments, the TSLP molecule is wild-type. In some embodiments, the TSLP molecule is a mutant (e.g., containing one or more insertions, deletions, and / or amino acid substitutions compared to the wild-type reference TSLP molecule).

[0081] In some embodiments, the anti-TSLP antibody portion is K D The antibody binds to the TSLP antigen at a concentration of ≤1 μM, for example, ≤100 nM, preferably ≤10 nM, and more preferably ≤1 nM. In some embodiments, the anti-TSLP antibody moiety binds to human TSLP antigen and / or monkey (e.g., cynomolgus monkey) TSLP antigen at a concentration of approximately 1 × 10⁻¹⁶. -14 M ~ approx. 1×10 -7 M, for example, about 1 × 10 -14 M ~ approx. 1×10 -10 M, about 1 x 10 -14 M ~ approx. 1×10 -11 M, about 1 x 10 -14 M ~ approx. 1×10 -12 M, about 1 x 10 -13 M ~ approx. 1×10 -10 M, about 1 x 10 -13 M ~ approx. 1×10 -11 M, about 1 x 10 -13 M ~ approx. 1×10 -12 M, about 1 x 10 -12 M ~ approx. 1×10 -10 M, about 1 x 10 -11 M ~ approx. 1×10 -10 M, about 1 x 10 -11 M ~ approx. 1×10 -9 M, about 1 x 10 -11 M ~ approx. 1×10 -8 M, about 1 x 10 -10M ~ approx. 1×10 -9 M, about 1 x 10 -10 M ~ approx. 1×10 -8 M, about 1 x 10 -10 M ~ approx. 1×10 -7 M, about 1 x 10 -9 M ~ approx. 1×10 -7 M, about 1 x 10 -9 M ~ approx. 1×10 -8 M, or approximately 1 x 10 -8 M ~ approx. 1×10 -7 M's K D It binds to human TSLP. In some embodiments, the anti-TSLP antibody moiety binds to human TSLP at a rate of approximately 1 × 10⁻⁶. -14 M ~ approx. 1×10 -10 M's K D It binds to the cynomolgus monkey TSLP at approximately 1 × 10⁶ times. In some embodiments, the anti-TSLP antibody moiety binds to the cynomolgus monkey TSLP at approximately 1 × 10⁶ times. -12 M ~ approx. 1×10 -9 M's K D They are joined together.

[0082] In some embodiments, the anti-TSLP antibody moiety has similar affinity (e.g., K) to the TSLP antigen (e.g., human TSLP and / or cyno-TSLP) compared to a reference anti-TSLP antibody such as the US-FDA approved anti-TSLP reference antibody #1 (Ref. Ab. #1) described herein. D The binding occurs within a difference of 2 times. In some embodiments, the anti-TSLP antibody moiety binds to the TSLP antigen (e.g., human TSLP and / or cyno TSLP) at a strength of at least about 2 times, 5 times, 10 times, 20 times, 100 times, 1000 times, or more compared to a reference anti-TSLP antibody such as the US-FDA approved anti-TSLP reference antibody #1 (Ref. Ab. #1) described herein. For example, in some embodiments, the binding of the reference anti-TSLP antibody (e.g., US-FDA approved anti-TSLP Ref. Ab. #1) to the TSLP antigen (e.g., human TSLP and / or cyno TSLP) is K D This refers to the K of the anti-TSLP antibody portion described herein. D It is at least approximately 2 times, 5 times, 10 times, 20 times, 100 times, 1000 times, or more.

[0083] In some embodiments, the anti-TSLP antibody moiety inhibits TSLP-induced CCL17 secretion by pro-inflammatory cells expressing the TSLP receptor (e.g., PBMCs or CD1c+ dendritic cells) in the presence of TSLP (e.g., human TSLP) by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%. In some embodiments, the inhibition of TSLP-induced CCL17 secretion by pro-inflammatory cells expressing the TSLP receptor (e.g., PBMCs or CD1c+ dendritic cells) by the anti-TSLP antibody moiety described herein is at least about 1.2 times (e.g., at least about 1.5 times, 2 times, 5 times, 10 times, 50 times, or more) than that by a reference anti-TSLP antibody (e.g., US-FDA approved anti-TSLP Ref. Ab. #1).

[0084] In some embodiments, the anti-TSLP antibody moiety specifically recognizes TSLP from both human and non-human primates (e.g., cynomolgus monkeys). Isolated anti-TSLP antibody constructs containing such an anti-TSLP antibody moiety with cross-reactivity to monkey TSLP can facilitate toxicity studies in non-human primates, which can provide a more relevant safety assessment for human clinical trial candidates without the need to conduct toxicity studies in chimpanzees or use surrogate molecules. In some embodiments, the anti-TSLP antibody moiety binds only to human TSLP. In some embodiments, the anti-TSLP antibody moiety binds more strongly to human TSLP than to non-human (e.g., cynomolgus monkey) TSLP (e.g., at least about 1.5 times, 2 times, 5 times, 10 times, 20 times, 50 times, 100 times, 1000 times, or more).

[0085] The anti-TSLP antibody moiety may completely or partially modulate, block, inhibit, reduce, antagonize, neutralize, or interfere with the functional activity of TSLP (e.g., binding to the TSLPR complex and / or induction of signaling). If the functional activity of TSLP is reduced by at least about 95% (e.g., at least about 96%, 97%, 98%, 99%, or 100%) in the presence of the anti-TSLP antibody moiety compared to when the anti-TSLP antibody moiety is not bound, then the anti-TSLP antibody moiety is considered to be able to completely modulate, block, inhibit, reduce, antagonize, neutralize, or interfere with the functional activity of TSLP. If the functional activity of TSLP is reduced by at least approximately 50% (e.g., at least approximately 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%) in the presence of the anti-TSLP antibody moiety compared to the case without the anti-TSLP antibody moiety, then the anti-TSLP antibody moiety is considered to be able to significantly modulate, block, inhibit, reduce, antagonize, neutralize, or interfere with the functional activity of TSLP. If the functional activity of TSLP is reduced by at least less than approximately 50% (e.g., at least approximately 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 45%) in the presence of the anti-TSLP antibody moiety compared to the case without the anti-TSLP antibody moiety, then the anti-TSLP antibody moiety is considered to be able to partially modulate, block, inhibit, reduce, antagonize, neutralize, or interfere with the functional activity of TSLP.

[0086] In some embodiments, inhibition or reduction of TSLP activity by the anti-TSLP antibody moiety includes inhibiting or reducing one or more of the following: i) binding of TSLP to its receptor, ii) proliferation, activation, and / or differentiation of TSLPR-expressing cells in the presence of TSLP, iii) production of Th2 cytokines (i.e., cytokines expressed by Th2 cells; e.g., IL-4, IL-5, IL-13, and IL-10) in a polarization assay in the presence of TSLP, iv) activation and / or maturation of dendritic cells in the presence of TSLP, and v) mast cell cytokine release in the presence of TSLP (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more).

[0087] In some embodiments, the anti-TSLP antibody moiety contains one or more mutations in its sequence. In some embodiments, amino acid mutations in the variant sequence (e.g., substitutions, e.g., conservative substitutions) do not substantially reduce the ability of the anti-TSLP antibody moiety to bind to TSLP (e.g., by any one of approximately 50%, 40%, 30%, 20%, 10%, 5%, 1%, or less). Modifications are also intended to substantially improve the binding affinity of the anti-TSLP antibody moiety to TSLP, or other properties such as specificity, immunogenicity, and / or cross-reactivity with TSLP epitope variants (e.g., by any one of at least approximately 1.2 times, 1.5 times, 2 times, 5 times, 10 times, 20 times, 50 times, or more).

[0088] In some embodiments, an anti-TSLP antibody moiety is provided comprising VH and VL, wherein VH comprises (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 2, or a variant thereof comprising up to about three (e.g., one, two, or three) amino acid mutations (e.g., insertions, deletions, and / or substitutions, e.g., conservative substitutions), (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 3, or a variant thereof comprising up to about three (e.g., one, two, or three) amino acid mutations (e.g., insertions, deletions, and / or substitutions, e.g., conservative substitutions), and (iii) the amino acid sequence of SEQ ID NO: 4, or a variant thereof comprising up to about three (e.g., one, two, or three) amino acid mutations (e.g., insertions, deletions, and / or substitutions, e.g., conservative substitutions). The VL comprises (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 6, or a variant thereof containing up to approximately three (e.g., one, two, or three) amino acid mutations (e.g., insertions, deletions, and / or substitutions, e.g., conservative substitutions); (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 7, or a variant thereof containing up to approximately three (e.g., one, two, or three) amino acid mutations (e.g., insertions, deletions, and / or substitutions, e.g., conservative substitutions); and (iii) CDR-L3 containing the amino acid sequence of SEQ ID NO: 8, or a variant thereof containing up to approximately three (e.g., one, two, or three) amino acid mutations (e.g., insertions, deletions, and / or substitutions, e.g., conservative substitutions). In some embodiments, the anti-TSLP antibody moiety comprises VH, which includes (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 2, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 3, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO: 4, and VL, which includes (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 6, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 7, and (iii) CDR-L3 containing the amino acid sequence of SEQ ID NO: 8. In some embodiments, the anti-TSLP antibody moiety comprises CDR-H1, CDR-H2, and CDR-H3 of VH, which includes the amino acid sequence of SEQ ID NO: 1, and CDR-L1, CDR-L2, and CDR-L3 of VL, which includes the amino acid sequence of SEQ ID NO: 5.In some embodiments, the anti-TSLP antibody moiety includes a VH containing an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 1, and a VL containing an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 5, or (b) a VH containing an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 9, and a VL containing an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 10. In some embodiments, the anti-TSLP antibody portion comprises (a) VH containing the amino acid sequence of SEQ ID NO: 1 and VL containing the amino acid sequence of SEQ ID NO: 5, or (b) VH containing the amino acid sequence of SEQ ID NO: 9 and VL containing the amino acid sequence of SEQ ID NO: 10.

[0089] In some embodiments, an anti-TSLP antibody moiety is provided comprising VH and VL, wherein VH comprises (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 12 or a variant thereof comprising up to about three (e.g., one, two, or three) amino acid mutations (e.g., insertions, deletions, and / or substitutions, e.g., conservative substitutions), (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13 or a variant thereof comprising up to about three (e.g., one, two, or three) amino acid mutations (e.g., insertions, deletions, and / or substitutions, e.g., conservative substitutions), and (iii) the amino acid sequence of SEQ ID NO: 4 or a variant thereof comprising up to about three (e.g., one, two, or three) amino acid mutations (e.g., insertions, deletions, and / or substitutions, e.g., conservative substitutions). The VL includes CDR-H3 containing a variant of (i) the amino acid sequence of SEQ ID NO: 6, or a variant thereof containing up to approximately three (e.g., one, two, or three) amino acid mutations (e.g., insertions, deletions, and / or substitutions, e.g., conservative substitutions), (ii) the amino acid sequence of SEQ ID NO: 7, or a variant thereof containing up to approximately three (e.g., one, two, or three) amino acid mutations (e.g., insertions, deletions, and / or substitutions, e.g., conservative substitutions), and (iii) the amino acid sequence of SEQ ID NO: 8, or a variant thereof containing up to approximately three (e.g., one, two, or three) amino acid mutations (e.g., insertions, deletions, and / or substitutions, e.g., conservative substitutions). In some embodiments, the anti-TSLP antibody moiety comprises VH, which includes (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 12, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 13, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO: 4, and VL, which includes (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 6, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 7, and (iii) CDR-L3 containing the amino acid sequence of SEQ ID NO: 8. In some embodiments, the anti-TSLP antibody moiety comprises CDR-H1, CDR-H2, and CDR-H3 of VH, which includes the amino acid sequence of SEQ ID NO: 11, and CDR-L1, CDR-L2, and CDR-L3 of VL, which includes the amino acid sequence of SEQ ID NO: 15.In some embodiments, the anti-TSLP antibody moiety includes a VH containing an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 11, and a VL containing an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 15. In some embodiments, the anti-TSLP antibody moiety includes a VH containing the amino acid sequence of SEQ ID NO: 11, and a VL containing the amino acid sequence of SEQ ID NO: 15.

