Anti-TSLP antibodies and uses thereof

Abstract

To provide antibodies against TSLP and use of the antibodies in the treatment of diseases.SOLUTION: The present invention relates to an anti-TSLP antibody or an antigen-binding fragment thereof, a nucleic acid molecule encoding the same, and a method for preparing the same. The anti-TSLP antibodies or antigen-binding fragments thereof have a high affinity for TSLP and are capable of effectively binding TSLP and blocking the proliferative effect of TSLP on Ba / F3 - hTSLPR - hIL7R alpha cells, as well as blocking the capacity of TSLP in the kinetics of activation and secretion on PBMCs. At the same time, it further relates to a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, and the use of the composition in the preparation of a medicament for preventing and / or treating asthma, allergic inflammation, allergic reaction or autoimmune disease.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention is in the field of therapeutic monoclonal antibodies, in particular antibodies against TSLP, and the use of antibodies in the treatment of disease. [Background technology]

[0002] Thymic stromal lymphopoietin (TSLP) is an IL-7-like proinflammatory cytokine primarily secreted by epithelial cells, such as those in the skin, lungs, thymus, and gastrointestinal tract, in response to microorganisms, physical injury, or proinflammatory cytokines (e.g., IL-1β and TNF). It is also secreted by stromal cells, such as keratinocytes, dendritic cells (DCs), and mast cells, under pathological conditions, such as inflammation. TSLP plays an important role in the initiation of allergic and adaptive airway inflammation. TSLP is highly expressed in the airways of patients with asthma compared with healthy controls, and its levels correlate with the expression of TH2 cytokines and chemokines and the severity of the disease. TSLP induces dendritic cell (DC) maturation and upregulates OX40L expression. OX40-OX40L interaction may be involved in the polarization of initial T cell-induced TH2 cells. After differentiation, these TH2 cells release cytokines such as IL-4, IL-5, and IL-13, contributing to the infiltration of mast cells and eosinophils, a series of allergic inflammatory responses, and airway lesions, resulting in asthma attacks. TSLP effectively activates mast cells and natural killer T (NKT) cells to produce TH2 cytokines such as IL-13, thereby exacerbating the onset and progression of airway inflammation.

[0003] The receptor for TSLP is a heterodimeric receptor complex consisting of IL-7Rα and a unique TSLPR chain (CRFL2). Binding of the TSLP heterodimeric receptor results in STAT5 activation and cell proliferation. TSLPR and IL-7Rα are highly expressed on DC cells.

[0004] Therefore, there is an urgent need to develop anti-TSLP antibodies that have high specificity and affinity, low toxicity and side effects, and excellent clinical efficacy, thereby providing more medical options for patients with asthma.

[0005] In this disclosure, the inventors first developed a chimeric antibody with excellent properties capable of binding to human TSLP. Based on this, the inventors developed a fully human version of the chimeric antibody by testing and modifying the chimeric antibody. The fully human antibody of the present invention has substantially the same (or even better) biological function as the chimeric antibody. The fully human antibody not only has high affinity for TSLP, but also effectively blocks TSLP-induced Ba / F3 cell proliferation and can block the ability of TSLP to activate PBMCs and inhibit cytokine secretion. Therefore, the present invention further relates to pharmaceutical compositions comprising the antibody or its antigen-binding fragment, and its use in preparing medicaments for the prevention and / or treatment of asthma, allergic inflammation, allergic reactions, or autoimmune diseases.

[0006] The antibodies of the present invention are highly humanized, even fully human, and therefore can be safely administered to human subjects without eliciting an immunogenic response, and are therefore of great clinical value.

[0007] Antibodies of the Invention In one aspect, the invention provides an antibody or antigen-binding fragment thereof that binds to TSLP, comprising the following complementarity determining regions (CDRs): (a) CDR-H1 or a sequence variant thereof, CDR-H2 or a sequence variant thereof, and CDR-H3 or a sequence variant thereof, which are contained in a heavy chain variable region (VH) set forth in SEQ ID NO: 1, 17, 30, 40, 53, or 68, and / or CDR-L1 or a sequence variant thereof, CDR-L2 or a sequence variant thereof, and CDR-L3 or a sequence variant thereof, which are contained in a light chain variable region (VL) set forth in SEQ ID NO: 2, 18, 31, 41, 54, or 69, or (b) CDR-H1 or a sequence variant thereof, CDR-H2 or a sequence variant thereof, and CDR-H3 or a sequence variant thereof contained in the VH set forth in SEQ ID NO: 1, and / or CDR-L1 or a sequence variant thereof, CDR-L2 or a sequence variant thereof, and CDR-L3 or a sequence variant thereof contained in the VL set forth in SEQ ID NO: 2. The present invention provides an antibody or antigen-binding fragment thereof comprising:

[0008] In certain embodiments, the antibody or antigen-binding fragment thereof comprises CDR-H1 or a sequence variant thereof, CDR-H2 or a sequence variant thereof, and CDR-H3 or a sequence variant thereof contained in the VH set forth in SEQ ID NO: 17, and / or CDR-L1 or a sequence variant thereof, CDR-L2 or a sequence variant thereof, and CDR-L3 or a sequence variant thereof contained in the VL set forth in SEQ ID NO: 18.

[0009] In certain embodiments, the antibody or antigen-binding fragment thereof comprises CDR-H1 or a sequence variant thereof, CDR-H2 or a sequence variant thereof, and CDR-H3 or a sequence variant thereof contained in the VH set forth in SEQ ID NO: 30, and / or CDR-L1 or a sequence variant thereof, CDR-L2 or a sequence variant thereof, and CDR-L3 or a sequence variant thereof contained in the VL set forth in SEQ ID NO: 31.

[0010] In certain embodiments, the antibody or antigen-binding fragment thereof comprises CDR-H1 or a sequence variant thereof, CDR-H2 or a sequence variant thereof, and CDR-H3 or a sequence variant thereof contained in the VH set forth in SEQ ID NO: 40, and / or CDR-L1 or a sequence variant thereof, CDR-L2 or a sequence variant thereof, and CDR-L3 or a sequence variant thereof contained in the VL set forth in SEQ ID NO: 41.

[0011] In certain embodiments, the antibody or antigen-binding fragment thereof comprises CDR-H1 or a sequence variant thereof, CDR-H2 or a sequence variant thereof, and CDR-H3 or a sequence variant thereof contained in the VH set forth in SEQ ID NO: 53, and / or CDR-L1 or a sequence variant thereof, CDR-L2 or a sequence variant thereof, and CDR-L3 or a sequence variant thereof contained in the VL set forth in SEQ ID NO: 54.

[0012] In certain embodiments, the antibody or antigen-binding fragment thereof comprises CDR-H1 or a sequence variant thereof, CDR-H2 or a sequence variant thereof, and CDR-H3 or a sequence variant thereof contained in the VH set forth in SEQ ID NO: 68, and / or CDR-L1 or a sequence variant thereof, CDR-L2 or a sequence variant thereof, and CDR-L3 or a sequence variant thereof contained in the VL set forth in SEQ ID NO: 69.