[0090] In some embodiments, an anti-TSLP antibody moiety is provided comprising VH and VL, wherein VH comprises (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 28 or a variant thereof comprising up to about three (e.g., one, two, or three) amino acid mutations (e.g., insertions, deletions, and / or substitutions, e.g., conservative substitutions), (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29 or a variant thereof comprising up to about three (e.g., one, two, or three) amino acid mutations (e.g., insertions, deletions, and / or substitutions, e.g., conservative substitutions), and (iii) the amino acid sequence of SEQ ID NO: 30 or a variant thereof comprising up to about three (e.g., one, two, or three) amino acid mutations (e.g., insertions, deletions, and / or substitutions, e.g., conservative substitutions). The VL comprises (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 32 or a variant thereof containing up to approximately three (e.g., one, two, or three) amino acid mutations (e.g., insertions, deletions, and / or substitutions, e.g., conservative substitutions), (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 33 or a variant thereof containing up to approximately three (e.g., one, two, or three) amino acid mutations (e.g., insertions, deletions, and / or substitutions, e.g., conservative substitutions), and (iii) CDR-L3 containing the amino acid sequence of SEQ ID NO: 34 or a variant thereof containing up to approximately three (e.g., one, two, or three) amino acid mutations (e.g., insertions, deletions, and / or substitutions, e.g., conservative substitutions). In some embodiments, the anti-TSLP antibody moiety comprises VH, which includes (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 28, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 29, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO: 30, and VL, which includes (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 32, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 33, and (iii) CDR-L3 containing the amino acid sequence of SEQ ID NO: 34. In some embodiments, the anti-TSLP antibody moiety comprises CDR-H1, CDR-H2, and CDR-H3 of VH, which includes the amino acid sequence of SEQ ID NO: 27, and CDR-L1, CDR-L2, and CDR-L3 of VL, which includes the amino acid sequence of SEQ ID NO: 31.In some embodiments, the anti-TSLP antibody moiety includes a VH containing an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 27, and a VL containing an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 31. In some embodiments, the anti-TSLP antibody moiety includes a VH containing the amino acid sequence of SEQ ID NO: 27, and a VL containing the amino acid sequence of SEQ ID NO: 31.

[0091] In some embodiments, an anti-TSLP antibody moiety is provided comprising VH and VL, wherein VH comprises (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 36 or a variant thereof comprising up to about three (e.g., one, two, or three) amino acid mutations (e.g., insertions, deletions, and / or substitutions, e.g., conservative substitutions), (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 37 or a variant thereof comprising up to about three (e.g., one, two, or three) amino acid mutations (e.g., insertions, deletions, and / or substitutions, e.g., conservative substitutions), and (iii) the amino acid sequence of SEQ ID NO: 38 or a variant thereof comprising up to about three (e.g., one, two, or three) amino acid mutations (e.g., insertions, deletions, and / or substitutions, e.g., conservative substitutions). The VL comprises (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, or a variant thereof containing up to approximately three (e.g., one, two, or three) amino acid mutations (e.g., insertions, deletions, and / or substitutions, e.g., conservative substitutions); (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, or a variant thereof containing up to approximately three (e.g., one, two, or three) amino acid mutations (e.g., insertions, deletions, and / or substitutions, e.g., conservative substitutions); and (iii) CDR-L3 containing the amino acid sequence of SEQ ID NO: 42, or a variant thereof containing up to approximately three (e.g., one, two, or three) amino acid mutations (e.g., insertions, deletions, and / or substitutions, e.g., conservative substitutions). In some embodiments, the anti-TSLP antibody moiety comprises VH, which includes (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 36, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 37, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO: 38, and VL, which includes (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and (iii) CDR-L3 containing the amino acid sequence of SEQ ID NO: 42. In some embodiments, the anti-TSLP antibody moiety comprises CDR-H1, CDR-H2, and CDR-H3 of VH, which includes the amino acid sequence of SEQ ID NO: 35, and CDR-L1, CDR-L2, and CDR-L3 of VL, which includes the amino acid sequence of SEQ ID NO: 39.In some embodiments, the anti-TSLP antibody moiety includes a VH containing an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 35, and a VL containing an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 39.

[0092] In some embodiments, an anti-TSLP antibody moiety comprising VH and VL is provided, wherein VH comprises (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 44 or a variant thereof comprising up to about three (e.g., one, two, or three) amino acid mutations (e.g., insertions, deletions, and / or substitutions, e.g., conservative substitutions), (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 45 or a variant thereof comprising up to about three (e.g., one, two, or three) amino acid mutations (e.g., insertions, deletions, and / or substitutions, e.g., conservative substitutions), and (iii) the amino acid sequence of SEQ ID NO: 46 or a variant thereof comprising up to about three (e.g., one, two, or three) amino acid mutations (e.g., insertions, deletions, and / or substitutions, e.g., conservative substitutions). The VL comprises (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 48 or a variant thereof containing up to approximately three (e.g., one, two, or three) amino acid mutations (e.g., insertions, deletions, and / or substitutions, e.g., conservative substitutions), (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 49 or a variant thereof containing up to approximately three (e.g., one, two, or three) amino acid mutations (e.g., insertions, deletions, and / or substitutions, e.g., conservative substitutions), and (iii) CDR-L3 containing the amino acid sequence of SEQ ID NO: 50 or a variant thereof containing up to approximately three (e.g., one, two, or three) amino acid mutations (e.g., insertions, deletions, and / or substitutions, e.g., conservative substitutions). In some embodiments, the anti-TSLP antibody moiety comprises VH, which includes (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 44, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 45, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO: 46, and VL, which includes (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 48, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 49, and (iii) CDR-L3 containing the amino acid sequence of SEQ ID NO: 50. In some embodiments, the anti-TSLP antibody moiety comprises CDR-H1, CDR-H2, and CDR-H3 of VH, which includes the amino acid sequence of SEQ ID NO: 43, and CDR-L1, CDR-L2, and CDR-L3 of VL, which includes the amino acid sequence of SEQ ID NO: 47.In some embodiments, the anti-TSLP antibody moiety includes a VH containing an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 43, and a VL containing an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 47. In some embodiments, the anti-TSLP antibody moiety includes a VH containing the amino acid sequence of SEQ ID NO: 43, and a VL containing the amino acid sequence of SEQ ID NO: 47.

[0093] In some embodiments, the anti-TSLP antibody moiety comprises VH and VL, where VH comprises (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 20 or a variant thereof comprising up to about three (e.g., one, two, or three) amino acid mutations (e.g., insertions, deletions, and / or substitutions, e.g., conservative substitutions), (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 21 or a variant thereof comprising up to about three (e.g., one, two, or three) amino acid mutations (e.g., insertions, deletions, and / or substitutions, e.g., conservative substitutions), and (iii) a variant thereof comprising the amino acid sequence of SEQ ID NO: 22 or a variant thereof comprising up to about three (e.g., one, two, or three) amino acid mutations (e.g., insertions, deletions, and / or substitutions, e.g., conservative substitutions). The VL comprises (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 24, or a variant thereof containing up to approximately three (e.g., one, two, or three) amino acid mutations (e.g., insertions, deletions, and / or substitutions, e.g., conservative substitutions); (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 25, or a variant thereof containing up to approximately three (e.g., one, two, or three) amino acid mutations (e.g., insertions, deletions, and / or substitutions, e.g., conservative substitutions); and (iii) CDR-L3 containing the amino acid sequence of SEQ ID NO: 26, or a variant thereof containing up to approximately three (e.g., one, two, or three) amino acid mutations (e.g., insertions, deletions, and / or substitutions, e.g., conservative substitutions). In some embodiments, the anti-TSLP antibody moiety comprises VH, which includes (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 20, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 21, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO: 22, and VL, which includes (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 24, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 25, and (iii) CDR-L3 containing the amino acid sequence of SEQ ID NO: 26. In some embodiments, the anti-TSLP antibody moiety comprises CDR-H1, CDR-H2, and CDR-H3 of VH, which includes the amino acid sequence of SEQ ID NO: 19, and CDR-L1, CDR-L2, and CDR-L3 of VL, which includes the amino acid sequence of SEQ ID NO: 23. In some embodiments, the anti-TSLP antibody moiety is(a) VH containing an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 19 (e.g., at least approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more), and VL containing an amino acid sequence having at least approximately 80% sequence identity with SEQ ID NO: 23 (e.g., at least approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more), (b) SEQ ID NO: 63 having at least approximately 80% sequence identity with SEQ ID NO: 63 (e.g., at least approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%) (c) VH containing an amino acid sequence having sequence identity of % or more, and VL containing an amino acid sequence having sequence identity of at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) with SEQ ID NO: 53, (c) VH containing an amino acid sequence having sequence identity of at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) with SEQ ID NO: 54 and at least about 8 (d) VL containing an amino acid sequence having 0% sequence identity (for example, at least approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more), VH containing an amino acid sequence having at least approximately 80% sequence identity (for example, at least approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) with SEQ ID NO: 56, and SEQ ID NO: 52 having at least approximately 80% sequence identity (for example, at least approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more). (e) VL containing an amino acid sequence having sequence identity with (a) 55, (f) VH containing an amino acid sequence having sequence identity with at least approximately 80% (e.g., at least approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) with 55, and VL containing an amino acid sequence having sequence identity with at least approximately 80% (e.g., at least approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) with 51, (f) VL containing an amino acid sequence having sequence identity with at least approximately 80% (e.g., at least approximately 85%,VH containing an amino acid sequence having sequence identity of 90%, 95%, 96%, 97%, 98%, 99%, or more; VL containing an amino acid sequence having sequence identity of at least approximately 80% (e.g., at least approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) with SEQ ID NO: 51; (g) an amino acid sequence having sequence identity of at least approximately 80% (e.g., at least approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) with SEQ ID NO: 63. VH containing an acid sequence, and VL containing an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 51, (h) VH containing an amino acid sequence having at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 51, (i) VL containing an amino acid sequence having sequence identity of 97%, 98%, 99%, or more; (i) VH containing an amino acid sequence having sequence identity of at least approximately 80% (e.g., at least approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) with SEQ ID NO: 55; and VL containing an amino acid sequence having sequence identity of at least approximately 80% (e.g., at least approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) with SEQ ID NO: 52; (j) VH containing an amino acid sequence having at least approximately 80% (e.g., at least approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 58, and VL containing an amino acid sequence having at least approximately 80% (e.g., at least approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 52, (k) containing an amino acid sequence having at least approximately 80% (e.g., at least approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%,VH containing an amino acid sequence having sequence identity of (1) or more, and VL containing an amino acid sequence having sequence identity of at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) with SEQ ID NO: 52, (1) VH containing an amino acid sequence having sequence identity of at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) with SEQ ID NO: 64 and VL contains an amino acid sequence having at least approximately 80% sequence identity (for example, at least approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more), VH contains an amino acid sequence having at least approximately 80% sequence identity (for example, at least approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) with (m) SEQ ID NO: 55, and SEQ ID NO: 53 contains an amino acid sequence having at least approximately 80% sequence identity (for example, at least approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, (n) VL containing an amino acid sequence having sequence identity of (n) or more, (n) VH containing an amino acid sequence having sequence identity of at least approximately 80% (e.g., at least approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) with SEQ ID NO: 57, and VL containing an amino acid sequence having sequence identity of at least approximately 80% (e.g., at least approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) with SEQ ID NO: 58 VH containing an amino acid sequence having at least approximately 80% sequence identity (for example, at least approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more), and VL containing an amino acid sequence having at least approximately 80% sequence identity (for example, at least approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) with SEQ ID NO: 53, (p) SEQ ID NO: 62 containing at least approximately 80% sequence identity (for example, at least approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%,VH containing an amino acid sequence having sequence identity of (q) or more, and VL containing an amino acid sequence having sequence identity of at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) with SEQ ID NO: 53, (q) VH containing an amino acid sequence having sequence identity of at least about 80% (e.g., at least about 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) with SEQ ID NO: 64 and VL containing an amino acid sequence having at least approximately 80% sequence identity (for example, at least approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more), VH containing an amino acid sequence having at least approximately 80% sequence identity (for example, at least approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) with SEQ ID NO: 55, and SEQ ID NO: 54 containing at least approximately 80% sequence identity (for example, at least approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, VL containing an amino acid sequence having sequence identity of (s) or more, VH containing an amino acid sequence having sequence identity of at least approximately 80% (e.g., at least approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) with SEQ ID NO: 61, and VL containing an amino acid sequence having sequence identity of at least approximately 80% (e.g., at least approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) with SEQ ID NO: 54, (t) SEQ ID NO: 62 VH containing an amino acid sequence having at least approximately 80% sequence identity (for example, at least approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more), and VL containing an amino acid sequence having at least approximately 80% sequence identity (for example, at least approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) with SEQ ID NO: 54, (u)SEQ ID NO: 64 containing at least approximately 80% sequence identity (for example, at least approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%,VH containing an amino acid sequence having sequence identity of (v) or more, and VL containing an amino acid sequence having sequence identity of at least approximately 80% (e.g., at least approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) with SEQ ID NO: 54, (v) containing an amino acid sequence having sequence identity of at least approximately 80% (e.g., at least approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) with SEQ ID NO: 63, VH containing an amino acid sequence having sequence identity with (k), and VL containing an amino acid sequence having at least approximately 80% (e.g., at least approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 52, (w) VH containing an amino acid sequence having at least approximately 80% (e.g., at least approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 188, and at least approximately 80% (e.g., at least approximately 85%, 90%, 9) (x) A VL containing an amino acid sequence having sequence identity of 5%, 96%, 97%, 98%, 99%, or more; or a VH containing an amino acid sequence having sequence identity of at least approximately 80% (for example, at least approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) with sequence number 188; and a VL containing an amino acid sequence having sequence identity of at least approximately 80% (for example, at least approximately 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) with sequence number 190. In some embodiments, the anti-TSLP antibody portion is: (a) VH containing the amino acid sequence of SEQ ID NO: 19 and VL containing the amino acid sequence of SEQ ID NO: 23; (b) VH containing the amino acid sequence of SEQ ID NO: 63 and VL containing the amino acid sequence of SEQ ID NO: 53; (c) VH containing the amino acid sequence of SEQ ID NO: 63 and VL containing the amino acid sequence of SEQ ID NO: 54; (d) VH containing the amino acid sequence of SEQ ID NO: 56 and VL containing the amino acid sequence of SEQ ID NO: 52; (e) VH containing the amino acid sequence of SEQ ID NO: 55 and VL containing the amino acid sequence of SEQ ID NO: 51. (f) VH containing the amino acid sequence of SEQ ID NO: 56, and VL containing the amino acid sequence of SEQ ID NO: 51, (g) VH containing the amino acid sequence of SEQ ID NO: 63, and VL containing the amino acid sequence of SEQ ID NO: 51, (h) VH containing the amino acid sequence of SEQ ID NO: 64, and VL containing the amino acid sequence of SEQ ID NO: 51, (i) VH containing the amino acid sequence of SEQ ID NO: 55, and VL containing the amino acid sequence of SEQ ID NO: 52, (j) VH containing the amino acid sequence of SEQ ID NO: 58, and VL containing the amino acid sequence of SEQ ID NO: 52, (k) VH containing the amino acid sequence of SEQ ID NO: 60,(l) VL containing the amino acid sequence of SEQ ID NO: 52, (m) VH containing the amino acid sequence of SEQ ID NO: 64, and VL containing the amino acid sequence of SEQ ID NO: 52, (n) VH containing the amino acid sequence of SEQ ID NO: 57, and VL containing the amino acid sequence of SEQ ID NO: 53, (o) VH containing the amino acid sequence of SEQ ID NO: 58, and VL containing the amino acid sequence of SEQ ID NO: 53, (p) VH containing the amino acid sequence of SEQ ID NO: 62, and VL containing the amino acid sequence of SEQ ID NO: 53, (q) VH containing the amino acid sequence of SEQ ID NO: 64, and VL containing the amino acid sequence of SEQ ID NO: 53, (r) amino acid sequence of SEQ ID NO: 55 (s) VH containing the sequence and VL containing the amino acid sequence of SEQ ID NO: 54, (t) VH containing the amino acid sequence of SEQ ID NO: 62 and VL containing the amino acid sequence of SEQ ID NO: 54, (u) VH containing the amino acid sequence of SEQ ID NO: 64 and VL containing the amino acid sequence of SEQ ID NO: 54, (v) VH containing the amino acid sequence of SEQ ID NO: 63 and VL containing the amino acid sequence of SEQ ID NO: 52, (w) VH containing the amino acid sequence of SEQ ID NO: 188 and VL containing the amino acid sequence of SEQ ID NO: 189, or (x) VH containing the amino acid sequence of SEQ ID NO: 188 and VL containing the amino acid sequence of SEQ ID NO: 190.

[0094] In some embodiments, (a) a reference VH containing the amino acid sequence of SEQ ID NO: 1 and a reference VL containing the amino acid sequence of SEQ ID NO: 5, (b) a reference VH containing the amino acid sequence of SEQ ID NO: 9 and a reference VL containing the amino acid sequence of SEQ ID NO: 10, (c) a reference VH containing the amino acid sequence of SEQ ID NO: 11 and a reference VL containing the amino acid sequence of SEQ ID NO: 15, (d) a reference VH containing the amino acid sequence of SEQ ID NO: 19 and a reference VL containing the amino acid sequence of SEQ ID NO: 23, (e) a reference VH containing the amino acid sequence of SEQ ID NO: 27 and a reference VL containing the amino acid sequence of SEQ ID NO: 31, (f) (g) Reference VH containing the amino acid sequence of sequence number 35, and reference VL containing the amino acid sequence of sequence number 39, (h) Reference VH containing the amino acid sequence of sequence number 43, and reference VL containing the amino acid sequence of sequence number 47, (i) Reference VH containing the amino acid sequence of sequence number 63, and reference VL containing the amino acid sequence of sequence number 53, (j) Reference VH containing the amino acid sequence of sequence number 56, and reference VL containing the amino acid sequence of sequence number 52, (k) Reference VH containing the amino acid sequence of sequence number 55 Reference VH, and reference VL containing the amino acid sequence of SEQ ID NO: 51, (l) Reference VH containing the amino acid sequence of SEQ ID NO: 56, and reference VL containing the amino acid sequence of SEQ ID NO: 51, (m) Reference VH containing the amino acid sequence of SEQ ID NO: 63, and reference VL containing the amino acid sequence of SEQ ID NO: 51, (n) Reference VH containing the amino acid sequence of SEQ ID NO: 64, and reference VL containing the amino acid sequence of SEQ ID NO: 51, (o) Reference VH containing the amino acid sequence of SEQ ID NO: 55, and reference VL containing the amino acid sequence of SEQ ID NO: 52, (p) Reference VH containing the amino acid sequence of SEQ ID NO: 58, and reference VL containing the amino acid sequence of SEQ ID NO: 52 Reference VL containing amino acid sequences, (q) Reference VH containing the amino acid sequence of SEQ ID NO: 60 and SEQ ID NO: 52, (r) Reference VH containing the amino acid sequence of SEQ ID NO: 64 and SEQ ID NO: 52, (s) Reference VH containing the amino acid sequence of SEQ ID NO: 55 and SEQ ID NO: 53, (t) Reference VH containing the amino acid sequence of SEQ ID NO: 57 and SEQ ID NO: 53, (u) Reference VH containing the amino acid sequence of SEQ ID NO: 58 and SEQ ID NO: 53,(v) Reference VH containing the amino acid sequence of SEQ ID NO: 62, and reference VL containing the amino acid sequence of SEQ ID NO: 53, (w) Reference VH containing the amino acid sequence of SEQ ID NO: 64, and reference VL containing the amino acid sequence of SEQ ID NO: 53, (x) Reference VH containing the amino acid sequence of SEQ ID NO: 55, and reference VL containing the amino acid sequence of SEQ ID NO: 54, (y) Reference VH containing the amino acid sequence of SEQ ID NO: 61, and reference VL containing the amino acid sequence of SEQ ID NO: 54, (z) Reference VH containing the amino acid sequence of SEQ ID NO: 62, and reference VL containing the amino acid sequence of SEQ ID NO: 54, (aa) Reference VH containing the amino acid sequence of SEQ ID NO: 64, and reference VL containing the amino acid sequence of SEQ ID NO: 54, (bb) sequence An anti-TSLP antibody moiety is provided that competes for binding to TSLP with a reference anti-TSLP antibody comprising a reference VH containing the amino acid sequence of sequence number 63 and a reference VL containing the amino acid sequence of sequence number 52, (cc) a reference VH containing the amino acid sequence of sequence number 188 and a reference VL containing the amino acid sequence of sequence number 189, or (dd) a reference VH containing the amino acid sequence of sequence number 188 and a reference VL containing the amino acid sequence of sequence number 190 (for example, competing for binding by at least about 50%, for example, at least about 55%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more).

[0095] In some embodiments, the anti-TSLP antibody moiety is selected from the group consisting of full-length antibody, Fab, Fab', F(ab')2, diabody, and scFv.

[0096] In some embodiments, the anti-TSLP antibody portion is a full-length antibody (anti-TSLP full-length antibody). In some embodiments, the anti-TSLP full-length antibody contains a CL comprising human kappa CL or human lambda CL, for example, one of the amino acid sequences from SEQ ID NOs. 73-75. In some embodiments, the anti-TSLP full-length antibody contains an Fc domain derived from human IgG, for example, human IgG1, IgG2, IgG3, or IgG4, for example, human IgG1. Any Fc domain described in the following “Fc Domains” subsection may be used herein. In some embodiments, the Fc domain comprises, according to EU numbering, i) L234A+L235A ("LALA") mutation, ii) M252Y+S254T+T256E ("YTE") mutation, and / or iii) M428L+N434S ("LS") mutation. In some embodiments, the Fc domain further includes a knob-in-hole mutation such as a T366W "knob" mutation in one Fc subunit and a T366S+L368A+Y407V "hole" mutation in another Fc subunit. In some embodiments, i) the first and second subunits of the Fc domain each contain one of the amino acid sequences of SEQ ID NOs. 76 to 79; ii) the first subunit of the Fc domain contains the amino acid sequence of SEQ ID NOs. 80 and the second subunit of the Fc domain contains the amino acid sequence of SEQ ID NOs. 81; or iii) the first subunit of the Fc domain contains the amino acid sequence of SEQ ID NOs. 81 and the second subunit of the Fc domain contains the amino acid sequence of SEQ ID NOs. 80. In some embodiments, the first and second subunits of the Fc domain each contain the amino acid sequence of SEQ ID NOs. 77 or 79.In some embodiments, the full-length anti-TSLP antibody comprises: i) two heavy chains, each containing a variant thereof having at least about 80% (e.g., at least about 85%, 90%, 95%, 99%, or more) sequence identity to the amino acid sequence of SEQ ID NO: 104; and two light chains, each containing a variant thereof having at least about 80% (e.g., at least about 85%, 90%, 95%, 99%, or more) sequence identity to the amino acid sequence of SEQ ID NO: 103; and ii) two heavy chains, each containing a variant thereof having at least about 80% (e.g., at least about 85%, 90%, 95%, 99%, or more) sequence identity to the amino acid sequence of SEQ ID NO: 105; and each containing at least about 80% ( For example, two light chains containing a variant of the sequence having at least approximately 85%, 90%, 95%, 99%, or more; or iii) a first heavy chain containing the amino acid sequence of SEQ ID NO: 136 (or a variant of the sequence having at least approximately 80% (e.g., at least approximately 85%, 90%, 95%, 99%, or more) of the amino acid sequence of SEQ ID NO: 136), a second heavy chain containing the amino acid sequence of SEQ ID NO: 137 (or a variant of the sequence having at least approximately 80% (e.g., at least approximately 85%, 90%, 95%, 99%, or more) of the amino acid sequence of SEQ ID NO: 137), and two light chains each containing the amino acid sequence of SEQ ID NO: 103 (or a variant of the sequence having at least approximately 80% (e.g., at least approximately 85%, 90%, 95%, 99%, or more) of the amino acid sequence of SEQ ID NO: 103).In some embodiments, the full-length anti-TSLP antibody comprises i) two heavy chains, each containing the amino acid sequence of SEQ ID NO: 104, and two light chains, each containing the amino acid sequence of SEQ ID NO: 103; ii) two heavy chains, each containing the amino acid sequence of SEQ ID NO: 105, and two light chains, each containing the amino acid sequence of SEQ ID NO: 103; or iii) a first heavy chain containing the amino acid sequence of SEQ ID NO: 136, a second heavy chain containing the amino acid sequence of SEQ ID NO: 137, and two light chains, each containing the amino acid sequence of SEQ ID NO: 103.