[0013] In certain embodiments, a sequence variant is a CDR that has one or more amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to the CDR from which it is derived.

[0014] In certain embodiments, the substitutions are conservative substitutions.

[0015] Preferably, the CDRs are defined according to the AbM, Chothia, Kabat or IMGT numbering systems.

[0016] In certain embodiments, the VH and / or VL of the antibody or antigen-binding fragment thereof comprises an immunoglobulin framework region (FR) of human origin.

[0017] In certain embodiments, the antibody or antigen-binding fragment thereof binds to human TSLP and / or monkey TSLP.

[0018] In one aspect, the invention provides an antibody or antigen-binding fragment thereof capable of binding to TSLP, comprising a heavy chain variable region (VH) and / or a light chain variable region (VL).

[0019] In certain embodiments, the antibody or antigen-binding fragment thereof according to the invention comprises the following heavy chain variable region (VH) and / or light chain variable region (VL), whose CDRs are defined according to the IMGT numbering system: (a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 3 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 3; CDR-H2 having the sequence set forth in SEQ ID NO: 4 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 4; and CDR-H3 having the sequence set forth in SEQ ID NO: 5 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 5; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 6 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 6; CDR-L2 having the sequence set forth in SEQ ID NO: 7 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 7; and CDR-L3 having the sequence set forth in SEQ ID NO: 8 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 8; (b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 19 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 19; CDR-H2 having the sequence set forth in SEQ ID NO: 20 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 20; and CDR-H3 having the sequence set forth in SEQ ID NO: 21 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 21; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 22 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 22; CDR-L2 having the sequence set forth in SEQ ID NO: 23 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 23; and CDR-L3 having the sequence set forth in SEQ ID NO: 24 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 24; (c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 32 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 32; CDR-H2 having the sequence set forth in SEQ ID NO: 33 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 33; and CDR-H3 having the sequence set forth in SEQ ID NO: 34 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 34; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 35 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 35; CDR-L2 having the sequence set forth in SEQ ID NO: 23 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 23; and CDR-L3 having the sequence set forth in SEQ ID NO: 24 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 24; (d) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 42 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 42; CDR-H2 having the sequence set forth in SEQ ID NO: 43 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 43; and CDR-H3 having the sequence set forth in SEQ ID NO: 44 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 44; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 45 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 45; CDR-L2 having the sequence set forth in SEQ ID NO: 46 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 46; and CDR-L3 having the sequence set forth in SEQ ID NO: 47 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 47; or (e) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 55 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 55; CDR-H2 having the sequence set forth in SEQ ID NO: 56 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 56; and CDR-H3 having the sequence set forth in SEQ ID NO: 57 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 57; and / or The light chain variable region (VL) comprises the following three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 58 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 58; CDR-L2 having the sequence set forth in SEQ ID NO: 59 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 59; and CDR-L3 having the sequence set forth in SEQ ID NO: 60 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 60.

[0020] In certain embodiments, the antibody or antigen-binding fragment thereof according to the invention comprises the following heavy chain variable region (VH) and / or light chain variable region (VL), whose CDRs are defined according to the IMGT numbering system: (a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 3, CDR-H2 having the sequence set forth in SEQ ID NO: 4, and CDR-H3 having the sequence set forth in SEQ ID NO: 5; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 6, CDR-L2 having the sequence set forth in SEQ ID NO: 7, and CDR-L3 having the sequence set forth in SEQ ID NO: 8; (b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 19, CDR-H2 having the sequence set forth in SEQ ID NO: 20, and CDR-H3 having the sequence set forth in SEQ ID NO: 21; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 22, CDR-L2 having the sequence set forth in SEQ ID NO: 23, and CDR-L3 having the sequence set forth in SEQ ID NO: 24; (c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 32, CDR-H2 having the sequence set forth in SEQ ID NO: 33, and CDR-H3 having the sequence set forth in SEQ ID NO: 34; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 35, CDR-L2 having the sequence set forth in SEQ ID NO: 23, and CDR-L3 having the sequence set forth in SEQ ID NO: 24; (d) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 42, CDR-H2 having the sequence set forth in SEQ ID NO: 43, and CDR-H3 having the sequence set forth in SEQ ID NO: 44; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 45, CDR-L2 having the sequence set forth in SEQ ID NO: 46, and CDR-L3 having the sequence set forth in SEQ ID NO: 47; or (e) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 55, CDR-H2 having the sequence set forth in SEQ ID NO: 56, and CDR-H3 having the sequence set forth in SEQ ID NO: 57; and / or It comprises a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 58, CDR-L2 having the sequence set forth in SEQ ID NO: 59, and CDR-L3 having the sequence set forth in SEQ ID NO: 60.

[0021] In certain embodiments, the antibody or antigen-binding fragment thereof according to the invention comprises the following heavy chain variable region (VH) and / or light chain variable region (VL), whose CDRs are defined according to the AbM numbering system: (a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 9 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 9; CDR-H2 having the sequence set forth in SEQ ID NO: 10 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 10; and CDR-H3 having the sequence set forth in SEQ ID NO: 11 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 11; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 12 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 12; CDR-L2 having the sequence set forth in SEQ ID NO: 13 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 13; and CDR-L3 having the sequence set forth in SEQ ID NO: 8 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 8; (b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 25 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 25; CDR-H2 having the sequence set forth in SEQ ID NO: 26 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 26; and CDR-H3 having the sequence set forth in SEQ ID NO: 27 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 27; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 28 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 28; CDR-L2 having the sequence set forth in SEQ ID NO: 29 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 29; and CDR-L3 having the sequence set forth in SEQ ID NO: 24 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 24; (c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 36 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 36; CDR-H2 having the sequence set forth in SEQ ID NO: 37 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 37; and CDR-H3 having the sequence set forth in SEQ ID NO: 38 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 38; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 39 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 39; CDR-L2 having the sequence set forth in SEQ ID NO: 29 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 29; and CDR-L3 having the sequence set forth in SEQ ID NO: 24 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 24; (d) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 48 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 48; CDR-H2 having the sequence set forth in SEQ ID NO: 49 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 49; and CDR-H3 having the sequence set forth in SEQ ID NO: 50 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 50; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 51 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 51; CDR-L2 having the sequence set forth in SEQ ID NO: 52 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 52; and CDR-L3 having the sequence set forth in SEQ ID NO: 47 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 47; or (e) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 61 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 61; CDR-H2 having the sequence set forth in SEQ ID NO: 62 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 62; and CDR-H3 having the sequence set forth in SEQ ID NO: 63 or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions) compared to SEQ ID NO: 63; and / or The light chain variable region (VL) comprises the following three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 64 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 64; CDR-L2 having the sequence set forth in SEQ ID NO: 65 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 65; and CDR-L3 having the sequence set forth in SEQ ID NO: 60 or a sequence having one or more amino acid substitutions, deletions or additions (e.g., one, two or three amino acid substitutions, deletions or additions) compared to SEQ ID NO: 60.