[0097] In some embodiments, the anti-TLSP antibody moiety is scFv (anti-TSLP scFv). In some embodiments, anti-TSLP scFv comprises VH and VL fused to each other via an optional linker. In some embodiments, anti-TSLP scFv comprises VH-optional linker-VL or VL-optional linker-VH from the N-terminus to the C-terminus. Any linker in the following “Linker” subsection can be used to link the VH and VL of anti-TSLP scFv. In some embodiments, the optional linker comprises GG and an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 16-18, 97-99, and 163-172, e.g., GG and any of SEQ ID NOs: 98-99. In some embodiments, anti-TSLP scFv comprises any one of SEQ ID NOs: 106, 107, 191, and 192.

[0098] In some embodiments, the anti-TSLP antibody moiety is a Fab fragment (anti-TSLP Fab fragment) comprising a first polypeptide containing VH and CH1, and a second polypeptide containing VL and CL. In some embodiments, the stereochemistry of the variable and constant regions within the anti-TSLP Fab fragment may differ from that found in the native anti-TSLP Fab fragment. For example, in some embodiments, the anti-TSLP Fab fragment comprises a first polypeptide containing VH and CL, and a second polypeptide containing VL and CH1 (see, for example, Shaefer et al. (2011), PNAS, 108:111870-92 (the entire content of which is incorporated herein by reference)).

[0099] In some embodiments, CH1 and VH heterodimerize with VL and CL in the anti-TSLP Fab and are covalently linked by disulfide bonds between the heavy chain constant region and the light chain constant region. In some embodiments, the anti-TSLP Fab fragment has the basic structure NH2-VL-CL-SS-CH1-VH-NH2. In some embodiments, CH1 and CL of the anti-TSLP Fab fragment are connected by one or more disulfide bonds. In some embodiments, the number of disulfide bonds between CH1 and CL of the anti-TSLP Fab fragment is at least one, e.g., 2, 3, 4, 5, or more. In some embodiments, cysteine ​​residues are manipulated in the anti-TSLP Fab fragment (e.g., the CH1 region and the CL region) to introduce disulfide bonds.

[0100] In some embodiments, the anti-TSLP Fab fragment does not contain a disulfide bond at the C-terminus. For example, the heavy and light chains of the anti-TSLP Fab fragment may be manipulated in such a way that they interact stably without requiring a disulfide bond. In some embodiments, the heavy or light chain may be manipulated to remove a cysteine ​​residue, and the heavy and light chains still interact stably and function as Fab. In some embodiments, mutations are made to promote stable interaction between the heavy and light chains. For example, a "knob-in-hole" manipulation strategy can be used to promote dimerization between the heavy and light chains of Fab (see, e.g., 1996 Protein Engineering, 9:617-621). Variant Fab fragments designed for specific purposes, e.g., amino acid changes in the constant domains of CH1 and / or CL, and removal of disulfide bonds or addition of tags for purification, are also intended for use herein. These mutations are described in more detail in the following subsection, “Constant Domain and Fc Domain.”

[0101] In some embodiments, the CH1 and CL of the anti-TSLP Fab fragment are linked by at least one or two disulfide bonds. In some embodiments, the anti-TSLP Fab fragment includes human immunoglobulin CH1, for example, the amino acid sequence of SEQ ID NO: 158. In some embodiments, the anti-TSLP Fab fragment includes a human lambda light chain constant region, for example, the amino acid sequence of SEQ ID NO: 74 or 75. In some embodiments, the anti-TSLP Fab fragment includes a human kappa light chain constant region, for example, the amino acid sequence of SEQ ID NO: 73.

[0102] In some embodiments, the anti-TSLP Fab fragment comprises a first polypeptide having the amino acid sequence of SEQ ID NO: 109 and a second polypeptide having the amino acid sequence of SEQ ID NO: 103.

[0103] Multispecific anti-TSLP antibody constructIn some embodiments, the isolated anti-TSLP antibody construct described herein is a multispecific anti-TSLP antibody construct comprising an anti-TSLP antibody moiety (e.g., any of the anti-TSLP antibody moieties described herein, e.g., full-length anti-TSLP antibody, anti-TSLP Fab, and / or anti-TSLP scFv) and a second antibody moiety that specifically recognizes a second target antigen (e.g., TSLP or IL-13). In some embodiments, the isolated anti-TSLP antibody construct comprises one or more anti-TSLP antibody moieties (e.g., any of the anti-TSLP antibody moieties described herein) and one or more antibody moieties that specifically recognize one or more target antigens (e.g., TSLP or IL-13). In some embodiments, the isolated anti-TSLP antibody construct is polyvalent but monospecific, i.e., all antibody moieties contained therein specifically recognize the same TSLP epitope. In some embodiments, the isolated anti-TSLP antibody construct is polyvalent and multispecific (e.g., bispecific). In some embodiments, the isolated anti-TSLP antibody construct specifically recognizes two or more epitopes of TSLP. In some embodiments, the isolated anti-TSLP antibody construct specifically recognizes TSLP via one or more anti-TSLP antibody moieties (e.g., any of the anti-TSLP antibody moieties described herein) and specifically recognizes one or more target antigens that are not TSLP. In some embodiments, the second target antigen is IL-13. In some embodiments, two or more anti-TSLP antibody moieties (e.g., scFv, Fab) have the same amino acid sequence. In some embodiments, two or more anti-TSLP antibody moieties (e.g., scFv, Fab) have different amino acid sequences. In some embodiments, two or more anti-TSLP antibody moieties bind to the same TSLP epitope. In some embodiments, two or more anti-TSLP antibody moieties bind to different TSLP epitopes. In some embodiments, the second antibody moiety is selected from the group consisting of full-length antibody, Fab, Fab', F(ab')2, sdAb, diabody, and scFv. In some embodiments, the second antibody moiety is scFv (e.g., anti-IL-13 scFv).In some embodiments, the second antibody moiety is a Fab (e.g., anti-IL-13 Fab). In some embodiments, the second antibody moiety is a full-length antibody (e.g., a full-length anti-IL-13 antibody). In some embodiments, one or more anti-TSLP antibody moieties (e.g., any of the anti-TSLP antibody moieties described herein) and one or more antibody moieties that specifically recognize one or more target antigens (e.g., TSLP or IL-13) are fused to each other via a linker. Any linker described in the following subsection "Linker" may be used herein. In some embodiments, the linker comprises GG and an amino acid sequence selected from any one of SEQ ID NOs: 14, 16-18, 97-99, and 163-172, for example, GG and any one of SEQ ID NOs: 98-99. If the isolated anti-TSLP antibody construct contains an Fc domain, any Fc domain described in the following subsection "Fc Domain" may be used herein.

[0104] FC Domain FC domains (e.g., each of two Fc subunits) and their variants that may be contained in any of the isolated anti-TSLP antibody constructs described herein (e.g., a full-length anti-TSLP antibody) are further described herein.

[0105] The Fc (fragment, crystallizable) domain is the tail region of an antibody that interacts with cell surface receptors called Fc receptors and several proteins of the complement system. The Fc-mediated effector function of antibodies mediates antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). This property is key to antibodies activating the immune system. Depending on the class of antibody, the Fc domain consists of two or three constant domains attached to the CH1 domain of each of the two heavy chains. Mutations in the CH3 subunit of the Fc domains on both heavy chains, called "knob-in-hole" mutations, can further improve heterodimerization when assembling two antibody polypeptides. For example, the CH3 domain of the Fc domain on the first heavy chain may contain a "knob" mutation (e.g., T366W) if the following "hole" substitutions are introduced at positions 366, 368, and 407 in the CH3 domain of the Fc domain on the second heavy chain: T366S, L368A, and Y407V. The positions of the knob mutation and the hole mutation can be swapped; for example, the knob mutation may be on the second heavy chain and the hole mutation may be on the first heavy chain. Additional Fc domain mutations, such as LALA mutations (e.g., L234A and L235A substitutions in the CH2 domain of the Fc domain), can be introduced to reduce FcγR activation and Fc-mediated toxicity, which can induce side effects such as cytokine release syndrome in individuals receiving the antibody. Furthermore, Fc domain mutations, such as LS mutations (e.g., M428L and N434S substitutions in the CH3 domain of the Fc domain), can be introduced to extend the antibody half-life without affecting efficacy. Fc variants that enhance FcRn affinity, such as the M252Y / S254T / T256E(YTE) mutation, which can extend the serum half-life when incorporated into the Fc domain, can also be used herein.Therefore, the described mutations can be used alone or in combination to improve antibody function and / or reduce toxicity to patients. In some embodiments, the Fc domain includes an LALA mutation. In some embodiments, the Fc domain includes an LS or YTE mutation. In some embodiments, the Fc domain includes both an LALA mutation and an LS mutation. In some embodiments, the Fc domain includes both an LALA mutation and a YTE mutation. In some embodiments, i) the first and second subunits of the Fc domain each include one of the amino acid sequences of SEQ ID NOs. 76 to 79; ii) the first subunit of the Fc domain includes the amino acid sequence of SEQ ID NOs. 80 and the second subunit of the Fc domain includes the amino acid sequence of SEQ ID NOs. 81; or iii) the first subunit of the Fc domain includes the amino acid sequence of SEQ ID NOs. 81 and the second subunit of the Fc domain includes the amino acid sequence of SEQ ID NOs. 80.

[0106] Linker The isolated anti-TSLP antibody constructs described herein (e.g., multispecific anti-TSLP antibody constructs, anti-TSLP scFv) may include one or more linkers (such as peptide linkers) connecting two or more domains or portions contained therein, for example, between the VH domain and the VL domain (e.g., within the scFv), between the anti-TSLP antibody portion and its fusion partner or a second antibody portion that specifically recognizes a second target antigen (e.g., scFv, Fab, or full-length antibody), or between two or more anti-TSLP antibody portions. In some embodiments, the two or more linkers are the same. In some embodiments, the two or more linkers are different. In some embodiments, the isolated anti-TSLP antibody construct does not contain linkers, i.e., the two or more domains or portions contained therein are directly linked to each other.

[0107] The linker can be a peptide linker of any length. In some embodiments, the peptide linker is about 1 to about 10 amino acids (aa) long, about 2 to about 15 aa long, about 3 to about 12 aa long, about 4 to about 10 aa long, about 5 to about 9 aa long, about 6 to about 8 aa long, about 1 to about 20 aa long, about 21 to about 30 aa long, about 1 to about 30 aa long, about 10 to about 30 aa long, or about 2 to about 5 aa long. In some embodiments, the peptide linker is any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid long. In some embodiments, the peptide linker is any of 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid long. In some embodiments, the peptide linker is about 2 to about 15 amino acids long.

[0108] Peptide linkers may have sequences that are naturally occurring or sequences that are not naturally occurring. For example, sequences derived from the hinge region of heavy chain-only antibodies can be used as linkers. See, for example, International Publication No. 1996 / 34103. In some embodiments, the peptide linker is a human IgG1 or IgG4 hinge. In some embodiments, the linker is a flexible linker. Exemplary flexible linkers include glycine polymers (G)n (e.g., GG), glycine-serine polymers (e.g., (GS)n, (GSGGS)n (SEQ ID NO: 14), (GGGS)n (SEQ ID NO: 16), or (GGGGS)n (SEQ ID NO: 17), where n is at least one integer), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured and can therefore function as neutral tethers between components. Glycine accesses far more phy-cy space than alanine and is far less restrictive than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11:173-142 (1992)). In some embodiments, the linker comprises an amino acid residue selected from the group consisting of glycine, serine, arginine, and alanine. Exemplary flexible linkers include, but are not limited to, Gly-Gly, GGSG (SEQ ID NO: 18), GGSGG (SEQ ID NO: 163), GGSSG (SEQ ID NO: 164), GSGGG (SEQ ID NO: 165), GGGSG (SEQ ID NO: 166), GSSSG (SEQ ID NO: 167), GGSGGS (SEQ ID NO: 168), SGGGGS (SEQ ID NO: 169), GRAGGGGAGGGG (SEQ ID NO: 170), GRAGGG (SEQ ID NO: 171), GGGGSGGGGSGGGGS (SEQ ID NO: 99), GGGG (SEQ ID NO: 98), GGGGS (SEQ ID NO: 172), and others. In some embodiments, the linker between VH and VL of scFv includes the sequence of sequence number 99.Those skilled in the art will recognize that the design of an isolated anti-TSLP antibody construct or anti-TSLP antibody moiety may include a linker that is all or partially flexible, and as a result the linker may include a flexible linker moiety as well as one or more moies that confer a less flexible structure, thereby providing a desired anti-TSLP antibody construct structure.

[0109] In some embodiments, the linker between the anti-TSLP antibody moiety and the second antibody moiety, or the linker within the anti-TSLP antibody moiety, is a stable linker (not cleavable by proteases, particularly MMPs).

[0110] In some embodiments, the linker is a cleavable linker. Substrate sequences that can be cleaved by MMPs have been extensively studied. For example, the sequence PLGLAG (sequence number 97) can be cleaved by most MMPs.

[0111] III.Preparation method Furthermore, a method for producing either an isolated anti-TSLP antibody construct or an anti-TSLP antibody moiety as described herein is also provided.

[0112] The isolated anti-TSLP antibody constructs or anti-TSLP antibody moieties described herein may be prepared by any of the protein expression and purification methods known in the art. The DNA sequences encoding the isolated anti-TSLP antibody constructs or anti-TSLP antibody moieties can be synthesized completely. After obtaining such sequences, they are cloned into a suitable expression vector and then transfected into suitable host cells. The transfected host cells are cultured, the supernatant is collected and purified to obtain the anti-TSLP antibody constructs or anti-TSLP antibody moieties described herein. In some embodiments, the host cells are lysed to obtain the expressed anti-TSLP antibody constructs or anti-TSLP antibody moieties.

[0113] In some embodiments, this application provides isolated nucleic acids encoding one or more polypeptides from any one of the isolated anti-TSLP antibody constructs described herein. The isolated nucleic acids may be DNA or RNA.

[0114] In some embodiments, isolated nucleic acids are inserted into vectors such as expression vectors, viral vectors, or cloning vectors. Therefore, vectors containing any of the isolated nucleic acids described herein are also provided. For nucleic acid expression, the vector may be introduced into host cells to enable nucleic acid expression within the host cells. Expression vectors may contain various elements for controlling expression, including, but are not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection markers, and signal sequences. These elements can be appropriately selected by those skilled in the art. For example, promoter sequences may be selected to promote the transcription of polynucleotides in the vector. Suitable promoter sequences include, but are not limited to, the T7 promoter, T3 promoter, SP6 promoter, beta-actin promoter, EF1a promoter, CMV promoter, and SV40 promoter. Enhancer sequences may be selected to enhance nucleic acid transcription. Selectable markers may be selected to enable selection of host cells into which the vector has been inserted from cells lacking the vector; for example, a selectable marker may be a gene conferring antibiotic resistance. Signal sequences may be selected to enable the transport of expressed polypeptides outside the host cell.

[0115] In some embodiments, isolated host cells are provided, comprising either isolated nucleic acids or vectors encoding any polypeptide portion of any of the isolated anti-TSLP antibody constructs described herein. In some embodiments, isolated host cells expressing any of the isolated anti-TSLP antibody constructs described herein are provided. In some embodiments, two or more polypeptides of any of the isolated anti-TSLP antibody constructs described herein (e.g., full-length antibodies, Fab fragments, or multispecific anti-TSLP antibody constructs) are encoded by a single vector. In some embodiments, two or more polypeptides of any of the isolated anti-TSLP antibody constructs described herein (e.g., full-length antibodies, Fab fragments, or multispecific isolated anti-TSLP antibody constructs) are encoded by two or more vectors. Host cells containing vectors may be useful in the expression or cloning of isolated nucleic acids. Suitable host cells include, but are not limited to, higher eukaryotic cells such as prokaryotic cells, fungal cells, yeast cells, or mammalian cells. The expression of antibodies and antigen-binding fragments in prokaryotic cells, such as Escherichia coli, is well established in the art. For an overview, see, for example, Pluckthun, A. BioTechnology 9:545-551 (1991). Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for the production of antibodies or their antigen-binding fragments; for an overview, see, for example, Ref, ME (1993) Curr. Opinion Biotech. 4:573-576, Trill J Jet al. (1995) Curr. Opinion Biotech 6:553-560. Higher eukaryotic cells, particularly cells derived from multicellular organisms, can be used for the expression of glycosylated polypeptides. Suitable higher eukaryotic cells include, but are not limited to, invertebrate cells and insect cells, as well as vertebrate cells. In some embodiments, the host cell is Escherichia coli. In some embodiments, the host cell is Chinese hamster ovary (CHO) cells or HEK293 cells.

[0116] The vectors can be introduced into host cells using any suitable method known in the art, including, but not limited to, DEAE-dextran-mediated delivery, calcium phosphate precipitation, cationic lipid-mediated delivery, liposome-mediated transfection, electroporation, microparticle guns, receptor-mediated gene delivery, and delivery mediated by polylysine, histones, chitosan, and peptides. Standard methods for transfection and transformation of cells for expression of the vector of interest are well known in the art. In some embodiments, the host cells contain two or more vectors, each encoding one of the polypeptides from the isolated anti-TSLP antibody constructs described herein. The two or more vectors can be introduced into the host cells in the same or different ratios. In some embodiments, the host cells contain a single vector containing isolated nucleic acids encoding two or more polypeptides from the isolated anti-TSLP antibody constructs described herein. The two or more nucleic acids encoding two or more polypeptides of the isolated anti-TSLP antibody constructs can be under the control of the same promoter or different promoters. For example, two or more nucleic acids under the control of the same promoter can be linked via an IRES sequence or a sequence encoding a self-cleaving peptide (e.g., P2A, T2A).

[0117] In some embodiments, the application provides a method for producing one of the isolated anti-TSLP antibody constructs described herein, comprising: i) culturing an isolated host cell containing one of the isolated nucleic acids or vectors described herein, or one of the isolated host cells described herein (for example, a host cell containing one of the isolated nucleic acids or vectors described herein), under conditions suitable for the expression of an anti-TSLP antibody construct; and ii) obtaining the expressed anti-TSLP antibody construct from the host cell (for example, from a cell culture or by lysing the host cell). The isolated host cell is cultured under conditions that enable the expression of the nucleic acid inserted into the vector. Suitable conditions for polynucleotide expression may include, but are not limited to, a suitable culture medium, a suitable density of host cells in the culture medium, the presence of necessary nutrients, the presence of cofactors, a suitable temperature and humidity, and the absence of microbial contaminants. Those skilled in the art can appropriately select suitable conditions depending on the purpose of expression. In some embodiments, the production method further comprises purifying one of the obtained anti-TSLP antibody constructs.

[0118] In some embodiments, polypeptides expressed by host cells can assemble together (e.g., to form polypeptide complexes such as dimers) to produce one of the isolated anti-TSLP antibody constructs described herein. In some embodiments, polypeptide complexes can be formed within host cells. For example, polypeptide complexes can be formed within host cells with the help of relevant enzymes and / or cofactors. In some embodiments, polypeptide complexes can be secreted from cells. In some embodiments, individual polypeptides can be secreted from host cells and then form polypeptide complexes (e.g., one of the isolated anti-TSLP antibody constructs described herein) outside the host cells.

[0119] In some embodiments, two or more polypeptides from any of the isolated anti-TSLP antibody constructs described herein may be expressed separately and allowed to form (e.g., dimerize) an isolated anti-TSLP antibody construct under preferred conditions. For example, the first polypeptide and the second polypeptide may be combined in a preferred buffer to allow the first protein monomer and the second protein monomer to dimerize via a suitable interaction, such as a hydrophobic interaction. In some embodiments, the first polypeptide and the second polypeptide may be combined in a preferred buffer containing an enzyme and / or cofactor that can promote the dimerization of the first polypeptide and the second polypeptide. In some embodiments, the first polypeptide and the second polypeptide may be combined in a preferred vehicle to allow them to react with each other in the presence of a preferred reagent and / or catalyst.

[0120] The expressed polypeptides and / or polypeptide complexes can be collected using any preferred method. Polypeptides and / or polypeptide complexes can be expressed intracellularly in the periplasmic space or secreted into the extracellular culture medium. If polypeptides and / or polypeptide complexes are expressed intracellularly, the host cells containing the polypeptides and / or polypeptide complexes may be lysed, and the polypeptides and / or polypeptide complexes may be isolated from the lysate by removing undesirable debris by centrifugation or ultrafiltration. If polypeptides and / or polypeptide complexes are secreted into the periplasmic space of E. coli cells, the cell paste may be thawed for about 30 minutes in the presence of agents such as sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF), and cell debris can be removed by centrifugation (Carter et al., BioTechnology 10:163-167 (1992)). If polypeptides and / or polypeptide complexes are secreted into the culture medium, the supernatant of the cell culture may be collected and concentrated using a commercially available protein concentration filter, such as an Amincon or Millipore Pellicon ultrafiltration unit. Protease inhibitors and / or antibiotics may be included in the collection and concentration steps to inhibit the proteolysis and / or growth of contaminating microorganisms.

[0121] The expressed polypeptides and / or polypeptide complexes can be further purified by preferred methods, for example, but not limited to affinity chromatography, hydroxyl apatite chromatography, size exclusion chromatography, gel electrophoresis, dialysis, ion exchange fractionation on an ion exchange column, ethanol precipitation, reversed-phase HPLC, chromatography on silica, chromatography on heparin Sepharose, chromatography on anion or cation exchange resin (e.g., polyaspartate column), isoelectric focusing, SDS-PAGE, and ammonium sulfate precipitation (see Bonner, PL, Protein purification, published by Taylor & Francis, 2007; Janson, JC, et al., Protein purification: principles, high resolution methods and applications, published by Wiley-VCH, 1998 for an overview).

[0122] In some embodiments, polypeptides and / or polypeptide complexes can be purified by affinity chromatography. In some embodiments, protein A chromatography or protein A / G (protein A and protein G fusion protein) chromatography can be useful for purifying polypeptides and / or polypeptide complexes containing components derived from antibody CH2 domains and / or CH3 domains (Lindmark et al., J.Immunol.Meth.62:1-13(1983)), Zettlit, KA, Antibody Engineering, Part V, 531-535, 2010). In some embodiments, protein G chromatography can be useful for purifying polypeptides and / or polypeptide complexes containing IgG γ triple chains (Guss et al., EMBO J.5:1567 1575(1986)). In some embodiments, protein L chromatography can be useful for purifying polypeptides and / or polypeptide complexes containing κ light chains (Sudhir, P., Antigen engineering protocols, Chapter 26, published by Humana Press, 1995; Nilson, BHK et al., J. Biol. Chem., 267, 2234-2239 (1992)). The matrix to which affinity ligands are attached is almost always agarose, but other matrices are also available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than those achievable with agarose. If any of the isolated anti-TSLP antibody constructs contain additional CH3 domains, Bakerbond ABX resin (JTBaker, Phillipsburg, NJ) is useful for purification.

[0123] IV. Pharmaceutical compositions, unit doses, products, and kits Further provided by this application are pharmaceutical compositions comprising the isolated anti-TSLP antibody construct of the present disclosure and an optionally pharmaceutically acceptable carrier.

[0124] The isolated anti-TSLP antibody construct or the pharmaceutical composition thereof may be suitable for various administration methods described herein, including, for example, systemic or topical administration. In some embodiments, the pharmaceutical composition is formulated for intravenous administration. In some embodiments, the pharmaceutical composition is formulated for subcutaneous injection.

[0125] As used herein, “carrier” includes pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to cells or mammals to which they are exposed at the doses and concentrations used. Often, physiologically acceptable carriers are pH-buffered aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to the recipient at the doses and concentrations used and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens (such as methyl or propylparaben); catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; and proteins (serum albumin, ze). This includes latin or immunoglobulins; hydrophilic polymers (such as polyvinylpyrrolidone); amino acids (such as glycine, glutamine, asparagine, histidine, arginine, or lysine); monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents (such as EDTA); sugars (such as sucrose, mannitol, trehalose, or sorbitol); salt-forming counterions (such as sodium); metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants (such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG)).

[0126] In some embodiments, the pharmaceutical composition is formulated to have a pH in the range of about 4.5 to about 9.0, including, for example, one of the pH ranges of about 5.0 to about 8.0, about 6.5 to about 7.5, about 6.5 to about 7.0, or about 7.0 to about 7.5. In some embodiments, the pharmaceutical composition can also be made isotonic with blood by adding a suitable isotonic agent such as glycerol.

[0127] Pharmaceutical compositions used for in vivo administration are generally sterile, substantially isotonic, and formulated in full accordance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration. Sterility is readily achieved by filtration through a sterile filtration membrane. In some embodiments, the composition contains no pathogens. For injection, the pharmaceutical composition may be in the form of a liquid solution in a physiologically compatible buffer, such as Hanks' solution or Ringer's solution. In addition, the pharmaceutical composition may be in solid form and may be redissolved or suspended immediately before use. Lyophilized compositions are also included.