[0022] In certain embodiments, the antibody or antigen-binding fragment thereof according to the invention comprises the following heavy chain variable region (VH) and / or light chain variable region (VL), whose CDRs are defined according to the AbM numbering system: (a) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 9, CDR-H2 having the sequence set forth in SEQ ID NO: 10, and CDR-H3 having the sequence set forth in SEQ ID NO: 11; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 12, CDR-L2 having the sequence set forth in SEQ ID NO: 13, and CDR-L3 having the sequence set forth in SEQ ID NO: 8; (b) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 25, CDR-H2 having the sequence set forth in SEQ ID NO: 26, and CDR-H3 having the sequence set forth in SEQ ID NO: 27; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 28, CDR-L2 having the sequence set forth in SEQ ID NO: 29, and CDR-L3 having the sequence set forth in SEQ ID NO: 24; (c) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 36, CDR-H2 having the sequence set forth in SEQ ID NO: 37, and CDR-H3 having the sequence set forth in SEQ ID NO: 38; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 39, CDR-L2 having the sequence set forth in SEQ ID NO: 29, and CDR-L3 having the sequence set forth in SEQ ID NO: 24; (d) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 48, CDR-H2 having the sequence set forth in SEQ ID NO: 49, and CDR-H3 having the sequence set forth in SEQ ID NO: 50; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 51, CDR-L2 having the sequence set forth in SEQ ID NO: 52, and CDR-L3 having the sequence set forth in SEQ ID NO: 47; or (e) a heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 having the sequence set forth in SEQ ID NO: 61, CDR-H2 having the sequence set forth in SEQ ID NO: 62, and CDR-H3 having the sequence set forth in SEQ ID NO: 63; and / or It comprises a light chain variable region (VL) comprising the following three CDRs: CDR-L1 having the sequence set forth in SEQ ID NO: 64, CDR-L2 having the sequence set forth in SEQ ID NO: 65, and CDR-L3 having the sequence set forth in SEQ ID NO: 60.

[0023] In certain embodiments, an antibody or antigen-binding fragment thereof according to the invention comprises a heavy chain variable region (VH) and / or a light chain variable region (VL) in which at least one CDR in the heavy chain variable region (VH) and / or light chain variable region (VL) comprises a mutation, compared to the CDRs defined by IMGT or AbM as described above, wherein the mutation is a substitution, deletion or addition of one or more amino acids, or any combination thereof (e.g., a substitution, deletion or addition of one, two or three amino acids, or any combination thereof).

[0024] Preferably, the substitutions of the present invention are conservative substitutions.

[0025] In certain embodiments, the VH of an antibody or antigen-binding fragment thereof according to the invention comprises a framework region (FR) derived from a heavy chain variable region (VH) of a human immunoglobulin, and / or the VL of an antibody or antigen-binding fragment thereof comprises a framework region (FR) derived from a light chain variable region (VL) of a human immunoglobulin. Thus, in certain embodiments, an antibody or antigen-binding fragment thereof according to the invention is humanized. In certain embodiments, an antibody or antigen-binding fragment thereof according to the invention is a fully human antibody or antigen-binding fragment thereof.

[0026] In certain embodiments, the antibody or antigen-binding fragment thereof according to the invention: (a) a heavy chain framework region of a human immunoglobulin, or a variant thereof, wherein the variant has up to 20 conservative substitutions of amino acids (e.g., up to 20, up to 15, up to 10, or up to 5 conservative substitutions of amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative substitutions) compared to the amino acid sequence encoded by the germline antibody gene from which it is derived; and / or (b) a light chain framework region of a human immunoglobulin, or a variant thereof, wherein the variant has up to 20 conservative substitutions of amino acids (e.g., up to 20, up to 15, up to 10, or up to 5 conservative substitutions of amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative substitutions) compared to the amino acid sequence encoded by the germline antibody gene sequence from which it is derived. Includes.

[0027] In certain embodiments, the degree of humanization of an antibody or antigen-binding fragment thereof of the invention is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.

[0028] In certain embodiments, the antibody or antigen-binding fragment thereof according to the invention: (a)(i) a sequence set forth in SEQ ID NO: 1, 17, 30, 40, 53, or 68; (ii) a sequence that contains one or more amino acid substitutions, deletions, or additions, or any combination thereof, compared to the sequence set forth in SEQ ID NO: 1, 17, 30, 40, 53, or 68 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, or additions, or any combination thereof); or (iii) a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence set forth in SEQ ID NO: 1, 17, 30, 40, 53, or 68 and / or a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of: (b)(iv) a sequence set forth in SEQ ID NO: 2, 18, 31, 41, 54, or 69; (v) a sequence that contains one or more amino acid substitutions, deletions, or additions, or any combination thereof, compared to the sequence set forth in SEQ ID NO: 2, 18, 31, 41, 54, or 69 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, or additions, or any combination thereof); or (vi) a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence set forth in SEQ ID NO: 2, 18, 31, 41, 54, or 69 The antibody comprises a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of:

[0029] In a particular embodiment, an antibody or antigen-binding fragment thereof according to the invention comprises a VH set forth in SEQ ID NO:1 and / or a VL set forth in SEQ ID NO:2.

[0030] In a particular embodiment, the antibody or antigen-binding fragment thereof according to the invention comprises a VH set forth in SEQ ID NO:17 and / or a VL set forth in SEQ ID NO:18.

[0031] In a particular embodiment, an antibody or antigen-binding fragment thereof according to the invention comprises a VH set forth in SEQ ID NO: 30 and / or a VL set forth in SEQ ID NO: 31.

[0032] In a particular embodiment, the antibody or antigen-binding fragment thereof according to the invention comprises a VH set forth in SEQ ID NO:40 and / or a VL set forth in SEQ ID NO:41.

[0033] In a particular embodiment, an antibody or antigen-binding fragment thereof according to the invention comprises a VH set forth in SEQ ID NO:53 and / or a VL set forth in SEQ ID NO:54.

[0034] In a particular embodiment, an antibody or antigen-binding fragment thereof according to the invention comprises a VH set forth in SEQ ID NO:68 and / or a VL set forth in SEQ ID NO:69.