[0128] In some embodiments, the pharmaceutical composition is formulated as a pharmaceutical composition adapted for parenteral (e.g., intravenous, intramuscular, or subcutaneous) administration according to routine procedures. Typically, the composition for injection is a solution in sterile aqueous isotonic buffer. If necessary, the composition may also include a solubilizer and a local anesthetic such as lidocaine to relieve pain at the injection site. Generally, the components are supplied individually or as a mixture in a unit dosage form, either as a dry, lyophilized powder or a water-free concentrate, in sealed containers such as ampoules or pouches indicating the amount of the active drug. When the composition is administered by injection, the composition can be dispensed in injection bottles containing pharmaceutical-grade sterile water or saline. When the composition is administered by injection, ampoules of sterile water or saline for injection can be provided so that the components can be mixed before administration.

[0129] In some embodiments, the pharmaceutical composition is suitable for administration to mammals such as humans. In some embodiments, the pharmaceutical composition is suitable for administration to rodents (e.g., mice, rats) or non-human primates (e.g., cynomolgus monkeys). In some embodiments, the pharmaceutical composition is contained in disposable vials, such as single-use sealed vials. In some embodiments, the pharmaceutical composition is contained in multi-dose vials. In some embodiments, the pharmaceutical composition is contained in large quantities in a container. In some embodiments, the pharmaceutical composition is stored by freezing.

[0130] Also provided herein are unit dosage forms of any of the isolated anti-TSLP antibody constructs described herein, or compositions thereof (such as pharmaceutical compositions). The term "unit dosage form" refers to a physically distinct unit suitable as a unit dose for an individual (e.g., a human), each unit containing a predetermined amount of active substance calculated to produce a desired therapeutic effect when combined with a suitable pharmaceutical carrier, diluent, or excipient. These unit dosage forms can be stored in suitable packaging in single or multiple unit doses, and can further be sterilized and sealed.

[0131] This application further provides products comprising the compositions described herein (such as pharmaceutical compositions) in suitable packaging. Suitable packaging for the compositions described herein (such as pharmaceutical compositions) is known in the art and includes, for example, vials (such as sealed vials), containers, ampoules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags). These products may be further sterilized and / or sealed.

[0132] This application also provides a kit comprising the compositions described herein (such as pharmaceutical compositions), and may further include instructions on how to use the compositions, such as the uses described herein. The kit described herein may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and / or applicators, and accompanying documentation having instructions for carrying out any of the methods described herein.

[0133] V. Methods for treating inflammatory diseases Also provided are methods for treating inflammatory diseases in an individual (e.g., a human), comprising administering to the individual an effective amount of any of the isolated anti-TSLP antibody constructs or pharmaceutical compositions described herein (e.g., intravenously or subcutaneously). In some embodiments, the inflammatory disease is asthma (e.g., severe asthma), atopic dermatitis (e.g., moderate to severe atopic dermatitis), or chronic obstructive pulmonary disease (COPD). In some embodiments, the method further comprises administering an effective amount of a corticosteroid simultaneously with or following the isolated anti-TSLP antibody construct or pharmaceutical composition thereof.

[0134] In some embodiments, the inflammatory disease is a TSLP-related disease. A TSLP-related inflammatory disease may be a disease caused by or associated with the expression of TSLP, for example, overexpression compared to a healthy state, or erroneous expression at a different location than a healthy state (e.g., on a different cell or tissue).

[0135] In some embodiments, the inflammatory disease is an autoimmune disease or autoimmune condition, such as atopic dermatitis, Netherton syndrome, lupus, Sjögren's syndrome, multiple sclerosis, myasthenia gravis, psoriasis, psoriatic arthritis, rheumatoid arthritis, sarcomatoid, ulcerative colitis, or inflammatory bowel disease. In some embodiments, the inflammatory disease is a disease involving immune function (e.g., asthma, endometriosis, fibrosis, or Hodgkin's lymphoma). In some embodiments, the inflammatory disease is an allergic reaction such as allergic asthma, allergic rhinitis, allergic sinusitis, allergic conjunctivitis, atopic dermatitis, or eosinophilic esophagitis. In some embodiments, the inflammatory disease is fibrosis, inflammatory bowel disease, or COPD. In some embodiments, the inflammatory disease is asthma or atopic dermatitis, such as asthma or atopic dermatitis of any severity.

[0136] In some embodiments, a method is provided for treating an inflammatory disease (e.g., atopic dermatitis, COPD, or asthma) in an individual (e.g., a human), comprising administering an effective amount of an isolated anti-TSLP antibody construct (or a pharmaceutical composition thereof) to the individual (e.g., intravenously or subcutaneously), wherein the isolated anti-TSLP antibody construct comprises anti-TSLP Fab, and anti-TSLP Fab comprises a first polypeptide comprising the amino acid sequence of SEQ ID NO: 109 and a second polypeptide comprising the amino acid sequence of SEQ ID NO: 103.

[0137] In some embodiments, a method is provided for treating an inflammatory disease (e.g., atopic dermatitis, COPD, or asthma) in an individual (e.g., a human), comprising administering an effective amount of an isolated anti-TSLP antibody construct (or a pharmaceutical composition thereof) to the individual (e.g., intravenously or subcutaneously), wherein the isolated anti-TSLP antibody construct comprises a full-length anti-TSLP antibody, the anti-TSLP full-length antibody comprising i) two heavy chains, each containing the amino acid sequence of SEQ ID NO: 104, and two light chains, each containing the amino acid sequence of SEQ ID NO: 103; ii) two heavy chains, each containing the amino acid sequence of SEQ ID NO: 105, and two light chains, each containing the amino acid sequence of SEQ ID NO: 103; or iii) a first heavy chain containing the amino acid sequence of SEQ ID NO: 136, a second heavy chain containing the amino acid sequence of SEQ ID NO: 137, and two light chains, each containing the amino acid sequence of SEQ ID NO: 103.

[0138] In some embodiments, a method is provided for treating an inflammatory disease (e.g., atopic dermatitis, COPD, or asthma) in an individual (e.g., a human), comprising administering an effective amount of an isolated anti-TSLP antibody construct (or a pharmaceutical composition thereof) to the individual (e.g., intravenously or subcutaneously), wherein the isolated anti-TSLP antibody construct comprises an anti-TSLP scFv having any of the amino acid sequences of SEQ ID NOs: 106, 107, 191, and 192.

[0139] In some embodiments, a method for treating an inflammatory disease involves the following biological activities: (1) inhibiting the maturation and / or activation of immune cells (e.g., B cells, mast cells, T helper 2 (Th2) cells, or APCs (e.g., dendritic cells) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%); and (2) inhibiting the proliferation of immune cells (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). (3) reducing systemic or local inflammation levels (e.g., by at least approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%), (4) reducing one or more symptoms in individuals with inflammatory disease (e.g., by at least approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%), (5) reducing systemic or local cytokine levels (e.g., Th2 cytokine levels, e.g., IL-13, IL-4, IL-5) (e.g., by a small amount) (6) Reduce the presentation, incidence, or burden of inflammatory diseases (for example, by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%), (7) Prolong the interval between inflammatory immune rednesses, for example, the time between the resumption of symptoms of an inflammatory disease and the reappearance of symptoms in the individual, to at least 1, 2, 4, 6, 12, 18, 20 hours, or longer. (8) Prolonging the recurrence of symptoms of an inflammatory disease by at least approximately 1, 2, 3, 4, 5, 6, 7 days or more, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, or 24 months or more, (8) Prolonging the recurrence of symptoms of an inflammatory disease by at least approximately 1, 2, 4, 6, 12, 18, 20 hours or more, 1, 2, 3, 4, 5, 6, 7 days or more, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months,(9) It may be possible to prevent (8, 9, 10, 11, 12 months, or longer) and to prevent, inhibit, or reduce the likelihood of relapse of inflammatory disease (for example, by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%).

[0140] The methods provided herein may be performed in a primary treatment setting, i.e., the method being performed is a primary / curative therapy. In some embodiments, the method may be performed before or in conjunction with a primary / curative therapy. In some embodiments, the method is used to treat an individual (such as a human) that has been treated previously. Any of the treatment methods provided herein may be used to treat an individual (such as a human) that has not been treated previously. In some embodiments, the method is used as a first-line therapy. In some embodiments, the method is used as a second-line therapy.

[0141] The methods described herein are suitable for treating a variety of inflammatory diseases. The methods are applicable to inflammatory diseases at all stages, including immediately after the onset of symptoms, or to inflammatory diseases in remission. The methods described herein may be used as a first therapy, a second therapy, a third therapy, or in combination with other types of therapies known in the art, such as anti-inflammatory agents (e.g., corticosteroids), gene therapy, immunotherapy, bone marrow transplantation, stem cell transplantation, targeted therapy, nutritional therapy, etc. In some embodiments, the inflammatory disease was unresponsive to the previous therapy.

[0142] Exemplary routes of administration for any of the isolated anti-TSLP antibody constructs (or their pharmaceutical compositions) described herein include, but are not limited to, intravenous, intracavitary, intraarterial, intramuscular, subcutaneous, parenteral, transdermal, or intraperitoneal routes, or for delivery to lymph nodes, body cavities, organs, or tissues affected by inflammatory disease. In some embodiments, the isolated anti-TSLP antibody construct or its pharmaceutical composition is administered intravenously, such as by infusion.

[0143] In some embodiments, the isolated anti-TSLP antibody construct (or its pharmaceutical composition) described herein is administered by intravenous infusion at any preferred rate.

[0144] The administration regimens of the isolated anti-TSLP antibody constructs or their pharmaceutical compositions described herein, administered to an individual (e.g., a human), may vary depending on the specific composition, method of administration, and the specific type of inflammatory disease being treated. In some embodiments, the effective dose of the isolated anti-TSLP antibody construct is below the level that induces a toxicological effect (i.e., an effect exceeding clinically acceptable toxicity levels), or at a level where potential side effects can be controlled or tolerated when the composition is administered to an individual.

[0145] The effective amount of the isolated anti-TSLP antibody construct (or its pharmaceutical composition) described herein may be administered in single or multiple doses. Examples of dosing frequencies for methods involving the administration of the isolated anti-TSLP antibody construct (or its pharmaceutical composition) in multiple doses include, but are not limited to, hourly, daily, daily without interruption, weekly, weekly without interruption, weekly for two weeks out of three, weekly for three weeks out of four, once every three weeks, once every two weeks, monthly, once every six months, and annually. In some embodiments, the isolated anti-TSLP antibody construct (or its pharmaceutical composition) is administered approximately once every two weeks, once every three weeks, once every four weeks, once every six weeks, or once every eight weeks. In some embodiments, the isolated anti-TSLP antibody construct (or its pharmaceutical composition) is administered at least approximately once, twice, three times, four times, five times, six times, or seven times per week (i.e., daily). In some embodiments, the interval between doses is less than one of the following: approximately 3 years, 2 years, 12 months, 11 months, 10 months, 9 months, 8 months, 7 months, 6 months, 5 months, 4 months, 3 months, 2 months, 1 month, 4 weeks, 3 weeks, 2 weeks, 1 week, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day. In some embodiments, the interval between doses is greater than one of the following: approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, or 3 years. In some embodiments, there is no interruption in the medication schedule.

[0146] The administration of the isolated anti-TSLP antibody construct (or its pharmaceutical composition) described herein can be extended over long periods, for example, from one day to about one week, from about one week to about one month, from about one month to about one year, or from about one year to about several years. In some embodiments, the isolated anti-TSLP antibody construct (or its pharmaceutical composition) is administered over a period of at least about one day, two days, three days, four days, five days, six days, seven days, eight days, one week, two weeks, three weeks, four weeks, five weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, one year, two years, three years, four years, or longer. [Examples]

[0147] The following examples are intended purely to illustrate the present invention and should not be considered to limit it in any way. The following examples and detailed description are provided as examples and not as limitations. For embodiments in which details of the experimental method are not described, such methods are carried out according to conventional conditions, such as those described in Sambrook et al. Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as suggested by the manufacturer.

[0148] Example 1: Production of exemplary anti-TSLP antibody.

[0149] Anti-TSLP antibodies were generated using human TSLP and cynomolgus monkey (hereinafter referred to as "cyno") TSLP as immunogens. After determining that each animal had a suitable antibody titer, single-cell suspensions of lymphocytes were obtained from the spleen and lymph nodes of immunized mice. Lymphocytes were fused with mouse myeloma cells using a standard method to generate hybridomas. The hybridoma supernatant was screened by ELISA for binding to human TSLP and cyno TSLP. Hybridomas that showed binding to human and cyno TSLP were further screened by reporter cell assays for anti-TSLP blocking antibodies.

[0150] TSLP Reporter Cell Assay: The TSLP reporter cell assay determines the degree of TSLP blockade by an anti-TSLP antibody in STAT5-luciferase HEK-293 reporter cells co-expressing TSLPR / IL-7Rα, where increased STAT5 signaling induces increased luciferase production. TSLP binding to the TSLP receptor complex is known to activate the STAT5 signaling pathway. Therefore, the degree of TSLP blockade can be determined by monitoring the activation of the STAT5 signaling pathway by measuring luciferase activity. STAT5-luciferase HEK-293 reporter cells co-expressing TSLPR / IL-7Rα were co-incubated with 10 ng / mL human TSLP and the anti-TSLP antibody described herein in a series of dilutions at 37°C for 5 hours. After 5 hours of co-incubation, bioluminescence signals were measured. Figure 1 shows the luciferase inhibition percentage (IC) for reporter cells analyzed using GraphPad Prism. 90 This demonstrates that all exemplary anti-TSLP antibody clones were effective in blocking the binding of free TSLP to the TSLP receptor complex.

[0151] Example 2: Measurement of binding affinity for exemplary anti-TSLP antibodies.

[0152] Recombinant chimeric anti-TSLP monoclonal antibodies (mAbs) containing a human constant domain were constructed based on exemplary mouse anti-TSLP mAbs. Both the exemplary mouse anti-TSLP mAb and the chimeric anti-TSLP mAb were purified.

[0153] Surface plasmon resonance (SPR) measurements of the binding affinity of exemplary hybridoma antibodies targeting TSLP were performed using Biacore.

[0154] In short, purified antibodies were captured using anti-Fc antibody chips (i.e., anti-human Fc for recombinant antibodies with human Fc, and anti-mouse Fc for hybridoma or recombinant mouse antibodies). Antigens were passed through the chips at different concentrations (a 1:3 dilution series starting at 48 nM and going down to 0.2 nM). The captured antigens were dissociated from the chips for 900-1200 seconds. The sensorgrams were fitted to a 1:1 binding model. The results are summarized in Tables 2-4 below.

[0155] As can be seen from Tables 2-4, all of the exemplary mouse anti-TSLP mAbs tested showed very strong binding to both human and cyno-TSLP (higher binding to human TSLP). Furthermore, chimerization did not significantly affect the binding affinity of the anti-TSLP antibodies (see, for example, comparison between 51A4 and Ch51A4).

[0156] [Table 3]

[0157] [Table 4]

[0158] [Table 5]

[0159] Determination of 51B2-binding epitope and potency compared to reference anti-TSLP antibody: Cross-competition between Ref.Ab.#1 and 51B2 was evaluated by pre-incubating Ref.Ab.#1 and biotinylated TSLP for 2 hours before adding 51B2 and US-FDA approved anti-TSLP reference antibody #1 ("Ref.Ab.#1") to ELISA plates pre-coated with Ref.Ab.#1. An inactive negative control antibody (Ab) was used as a non-competitive Ab. After short incubation and washing of the ELISA plates, biotin-TSLP captured on the plates was detected by HRP-streptavidin. The results in Figure 2 show that the inactive negative control Ab did not bind to TSLP, and Ref.Ab.#1 completely competed with itself for TSLP binding. Figure 2 demonstrates that 51B2 does not completely compete with Ref.Ab.#1 and does not prevent Ref.Ab.#1 from binding to TSLP. These results indicate that 51B2 has a unique binding epitope that differs from Ref.Ab.#1.

[0160] The efficacy of 51B2 in inhibiting TSLP signaling in primary human cells was tested in human PBMCs and isolated CD1c+ dendritic cells. Briefly, human PBMCs and isolated CD1c+ dendritic cells were seeded in 96-well plates and incubated with either 50 ng / ml or 5 ng / ml of TSLP in the presence of multiple concentrations of 51B2 or Ref. Ab #1, respectively. Cell culture supernatants were collected after either 48 hours (PBMCs) or 24 hours (dendritic cells) and analyzed for TSLP-induced CCL17 secretion using a commercially available ELISA kit according to the manufacturer's protocol. Since increased TSLP signaling induces increased levels of secreted CCL17, reduction or inhibition of CCL17 secretion by pro-inflammatory cells expressing the TSLP receptor upon treatment with TSLP cytokines serves as read-through information about the inhibition of TSLP binding to the TSLPR complex. Data were analyzed and presented using GraphPad Prism. As shown in Figure 3A (human PBMCs) and Figure 3B (CD1c+ dendritic cells), 51B2 showed much stronger inhibition of CCL17 in both human PBMCs and isolated CD1c+ dendritic cells across all tested antibody concentrations compared to Ref. Ab. #1. Therefore, these results demonstrate that 51B2 is far more potent and effective than the US-FDA approved Ref. Ab. #1 in blocking TSLP signaling in pro-inflammatory immune cells.

[0161] Humanization of 51B2 and humanized 51B2 variants: The parental clone 51B2 was humanized by transplanting CDRs derived from the heavy chain (HC) and light chain (LC) onto human HC and LC framework sequences. In short, the HC and LC CDRs of chimeric 51B2 were transplanted onto the closest HC and LC human germlines. The sequences were visually examined to identify framework residues important for CDR binding and structural stability. These framework residues were mutated individually or in combination. Refer to SEQ ID NOs. 51-54 for VL sequences and SEQ ID NOs. 55-64 for VH sequences. Humanized variants that retained binding and stability were selected as the final lead clones.

[0162] Humanized 51B2 antibodies were tested for binding affinity using Biacore and ranked by activity (similar to the TSLP reporter assay described in Example 1) and expression quality / purification behavior. Based on Biacore binding results (see Tables 5-6) and results from activity assays (see Figures 4 and 5A), the humanized 51B2 antibody, L3H9 (i.e., having pairings of VL-3 (SEQ ID NO: 53) and VH-9 (SEQ ID NO: 63)), was selected as the lead humanized 51B2 clone.

[0163] Humanized 51B2 variants binding to huTSLP in Biacore: Purified humanized or chimeric antibodies were captured on an anti-human Fc antibody chip, and different concentrations of antigen (human TSLP) (a 1:3 dilution series starting at 48 nM and going down to 0.2 nM) were passed over the chip together with the captured antibody, as described above. The captured antigen was then dissociated from the chip for 1200 seconds. The sensorgrams were fitted to a 1:1 binding model and summarized in Table 5 below.

[0164] As shown in Table 5, with the exception of the L1H2 and L1H9 variants, almost all humanized 51B2 variants maintained similar or stronger binding affinity to human TSLP.

[0165] [Table 6]

[0166] Characterization of the anti-TSLP antibody hz51B2 L3H9: The binding affinity of hz51B2-L3H9 to human and cyno-TSLP was measured using a Biacore T200 with different concentrations of anti-human Fc capture chips and purified TSLP as analytes. Binding sensorgrams were fitted to a 1:1 binding kinetics model. The binding constants for on-rate, off-rate, and equilibrium binding KD are summarized in Table 6 below.

[0167] [Table 7]

[0168] The results summarized in Table 6 demonstrate strong binding of the humanized (hz)51B2 L3H9 antibody to both human TSLP and cynomolgus monkey TSLP, which is useful for extrapolating results from toxicity and efficacy studies conducted in cynomolgus monkeys to human clinical studies in order to evaluate anti-TSLP antibodies or constructs containing them (e.g., any of the multispecific anti-TSLP antibody constructs described herein).

[0169] The binding of hz51B2 L3H9 to human TSLP was characterized by measuring the concentration of free TSLP in the equilibrium binding reaction mixture using a Biacore T200 instrument. Briefly, 1 nM human TSLP was incubated overnight at room temperature with 3 × serial dilutions of hz51B2 L3H9 solution in Fab Arm ranging from 96 nM to 44 pM in PBS solution containing 0.1 mg / ml BSA and 0.005% polysorbate 20. Unbound TSLP in each binding reaction was measured by injecting the binding solution onto the amine-immobilized hz51B2 L3H9 surface. The percentage of free TSLP concentration in each reaction mixture was fitted to a 1:1 equilibrium Ab-Ag binding model using GraphPad, and K D This was derived from curve fitting (Figure 5B).

[0170] In summary, lead 51B2 antibodies (for example, hz51B2 Ab such as hz51B2 L3H9) are K D It is a robust binder to human TSLP at <10 pM. While not theoretically bound, such properties allow for complete neutralization of TSLP-inducible CCL17 in PBMC and dendritic cell assays compared to the incomplete inhibition of TSLP-inducible CCL17 by US-FDA approved Ref. Ab. #1 (see Figures 3A-3B).

[0171] All references cited herein are incorporated herein by reference as if each of those references were incorporated individually by reference. Although the description has referred to specific embodiments, it will be apparent to those skilled in the art that the present invention can be carried out in variations of these specific details. Therefore, the present invention should not be construed as being limited to the embodiments described herein.

[0172] [Table 8-1]

[0173] [Table 8-2]

[0174] [Table 8-3]

[0175] [Table 8-4]

[0176] [Table 8-5]

[0177] [Table 8-6]

[0178] Table 8-7

Claims

1. An isolated antibody construct ("anti-TSLP antibody construct") comprising an antibody moiety that specifically recognizes thymic interstitial lymphocyte necrosis factor (TSLP) ("anti-TSLP antibody moiety"), wherein the anti-TSLP antibody moiety comprises a heavy chain variable region (VH) and a light chain variable region (VL), (a) The VH comprises (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 2 or a variant thereof containing up to three amino acid mutations, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 3 or a variant thereof containing up to three amino acid mutations, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 4 or a variant thereof containing up to three amino acid mutations, and the VL comprises (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 6 or a variant thereof containing up to three amino acid mutations, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 7 or a variant thereof containing up to three amino acid mutations, and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8 or a variant thereof containing up to three amino acid mutations. (b) The VH comprises (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 12 or a variant thereof containing up to three amino acid mutations, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 13 or a variant thereof containing up to three amino acid mutations, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 4 or a variant thereof containing up to three amino acid mutations, and the VL comprises (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 6 or a variant thereof containing up to three amino acid mutations, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 7 or a variant thereof containing up to three amino acid mutations, and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8 or a variant thereof containing up to three amino acid mutations. (c) The VH comprises (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 20 or a variant thereof containing up to three amino acid mutations, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 21 or a variant thereof containing up to three amino acid mutations, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 22 or a variant thereof containing up to three amino acid mutations, and the VL comprises (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 24 or a variant thereof containing up to three amino acid mutations, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 25 or a variant thereof containing up to three amino acid mutations, and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 26 or a variant thereof containing up to three amino acid mutations. (d) The VH comprises (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 28 or a variant thereof containing up to three amino acid mutations, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29 or a variant thereof containing up to three amino acid mutations, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30 or a variant thereof containing up to three amino acid mutations, and the VL comprises (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 32 or a variant thereof containing up to three amino acid mutations, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 33 or a variant thereof containing up to three amino acid mutations, and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 34 or a variant thereof containing up to three amino acid mutations. (e) The VH includes (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 36 or a variant thereof containing up to three amino acid mutations, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 37 or a variant thereof containing up to three amino acid mutations, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO: 38 or a variant thereof containing up to three amino acid mutations, and the VL includes (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 40 or a variant thereof containing up to three amino acid mutations, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 41 or a variant thereof containing up to three amino acid mutations, and (iii) CDR-L3 containing the amino acid sequence of SEQ ID NO: 42 or a variant thereof containing up to three amino acid mutations, or (f) An isolated antibody construct in which VH comprises (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 44 or a variant thereof containing up to three amino acid mutations, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 45 or a variant thereof containing up to three amino acid mutations, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 46 or a variant thereof containing up to three amino acid mutations, and VL comprises (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 48 or a variant thereof containing up to three amino acid mutations, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 49 or a variant thereof containing up to three amino acid mutations, and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 50 or a variant thereof containing up to three amino acid mutations.