[0035] In certain embodiments, the antibody or antigen-binding fragment thereof according to the invention: (a) VH set forth in SEQ ID NO: 1 and VL set forth in SEQ ID NO: 2; (b) VH set forth in SEQ ID NO: 17 and VL set forth in SEQ ID NO: 18; (c) VH set forth in SEQ ID NO: 30 and VL set forth in SEQ ID NO: 31; (d) VH set forth in SEQ ID NO: 40 and VL set forth in SEQ ID NO: 41; (e) VH set forth in SEQ ID NO: 53 and VL set forth in SEQ ID NO: 54; (f) VH set forth in SEQ ID NO: 68 and VL set forth in SEQ ID NO: 69; (g) a heavy chain variable region (VH) and a light chain variable region (VL) that independently have at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the VH and VL in any one of (a) to (f); or (h) A heavy chain variable region (VH) and a light chain variable region (VL) each independently having one or more amino acid substitutions, deletions, or additions, or any combination thereof (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, or additions, or any combination thereof) compared to the VH and VL in any one of (a) to (f). Preferably, the substitutions are conservative substitutions.

[0036] In certain embodiments, the heavy chain of an antibody or antigen-binding fragment thereof of the present invention comprises a human immunoglobulin heavy chain constant region (CH) or a variant thereof, wherein the variant has up to 50 conservative substitutions of amino acids (e.g., up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 conservative substitutions of amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative substitutions) compared to the wild-type sequence from which it is derived. In certain embodiments, the light chain of an antibody or antigen-binding fragment thereof of the invention comprises a human immunoglobulin light chain constant region (CL) or a variant thereof, wherein the variant has up to 50 amino acid substitutions (e.g., up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid conservative substitutions, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid conservative substitutions) compared to the wild-type sequence from which it is derived.

[0037] In certain embodiments, the constant region is altered, e.g., mutated, to modify the characteristics of the anti-TSLP antibody molecule (e.g., to change one or more of the following properties: Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function). Replacing at least one amino acid residue in the antibody constant region with a different residue can alter function, for example, to change the affinity of the antibody for an effector ligand (e.g., FcR or complement C1q), thereby altering (e.g., decreasing) effector function. The Fc region of an antibody mediates several important effector functions, such as ADCC, phagocytosis (ADCP), and CDC.

[0038] In certain embodiments, antibodies or antigen-binding fragments thereof according to the invention comprise a heavy chain constant region (Fc), which is chosen from, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE, particularly IgG1, IgG2, IgG3 and IgG4, more particularly IgG1 (e.g., human IgG1). In some embodiments, the heavy chain constant region of human IgG1 is set forth in SEQ ID NO: 14. In some embodiments, antibodies or antigen-binding fragments thereof according to the invention comprise a light chain constant region, which is chosen from, for example, a kappa or lambda light chain constant region, preferably a kappa light chain constant region (e.g., a human kappa light chain constant region).

[0039] In some embodiments, the antibody or antigen-binding fragment thereof comprises the heavy chain constant region of human IgG1. In some preferred embodiments, the antibody or antigen-binding fragment thereof comprises the constant region of human IgG1 (SEQ ID NO: 74) set forth in uniprotID P01857.

[0040] In some embodiments, the antibody or antigen-binding fragment thereof comprises a human IgG1 heavy chain constant region (e.g., SEQ ID NO: 74) or a variant thereof, wherein the variant is mutated at at least one of positions 234, 235, 237, 265, 297, 331, 329, and 434 according to the EU numbering system. In some embodiments, the variant comprises at least one of the following mutations: L234A, L235A, D265A, N297A, L234F, L235E, P331S, P329G, N434A, N434Y, N434F, N434W, N434S, N434G, N434H, and N434Q. In some embodiments, the variant comprises at least one of the following mutations: L234A, L235A, G237A, and N434A. In some embodiments, the variant of the IgG1 heavy chain constant region comprises L234A, L235A, and G237A, hi some embodiments, the variant of the IgG1 heavy chain constant region comprises L234A, L235A, G237A, and N434A.

[0041] In some embodiments, the antibody or antigen-binding fragment thereof comprises a CH set forth in SEQ ID NO: 14 or a variant thereof, wherein the variant comprises up to 20 amino acid conservative substitutions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid conservative substitutions, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid conservative substitutions) compared to SEQ ID NO: 14, or has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 14. The variant comprises N297A and / or N434A according to the EU numbering system. In some embodiments, the variant comprises N434A.

[0042] In some embodiments, the antibody or antigen-binding fragment thereof comprises a CH set forth in SEQ ID NO: 15 or a variant thereof, wherein the variant has up to 20 amino acid conservative substitutions compared to SEQ ID NO: 15 (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid conservative substitutions, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid conservative substitutions), or has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 15.

[0043] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region of human IgG4 (e.g., SEQ ID NO: 75) or a variant thereof, wherein the variant is mutated at at least one of positions 228 and / or 434 according to the EU numbering system.

[0044] In some embodiments, the mutation comprises S228P and / or N434A. In some embodiments, the variant of the heavy chain constant region of human IgG4 comprises S228P and N434A.

[0045] In some embodiments, the antibody or antigen-binding fragment thereof comprises a CH set forth in SEQ ID NO: 70 or a variant thereof, wherein the variant has up to 20 amino acid conservative substitutions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid conservative substitutions, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid conservative substitutions) compared to SEQ ID NO: 70, or has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 70.

[0046] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain constant region or a variant thereof. In some embodiments, the light chain constant region comprises a kappa light chain constant region. In some embodiments, the light chain constant region comprises the light chain constant region (CL) set forth in SEQ ID NO: 16 or a variant thereof, wherein the variant comprises up to 20 amino acid conservative substitutions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid conservative substitutions, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid conservative substitutions) compared to SEQ ID NO: 16, or has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 16.

[0047] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) set forth in SEQ ID NO:14 and a light chain constant region (CL) set forth in SEQ ID NO:16.

[0048] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) set forth in SEQ ID NO:15 and a light chain constant region (CL) set forth in SEQ ID NO:16.

[0049] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) set forth in SEQ ID NO:70 and a light chain constant region (CL) set forth in SEQ ID NO:16.

[0050] In some embodiments, the above mutation(s) result in the antibody or antigen-binding fragment thereof having no or reduced ADCP, ADCC and / or CDC activity compared to a corresponding antibody or antigen-binding fragment thereof comprising a human IgG4 or an IgG4 heavy chain constant region.

[0051] In some embodiments, the mutation(s) described above result in the antibody or antigen-binding fragment thereof having no or reduced ADCP, ADCC and / or CDC activity compared to a corresponding antibody or antigen-binding fragment thereof without the mutation or substitution described above.

[0052] In certain embodiments, the antibody or antigen-binding fragment thereof according to the invention: (a)(i) a sequence comprising the VH sequence set forth in SEQ ID NO: 1 and the CH sequence set forth in SEQ ID NO: 14, 15, or 70; (ii) a sequence having one or more amino acid substitutions, deletions or additions, or any combination thereof, compared to the sequence of (i) (e.g., up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions, or any combination thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions, or any combination thereof); or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of (i). a heavy chain comprising an amino acid sequence selected from the group consisting of: (b)(iv) a sequence comprising the VL sequence set forth in SEQ ID NO: 2 and the CL sequence set forth in SEQ ID NO: 16; (v) a sequence having one or more amino acid substitutions, deletions or additions, or any combination thereof, compared to the sequence of (iv) (e.g., up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions, or any combination thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions, or any combination thereof); or (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of (iv). and a light chain comprising an amino acid sequence selected from the group consisting of:

[0053] In certain embodiments, the substitution in (ii) or (v) is a conservative substitution.