2. (a) The VH comprises (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 2, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 3, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO: 4, and the VL comprises (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 6, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 7, and (iii) CDR-L3 containing the amino acid sequence of SEQ ID NO:

8. (b) The VH comprises (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 12, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 13, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO: 4, and the VL comprises (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 6, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 7, and (iii) CDR-L3 containing the amino acid sequence of SEQ ID NO:

8. (c) The VH comprises (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 20, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 21, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO: 22, and the VL comprises (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 24, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 25, and (iii) CDR-L3 containing the amino acid sequence of SEQ ID NO:

26. (d) The VH comprises (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 28, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 29, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO: 30, and the VL comprises (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 32, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 33, and (iii) CDR-L3 containing the amino acid sequence of SEQ ID NO:

34. (e) The VH comprises (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 36, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 37, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO: 38, and the VL comprises (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 40, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 41, and (iii) CDR-L3 containing the amino acid sequence of SEQ ID NO: 42, or (f) The isolated anti-TSLP antibody construct according to claim 1, wherein VH comprises (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 44, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 45, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO: 46, and VL comprises (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 48, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 49, and (iii) CDR-L3 containing the amino acid sequence of SEQ ID NO:

50.

3. (a) The VH comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 1, and the VL comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:

5. (b) The VH comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 9, and the VL comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:

10. (c) The VH comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 11, and the VL comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:

15. (d) The VH comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 19, and the VL comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:

23. (e) The VH comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 27, and the VL comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:

31. (f) The VH comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 35, and the VL comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:

39. (g) The VH comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 43, and the VL comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:

47. (h) The VH comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 63, and the VL comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:

53. (i) The VH comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 63, and the VL comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:

54. (j) The VH comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 56, and the VL comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:

52. (k) The VH comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 55, and the VL comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:

51. (l) The VH comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 56, and the VL comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:

51. (m) The VH comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 63, and the VL comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:

51. (n) The VH comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 64, and the VL comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:

51. (o) The VH comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 55, and the VL comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:

52. (p) The VH comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 58, and the VL comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:

52. (q) The VH comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 60, and the VL comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:

52. (r) The VH comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 64, and the VL comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:

52. (s) The VH comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 55, and the VL comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:

53. (t) The VH comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 57, and the VL comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:

53. (u) The VH comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 58, and the VL comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:

53. (v) The VH comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 62, and the VL comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:

53. (w) The VH comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 64, and the VL comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:

53. (x) The VH comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 55, and the VL comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:

54. (y) The VH comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 61, and the VL comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:

54. (z) The VH comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 62, and the VL comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:

54. (aa) The VH comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 64, and the VL comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:

54. (bb) The VH comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 63, and the VL comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:

52. (cc) The VH contains an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 188, and the VL contains an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 189, or (dd) The isolated anti-TSLP antibody construct according to claim 1 or 2, wherein VH comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO: 188, and VL comprises an amino acid sequence having at least about 80% sequence identity with SEQ ID NO:

190.

4. (a) The VH comprises the amino acid sequence of SEQ ID NO: 1, and the VL comprises the amino acid sequence of SEQ ID NO: 5 (b) The VH comprises the amino acid sequence of SEQ ID NO: 9, and the VL comprises the amino acid sequence of SEQ ID NO:

10. (c) The VH comprises the amino acid sequence of SEQ ID NO: 11, and the VL comprises the amino acid sequence of SEQ ID NO:

15. (d) The VH comprises the amino acid sequence of SEQ ID NO: 19, and the VL comprises the amino acid sequence of SEQ ID NO:

23. (e) The VH comprises the amino acid sequence of SEQ ID NO: 27, and the VL comprises the amino acid sequence of SEQ ID NO:

31. (f) The VH comprises the amino acid sequence of SEQ ID NO: 35, and the VL comprises the amino acid sequence of SEQ ID NO:

39. (g) The VH comprises the amino acid sequence of SEQ ID NO: 43, and the VL comprises the amino acid sequence of SEQ ID NO:

47. (h) The VH comprises the amino acid sequence of SEQ ID NO: 63, and the VL comprises the amino acid sequence of SEQ ID NO:

53. (i) The VH comprises the amino acid sequence of SEQ ID NO: 63, and the VL comprises the amino acid sequence of SEQ ID NO: 54 (j) The VH comprises the amino acid sequence of SEQ ID NO: 56, and the VL comprises the amino acid sequence of SEQ ID NO:

52. (k) The VH comprises the amino acid sequence of SEQ ID NO: 55, and the VL comprises the amino acid sequence of SEQ ID NO:

51. (l) The VH comprises the amino acid sequence of SEQ ID NO: 56, and the VL comprises the amino acid sequence of SEQ ID NO:

51. (m) The VH comprises the amino acid sequence of SEQ ID NO: 63, and the VL comprises the amino acid sequence of SEQ ID NO:

51. (n) The VH comprises the amino acid sequence of SEQ ID NO: 64, and the VL comprises the amino acid sequence of SEQ ID NO:

51. (o) The VH comprises the amino acid sequence of SEQ ID NO: 55, and the VL comprises the amino acid sequence of SEQ ID NO:

52. (p) The VH comprises the amino acid sequence of SEQ ID NO: 58, and the VL comprises the amino acid sequence of SEQ ID NO:

52. (q) The VH comprises the amino acid sequence of SEQ ID NO: 60, and the VL comprises the amino acid sequence of SEQ ID NO:

52. (r) The VH comprises the amino acid sequence of SEQ ID NO: 64, and the VL comprises the amino acid sequence of SEQ ID NO:

52. (s) The VH comprises the amino acid sequence of SEQ ID NO: 55, and the VL comprises the amino acid sequence of SEQ ID NO:

53. (t) The VH comprises the amino acid sequence of SEQ ID NO: 57, and the VL comprises the amino acid sequence of SEQ ID NO:

53. (u) The VH comprises the amino acid sequence of SEQ ID NO: 58, and the VL comprises the amino acid sequence of SEQ ID NO:

53. (v) The VH comprises the amino acid sequence of SEQ ID NO: 62, and the VL comprises the amino acid sequence of SEQ ID NO:

53. (w) The VH comprises the amino acid sequence of SEQ ID NO: 64, and the VL comprises the amino acid sequence of SEQ ID NO:

53. (x) The VH comprises the amino acid sequence of SEQ ID NO: 55, and the VL comprises the amino acid sequence of SEQ ID NO:

54. (y) The VH comprises the amino acid sequence of SEQ ID NO: 61, and the VL comprises the amino acid sequence of SEQ ID NO:

54. (z) The VH comprises the amino acid sequence of SEQ ID NO: 62, and the VL comprises the amino acid sequence of SEQ ID NO:

54. (aa) The VH comprises the amino acid sequence of SEQ ID NO: 64, and the VL comprises the amino acid sequence of SEQ ID NO:

54. (bb) The VH comprises the amino acid sequence of SEQ ID NO: 63, and the VL comprises the amino acid sequence of SEQ ID NO:

52. (cc) The VH contains the amino acid sequence of SEQ ID NO: 188, and the VL contains the amino acid sequence of SEQ ID NO: 189, or (dd) The isolated anti-TSLP antibody construct according to any one of claims 1 to 3, wherein VH comprises the amino acid sequence of SEQ ID NO: 188 and VL comprises the amino acid sequence of SEQ ID NO:

190.

5. The anti-TSLP antibody portion consists of a full-length antibody, Fab, Fab', and F(ab'). ２ An isolated anti-TSLP antibody construct according to any one of claims 1 to 4, selected from the group consisting of diabody and scFv.

6. The isolated anti-TSLP antibody construct according to any one of claims 1 to 5, wherein the anti-TSLP antibody portion is a full-length antibody ("anti-TSLP full-length antibody").

7. The isolated anti-TSLP antibody construct according to claim 6, wherein the full-length anti-TSLP antibody comprises a CL containing any one amino acid sequence of SEQ ID NOs. 73 to 75.

8. The isolated anti-TSLP antibody construct according to claim 6 or 7, wherein the full-length anti-TSLP antibody comprises an Fc domain derived from human IgG1, IgG2, or IgG4.

9. The Fc domain is derived from human IgG1, and i) The first subunit and the second subunit of the Fc domain each contain one amino acid sequence from among sequence numbers 76 to 79, ii) The first subunit of the Fc domain includes the amino acid sequence of SEQ ID NO: 80, and the second subunit of the Fc domain includes the amino acid sequence of SEQ ID NO:

81. iii) The first subunit of the Fc domain includes the amino acid sequence of SEQ ID NO: 81, and the second subunit of the Fc domain includes the amino acid sequence of SEQ ID NO:

80. iv) The first subunit of the Fc domain contains the amino acid sequence of SEQ ID NO: 95, and the second subunit of the Fc domain contains the amino acid sequence of SEQ ID NO: 96, or v) The isolated anti-TSLP antibody construct according to claim 8, wherein the first subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO: 96, and the second subunit of the Fc domain comprises the amino acid sequence of SEQ ID NO:

95.

10. The isolated anti-TSLP antibody construct according to claim 8 or 9, wherein the first subunit and the second subunit of the Fc domain each comprise the amino acid sequence of SEQ ID NO: 77 or 79.

11. The aforementioned full-length anti-TSLP antibody i) Two heavy chains, each containing the amino acid sequence of SEQ ID NO: 104, and two light chains, each containing the amino acid sequence of SEQ ID NO: 103, ii) Two heavy chains, each containing the amino acid sequence of SEQ ID NO: 105, and two light chains, each containing the amino acid sequence of SEQ ID NO: 103, or iii) An isolated anti-TSLP antibody construct according to any one of claims 6 to 10, comprising a first heavy chain containing the amino acid sequence of SEQ ID NO: 136, a second heavy chain containing the amino acid sequence of SEQ ID NO: 137, and two light chains, each containing the amino acid sequence of SEQ ID NO:

103.

12. The isolated anti-TSLP antibody construct according to any one of claims 1 to 5, wherein the anti-TSLP antibody portion is scFv ("anti-TSLP scFv").

13. The isolated anti-TSLP antibody construct according to claim 12, wherein the anti-TSLP scFv comprises one amino acid sequence of sequence numbers 106, 107, 191, and 192.

14. The isolated anti-TSLP antibody construct according to any one of claims 1 to 5, wherein the anti-TSLP antibody portion is a Fab ("anti-TSLP Fab").

15. The isolated anti-TSLP antibody construct according to claim 14, wherein the anti-TSLP Fab comprises a first polypeptide having the amino acid sequence of SEQ ID NO: 109 and a second polypeptide having the amino acid sequence of SEQ ID NO:

103.

16. The isolated anti-TSLP antibody construct according to any one of claims 1 to 15, wherein the isolated anti-TSLP antibody construct is monospecific.

17. The isolated anti-TSLP antibody construct according to any one of claims 1 to 15, wherein the isolated anti-TSLP antibody construct is a multispecific anti-TSLP antibody construct comprising a second antibody moiety that specifically recognizes a second target antigen.

18. The second antibody portion is full-length antibody, Fab, Fab', F(ab') ２ An isolated anti-TSLP antibody construct according to claim 17, selected from the group consisting of sdAb, diabody, and scFv.

19. The isolated anti-TSLP antibody construct according to claim 18, wherein the second antibody portion is scFv.

20. The isolated anti-TSLP antibody construct according to claim 18, wherein the second antibody portion is Fab.

21. The isolated anti-TSLP antibody construct according to claim 18, wherein the second antibody portion is a full-length antibody.

22. The isolated anti-TSLP antibody construct according to any one of claims 17 to 21, wherein the anti-TSLP antibody portion and the second antibody portion are fused to each other via a linker.

23. The isolated anti-TSLP antibody construct according to claim 22, wherein the linker comprises GG and an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 16-18, 97-99, and 163-172.

24. A pharmaceutical composition comprising an isolated anti-TSLP antibody construct according to any one of claims 1 to 23 and a pharmaceutically acceptable carrier.

25. An isolated nucleic acid encoding the polypeptide portion of an isolated anti-TSLP antibody construct according to any one of claims 1 to 23.

26. A vector comprising the isolated nucleic acid described in claim 25.

27. A host cell comprising the isolated nucleic acid described in claim 25 or the vector described in claim 26.

28. A method for treating an inflammatory disease in an individual, comprising administering to the individual an effective amount of an isolated anti-TSLP antibody construct according to any one of claims 1 to 23 or a pharmaceutical composition according to claim 24.

29. The method according to claim 28, wherein the inflammatory disease is asthma, atopic dermatitis, or chronic obstructive pulmonary disease.

30. The method according to claim 28 or 29, wherein the individual is a human.

31. A method for producing an anti-TSLP antibody construct, i) Culturing a host cell containing the isolated nucleic acid described in claim 25 or the vector described in claim 26, or the host cell described in claim 27, under conditions suitable for the expression of an anti-TSLP antibody construct, ii) A method comprising obtaining the expressed anti-TSLP antibody construct.

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