[0054] In certain embodiments, the antibody or antigen-binding fragment thereof according to the invention: (a)(i) a sequence comprising the VH sequence set forth in SEQ ID NO: 17 and the CH sequence set forth in SEQ ID NO: 14, 15, or 70; (ii) a sequence having one or more amino acid substitutions, deletions or additions, or any combination thereof, compared to the sequence of (i) (e.g., up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions, or any combination thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions, or any combination thereof); or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of (i). a heavy chain comprising an amino acid sequence selected from the group consisting of: (b)(iv) a sequence comprising the VL sequence set forth in SEQ ID NO: 18 and the CL sequence set forth in SEQ ID NO: 16; (v) a sequence having one or more amino acid substitutions, deletions or additions, or any combination thereof, compared to the sequence of (iv) (e.g., up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions, or any combination thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions, or any combination thereof); or (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of (iv). and a light chain comprising an amino acid sequence selected from the group consisting of:

[0055] In certain embodiments, the substitution in (ii) or (v) is a conservative substitution.

[0056] In certain embodiments, the antibody or antigen-binding fragment thereof according to the invention: (a)(i) a sequence comprising the VH sequence set forth in SEQ ID NO: 30 and the CH sequence set forth in SEQ ID NO: 14, 15, or 70; (ii) a sequence having one or more amino acid substitutions, deletions or additions, or any combination thereof, compared to the sequence of (i) (e.g., up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions, or any combination thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions, or any combination thereof); or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of (i). a heavy chain comprising an amino acid sequence selected from the group consisting of: (b)(iv) a sequence comprising the VL sequence set forth in SEQ ID NO: 31 and the CL sequence set forth in SEQ ID NO: 16; (v) a sequence having one or more amino acid substitutions, deletions or additions, or any combination thereof, compared to the sequence of (iv) (e.g., up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions, or any combination thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions, or any combination thereof); or (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of (iv). and a light chain comprising an amino acid sequence selected from the group consisting of:

[0057] In certain embodiments, the substitution in (ii) or (v) is a conservative substitution.

[0058] In certain embodiments, the antibody or antigen-binding fragment thereof according to the invention: (a)(i) a sequence comprising the VH sequence set forth in SEQ ID NO: 40 and the CH sequence set forth in SEQ ID NO: 14, 15, or 70; (ii) a sequence having one or more amino acid substitutions, deletions or additions, or any combination thereof, compared to the sequence of (i) (e.g., up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions, or any combination thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions, or any combination thereof); or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of (i). a heavy chain comprising an amino acid sequence selected from the group consisting of: (b)(iv) a sequence comprising the VL sequence set forth in SEQ ID NO: 41 and the CL sequence set forth in SEQ ID NO: 16; (v) a sequence having one or more amino acid substitutions, deletions or additions, or any combination thereof, compared to the sequence of (iv) (e.g., up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions, or any combination thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions, or any combination thereof); or (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of (iv). and a light chain comprising an amino acid sequence selected from the group consisting of:

[0059] In certain embodiments, the substitution in (ii) or (v) is a conservative substitution.

[0060] In certain embodiments, the antibody or antigen-binding fragment thereof according to the invention: (a)(i) a sequence comprising the VH sequence set forth in SEQ ID NO: 53 and the CH sequence set forth in SEQ ID NO: 14, 15, or 70; (ii) a sequence having one or more amino acid substitutions, deletions or additions, or any combination thereof, compared to the sequence of (i) (e.g., up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions, or any combination thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions, or any combination thereof); or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of (i). a heavy chain comprising an amino acid sequence selected from the group consisting of: (b)(iv) a sequence comprising the VL sequence set forth in SEQ ID NO: 54 and the CL sequence set forth in SEQ ID NO: 16; (v) a sequence having one or more amino acid substitutions, deletions or additions, or any combination thereof, compared to the sequence of (iv) (e.g., up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions, or any combination thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions, or any combination thereof); or (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of (iv). and a light chain comprising an amino acid sequence selected from the group consisting of:

[0061] In certain embodiments, the substitution in (ii) or (v) is a conservative substitution.

[0062] In certain embodiments, the antibody or antigen-binding fragment thereof according to the invention: (a)(i) a sequence comprising the VH sequence set forth in SEQ ID NO: 68 and the CH sequence set forth in SEQ ID NO: 14, 15, or 70; (ii) a sequence having one or more amino acid substitutions, deletions or additions, or any combination thereof, compared to the sequence of (i) (e.g., up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions, or any combination thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions, or any combination thereof); or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of (i). a heavy chain comprising an amino acid sequence selected from the group consisting of: (b)(iv) a sequence comprising the VL sequence set forth in SEQ ID NO: 69 and the CL sequence set forth in SEQ ID NO: 16; (v) a sequence having one or more amino acid substitutions, deletions or additions, or any combination thereof, compared to the sequence of (iv) (e.g., up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions, or any combination thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions, or any combination thereof); or (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of (iv). and a light chain comprising an amino acid sequence selected from the group consisting of:

[0063] In certain embodiments, the substitution in (ii) or (v) is a conservative substitution.

[0064] In certain embodiments, an antibody of the invention comprises a heavy chain comprising a VH set forth in SEQ ID NO: 1 and a heavy chain constant region (CH) set forth in SEQ ID NO: 14, 15 or 70, and a light chain comprising a VL set forth in SEQ ID NO: 2 and a light chain constant region (CL) set forth in SEQ ID NO: 16.

[0065] In certain embodiments, an antibody of the invention comprises a heavy chain comprising a VH set forth in SEQ ID NO: 17 and a heavy chain constant region (CH) set forth in SEQ ID NO: 14, 15 or 70, and a light chain comprising a VL set forth in SEQ ID NO: 18 and a light chain constant region (CL) set forth in SEQ ID NO: 16.

[0066] In certain embodiments, an antibody of the invention comprises a heavy chain comprising a VH set forth in SEQ ID NO: 30 and a heavy chain constant region (CH) set forth in SEQ ID NO: 14, 15 or 70, and a light chain comprising a VL set forth in SEQ ID NO: 31 and a light chain constant region (CL) set forth in SEQ ID NO: 16.

[0067] In certain embodiments, an antibody of the invention comprises a heavy chain comprising a VH set forth in SEQ ID NO: 40 and a heavy chain constant region (CH) set forth in SEQ ID NO: 14, 15 or 70, and a light chain comprising a VL set forth in SEQ ID NO: 41 and a light chain constant region (CL) set forth in SEQ ID NO: 16.

[0068] In certain embodiments, an antibody of the invention comprises a heavy chain comprising a VH set forth in SEQ ID NO: 53 and a heavy chain constant region (CH) set forth in SEQ ID NO: 14, 15 or 70, and a light chain comprising a VL set forth in SEQ ID NO: 54 and a light chain constant region (CL) set forth in SEQ ID NO: 16.

[0069] In certain embodiments, an antibody of the invention comprises a heavy chain comprising a VH set forth in SEQ ID NO: 68 and a heavy chain constant region (CH) set forth in SEQ ID NO: 14, 15 or 70, and a light chain comprising a VL set forth in SEQ ID NO: 69 and a light chain constant region (CL) set forth in SEQ ID NO: 16.

[0070] In certain embodiments, the antibody or antigen-binding fragment thereof according to the invention comprises a heavy chain and a light chain, wherein the heavy chain comprises: (i) a sequence set forth in SEQ ID NO: 66 or 73; (ii) a sequence having one or more amino acid substitutions, deletions or additions, or any combination thereof, compared to the sequence of (i) (e.g., up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions, or any combination thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions, or any combination thereof); or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of (i); The light chain (iv) the sequence set forth in SEQ ID NO: 67; (v) a sequence having one or more amino acid substitutions, deletions or additions, or any combination thereof, compared to the sequence of (iv) (e.g., up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions, or any combination thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions, or any combination thereof); or (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of (iv); Preferably, the substitution (ii) or (v) is a conservative substitution.

[0071] In certain embodiments, the antibody or antigen-binding fragment thereof according to the invention comprises a heavy chain and a light chain, wherein the heavy chain comprises: (i) the sequence set forth in SEQ ID NO: 71; (ii) a sequence having one or more amino acid substitutions, deletions or additions, or any combination thereof, compared to the sequence of (i) (e.g., up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions, or any combination thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions, or any combination thereof); or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of (i); The light chain (iv) the sequence set forth in SEQ ID NO: 72; (v) a sequence having one or more amino acid substitutions, deletions or additions, or any combination thereof, compared to the sequence of (iv) (e.g., up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions, or any combination thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions, or any combination thereof); or (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of (iv); Preferably, the substitution (ii) or (v) is a conservative substitution.

[0072] In certain embodiments, the antibodies of the invention are chimeric, humanized, or fully human antibodies. In certain embodiments, the antibodies or antigen-binding fragments thereof according to the invention are selected from the group consisting of scFv, Fab, Fab', F(ab')2, Fv fragments, disulfide-linked Fv (dsFv), and diabodies.

[0073] In certain embodiments, the antibodies or antigen-binding fragments thereof according to the invention have the following characteristics: (a) binding to TSLP (e.g., human TSLP) with a KD of less than about 50 nM, e.g., about 40 nM, 30 nM, 20 nM, 10 nM, 1 nM, 0.1 nM, 1 pM, 0.1 pM, or less, where the KD is measured by techniques well known in the art, e.g., Fortebio or ELISA; (b) binding to TSLP (e.g., human TSLP) with an EC50 of less than about 50 nM, e.g., about 40 nM, 30 nM, 20 nM, 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.01 nM, 1 pM, 0.1 pM or less, where the EC50 is measured by techniques well known in the art, e.g., flow cytometry or ELISA, e.g., affinity ELISA or cell competition ELISA; (c) inhibition of TSLP binding to IL7Rα / TSLPR with an IC50 of about 50 nM, e.g., about 50 nM, 20 nM, 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.01 nM, 1 pM, 0.1 pM or less, wherein the IC50 is measured by ELISA; (d) inhibiting or blocking TSLP-induced OX40L expression; (e) inhibiting or blocking TSLP-induced activity and / or proliferation of mast cells, DCs, and NKT cells; (f) inhibiting or blocking TSLP-induced osteoprotegerin (OPG) secretion; (g) inhibiting or blocking the secretion of Th2 cytokines, such as TARC, CCL22, IL-4, IL-13, or IL-5; (h) good affinity for binding to FcRn; (i) an isoelectric point (PI) of about 6.5 to about 8.5, for example, about 6.5, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.7, about 7.9, about 8.0, about 8.2, or about 8.5; may indicate at least one of:

[0074] In addition, the antibodies of the present invention have good affinity for FcRn. In a specific embodiment, the KD(M) value of the affinity for FcRn is 10 -9 The antibody of the present invention has a long half-life in vivo. The antibody of the present invention also has good hydrophilicity. In a specific embodiment, the hydrophobic time of the antibody of the present invention is determined to be between 8 and 14 minutes using a chromatography column. antibody derivative Antibodies or antigen-binding fragments thereof according to the invention can be derivatized, for example, by conjugation to another molecule (e.g., another polypeptide or protein). Typically, derivatization (e.g., labeling) of antibodies or antigen-binding fragments thereof does not adversely affect binding to TSLP (particularly human TSLP). Thus, antibodies or antigen-binding fragments thereof according to the invention are intended to encompass such derivative forms. For example, antibodies or antigen-binding fragments thereof according to the invention can be conjugated (by chemical coupling, genetic fusion, noncovalent or other means) to one or more other molecular moieties, e.g., another antibody (e.g., to form a bispecific antibody), a detection reagent, a pharmaceutical reagent, and / or a protein or polypeptide capable of mediating binding of the antibody or antigen-binding fragment thereof to another molecule (e.g., avidin or polyhistidine tag).

[0075] One type of derivatized antibody (e.g., bispecific antibody) is produced by crosslinking two or more antibodies (belonging to the same or different species). Methods for obtaining bispecific antibodies are well known in the art; exemplary methods include, but are not limited to, chemical crosslinking, cell modification (hybridomas), or genetic modification.

[0076] Another type of derivatized antibody is a labeled antibody. For example, an antibody or antigen-binding fragment thereof according to the invention can be conjugated to a detectable marker. A detectable marker of the invention can be any substance that is detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, light, or chemistry. Such markers are well known in the art and include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., H, I, S, C, or P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa750)), acridine ester compounds, magnetic beads (e.g., Dynabeads®), calorimetric markers, such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin used to bind to avidin (e.g., streptavidin) modified with the above markers. The use of such markers is taught in patents, including, but not limited to, U.S. Patent Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241, all of which are incorporated herein by reference. The above-described detectable markers can be detected by methods known in the art. For example, radioactive markers may be detected using photographic film or a scintillation counter, and fluorescent markers may be detected using a photodetector that detects emitted light. Enzymatic markers may generally be detected by providing a substrate for the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and calorimetric markers may be detected using a single visual color-developing marker.In certain embodiments, such markers may be useful in immunoassays (e.g., enzyme-linked immunoassays, radioimmunoassays, fluorescent immunoassays, chemiluminescent immunoassays, etc.) In certain embodiments, the above-described detectable markers may be attached to the antibodies or antigen-binding fragments thereof according to the invention by linkers of various lengths to reduce potential steric hindrance.

[0077] In addition, antibodies or antigen-binding fragments thereof according to the invention can also be derivatized using chemical groups such as polyethylene glycol (PEG), methyl or ethyl, or glycosyl groups, which can be used to improve the biological properties of the antibody, for example, to increase its serum half-life.

[0078] Thus, in one aspect, the present invention provides a conjugate comprising a monoclonal antibody or antigen-binding fragment thereof according to the invention and a coupling moiety, wherein the coupling moiety is a detectable marker as described above, such as a radioisotope, a fluorescent substance, a luminescent substance, a chromogenic substance, or an enzyme. The coupling moiety may also be a therapeutic agent.

[0079] As one type of antibody derivative, the present invention provides a multispecific antibody comprising a primary antibody or fragment thereof and an additional antibody or fragment thereof, or antibody mimetic, wherein the primary antibody or fragment thereof, the additional antibody or fragment thereof, or antibody mimetic retains its original binding specificity. The primary antibody or fragment thereof is any one of the (monoclonal) antibodies or antigen-binding fragments thereof of the present invention that bind to TSLP. As used herein, "antibody mimetic" refers to a substance that specifically binds to an antigen in the same way as an antibody but does not have an antibody structure. These are typically artificial peptides or proteins with a molar mass of approximately 3 to 20 kDa, such as designed ankyrin repeat proteins (DARPins) and fynomers. Designed ankyrin repeat proteins (DARPins) can be conjugated to IgG antibodies, scFv-Fc antibody fragments, or combinations thereof, as described in Chinese Patent Application Publication No. 104341529. The bispecific fusion polypeptide is generated by fusing a phenomer against IL-17a to an anti-IL-6R antibody, as described in WO2015141862.

[0080] In certain embodiments, a multispecific antibody is formed by coupling a primary antibody or antibody-binding fragment thereof with another antibody or antigen-binding fragment thereof, or antibody mimetic, where each antibody or antigen-binding fragment thereof, or antibody mimetic, retains its original binding specificity, and the primary antibody or antibody-binding fragment thereof is an antibody or antibody-binding fragment thereof according to the invention. In certain embodiments, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody. Antibody preparation The antibodies of the present invention can be prepared by various methods known in the art, such as recombinant genetic engineering techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the present invention can be obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into an expression vector and then transfected into host cells. The transfected host cells are then cultured under specific conditions to express the antibodies of the present invention.

[0081] Antigen-binding fragments of the present invention can be obtained by hydrolysis of intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). In addition, these antigen-binding fragments can also be produced directly by recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000)). For example, Fab' fragments can be obtained directly from host cells, and Fab' fragments can be chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology, 10:163-167 (1992)). In addition, Fv, Fab or F(ab')2 fragments can also be isolated directly from recombinant host cell culture. Other techniques for preparing these antigen-binding fragments are known to those skilled in the art.

[0082] Therefore, in another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof according to the present invention, or the heavy chain variable region and / or light chain variable region thereof, or one or more CDRs thereof. Taking into account codon degeneracy known in the art, in some embodiments, the nucleotide sequence may vary depending on the codon degeneracy. In certain embodiments, the nucleotide sequence is codon-optimized.

[0083] In certain embodiments, an isolated nucleic acid molecule according to the present invention comprises (i) a first nucleic acid and a second nucleic acid encoding, respectively, a heavy chain variable region and a light chain variable region of an antibody or antigen-binding fragment thereof according to the present invention, or (ii) a first nucleic acid encoding a heavy chain variable region and a heavy chain constant region and a second nucleic acid encoding a light chain variable region and a light chain constant region of an antibody or antigen-binding fragment thereof according to the present invention, or (iii) a first nucleic acid and a second nucleic acid encoding, respectively, a heavy chain and a light chain of an antibody or antigen-binding fragment thereof according to the present invention. In certain embodiments, the first and second nucleic acids comprise a nucleic acid having a degenerate sequence of, or a substantially identical sequence to, any one of the first and second nucleic acids (i) to (iii) above. In certain embodiments, a degenerate sequence or a substantially identical sequence refers to a sequence having at least 85%, 90%, 95%, 99%, or more sequence identity, or one or more nucleotide substitutions, or no more than 3, 6, 15, 30, or 45 nucleotide differences, compared to the nucleic acid molecule of (i) to (iii).

[0084] In another aspect, there is provided a vector (e.g., a cloning vector or an expression vector) comprising an isolated nucleic acid molecule according to the invention. In certain embodiments, a vector according to the invention is, for example, a plasmid, cosmid, phage, lentivirus, etc. In certain embodiments, the vector is capable of expressing an antibody or antigen-binding fragment thereof according to the invention in vivo in a subject (e.g., a mammal, e.g., a human).

[0085] In another aspect, there is provided a host cell comprising an isolated nucleic acid molecule of the invention, or a vector of the invention. The host cell can be a eukaryotic cell (e.g., a mammalian cell, an insect cell, a yeast cell) or a prokaryotic cell (e.g., Escherichia coli). Suitable eukaryotic cells include, but are not limited to, NSO cells, Vero cells, Hela cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells. In a specific embodiment, the host cell of the invention is a mammalian cell, such as a CHO (e.g., CHO-K1, CHO-S, CHO DXB11, CHO DG44).

[0086] In another aspect, there is provided a method for preparing an antibody or antigen-binding fragment thereof according to the invention, comprising culturing a host cell according to the invention under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture. Uses, Methods of Treatment, and Pharmaceutical Compositions In another aspect of the invention, there is provided a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof, a nucleic acid, a vector, a host cell, a multispecific antibody, and / or a conjugate according to the invention, and a pharmaceutically acceptable carrier and / or excipient.

[0087] In certain embodiments, a pharmaceutical composition according to the invention comprises an antibody or antigen-binding fragment thereof according to the invention, and a pharmaceutically acceptable carrier and / or excipient.

[0088] In certain embodiments, a pharmaceutical composition according to the invention comprises a host cell according to the invention and a pharmaceutically acceptable carrier and / or excipient, wherein the host cell comprises an isolated nucleic acid molecule or vector as described above.

[0089] In certain embodiments, a pharmaceutical composition according to the invention comprises a multispecific antibody according to the invention and a pharmaceutically acceptable carrier and / or excipient.

[0090] In certain embodiments, a pharmaceutical composition according to the invention comprises a conjugate according to the invention and a pharmaceutically acceptable carrier and / or excipient.

[0091] In another aspect, the antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, multispecific antibody, or conjugate in the pharmaceutical composition according to the invention has the following biological activity in a subject: (1) Inhibition or blocking of TSLP binding to TSLPR / IL7Rα; (2) downregulation or ablation of TSLP activity; (3) downregulation or blocking of OX40L expression; (4) inhibiting or blocking TSLP-induced osteoprotegerin (OPG) secretion; (5) Inhibition or blocking of the secretion of Th2-like cytokines, such as TARC, CCL22, IL-4, IL-13, or IL-5; (6) Inhibition or blocking of TSLP-induced activation and / or proliferation of mast cells, DCs, and NKT cells It shall result in at least one of the following:

[0092] The antibodies or antigen-binding fragments thereof, nucleic acids, vectors, host cells, multispecific antibodies, or conjugates in the pharmaceutical compositions of the present invention can inhibit or block the binding of TSLP to TSLPR / IL7Rα. Binding of TSLP to TSLPR / IL7Rα can result in a variety of allergic inflammatory diseases, including allergic and non-allergic diseases. These diseases include asthma (including severe asthma), idiopathic pulmonary fibrosis, atopic dermatitis (AD), allergic conjunctivitis, allergic rhinitis (AR), Netherton syndrome (NS), eosinophilic esophagitis (EoE), food allergy, allergic diarrhea, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis (ABPA), allergic fungal sinusitis, rheumatoid arthritis, COPD, systemic sclerosis, and keloids. These diseases include, but are not limited to, ulcerative colitis, chronic rhinosinusitis (CRS) and nasal polyps, chronic eosinophilic pneumonia, and eosinophilic bronchitis; abdominal diseases such as eosinophilic gastroenteritis and Churg-Strauss syndrome; eosinophil-associated gastrointestinal diseases such as eosinophilia / eosinophilic granulomatosis with polyangiitis, eosinophilic esophagitis, and inflammatory bowel disease; urticaria, systemic mastocytosis, cutaneous mastocytosis, and recurrent idiopathic angioedema. Therefore, the antibodies or antigen-binding fragments thereof, nucleic acids, vectors, host cells, multispecific antibodies, or conjugates in the pharmaceutical compositions of the present invention can be used to prevent or treat the above-mentioned diseases.

[0093] Furthermore, binding of TSLP to TSLPR / IL7Rα is associated with autoimmune diseases. Therefore, the antibodies or antigen-binding fragments thereof, nucleic acids, vectors, host cells, multispecific antibodies, or conjugates in the pharmaceutical compositions of the present invention can prevent or treat autoimmune diseases, such as diabetes, myasthenia gravis, gastritis, pemphigus, primary biliary cirrhosis, multiple sclerosis, lupus, colitis, rheumatic diseases, psoriasis, and thyroid diseases.

[0094] In certain embodiments, the antibody or antigen-binding fragment thereof and the additional pharmaceutically active agent in the pharmaceutical composition of the invention are provided as separate components or as components of a single composition. Thus, the antibody or antigen-binding fragment thereof according to the invention can be administered in combination with the additional pharmaceutically active agent or separately, simultaneously or sequentially.

[0095] In certain embodiments, the pharmaceutical composition may also include an additional pharmaceutically active agent.

[0096] Anti-TSLP antibodies or antigen-binding fragments thereof according to the invention can be administered alone or in combination with other active agents. Anti-TSLP antibodies or antigen-binding fragments thereof according to the invention can be administered separately, simultaneously, or sequentially with one or more other active agents.

[0097] Anti-TSLP antibodies or antigen-binding fragments thereof according to the invention can be administered in combination with any suitable immunosuppressant, including, but not limited to, inhaled, intranasal, or parenterally administered anti-inflammatory agents, particularly corticosteroids such as budesonide, beclomethasone dipropionate, fluticasone propionate, ciclesonide, mometasone furoate, fluticasone furoate, fluticasone propionate, budesonide, ciclesonide, beclomethasone propionate, mometasone furoate, triamcinolone acetonide, and prednisolone. In one embodiment, anti-TSLP antibodies or antigen-binding fragments thereof according to the invention can be administered in combination with a fixed dose of an inhaled corticosteroid, such as a fixed dose of fluticasone furoate or fluticasone propionate. In one embodiment, an anti-TSLP antibody or antigen-binding fragment thereof according to the invention can be administered in combination with a nonsteroidal glucocorticoid receptor agonist: an LTD4 antagonist or an LTB4 antagonist, including montelukast, pranlukast, zafirlukast, Accolate, etc.; an A2A agonist; an A2B antagonist; a dopamine receptor agonist; or a PDE4 inhibitor. In one embodiment, an anti-TSLP antibody or antigen-binding fragment thereof according to the invention can be administered in combination with pirfenidone, nintedanib, or an avB6 antagonist.

[0098] The anti-TSLP antibody or antigen-binding fragment thereof of the present invention can be administered in combination with a bronchodilator, such as a beta2 adrenergic receptor agonist and / or a muscarinic antagonist. Suitable beta2 adrenergic receptor agonists include vilanterol, salmeterol, salbutamol, formoterol, salmefamol, fenoterol, carmoterol, etanterol, naminterol, clenbuterol, pirbuterol, flerobuterol, reproterol, bambuterol, indacaterol, terbutaline, and salts thereof. Suitable muscarinic antagonists include umeclidinium bromide, tiotropium bromide, glycopyrronium bromide, ipratropium, and salts thereof, such as umeclidinium bromide hydrobromide. In one embodiment, an anti-TSLP antibody or antigen-binding fragment thereof according to the invention can be administered in combination with a dose of a beta-2 adrenergic receptor agonist and / or a muscarinic antagonist, such as a dose of vilanterol triphenylacetate or umeclidinium bromide, or both vilanterol triphenylacetate and umeclidinium bromide.

[0099] Anti-TSLP antibodies or antigen-binding fragments thereof according to the invention can be administered in combination with one or more bronchodilators and inhaled steroids. Such combinations can include dual combinations, such as fluticasone furoate and vilanterol triphenylacetate, fluticasone furoate and umeclidinium bromide, fluticasone propionate and salmeterol, budesonide and formoterol, or mometasone and formoterol, as well as triple therapy, such as fluticasone furoate, vilanterol triphenylacetate, and umeclidinium bromide. In one embodiment, an anti-TSLP antibody or antigen-binding fragment thereof according to the invention can be administered in combination with a dose of an inhaled corticosteroid and one or more bronchodilators, such as a dose of fluticasone furoate and vilanterol triphenylacetate, or fluticasone propionate and salmeterol, or fluticasone furoate and umeclidinium bromide, or fluticasone furoate, vilanterol triphenylacetate, and umeclidinium bromide.

[0100] In one embodiment, an anti-TSLP antibody or antigen-binding fragment thereof according to the invention can be administered in combination with a cytokine receptor antagonist, e.g., an antagonist of CCR-1, CCR-3, CCR-4, CCR-5, CCR-6, CCR-7, CCR-8, CCR-9, and CCR10, CXCR1, CXCR2, CXCR3, CXCR4, or CXCR5. In one embodiment, an anti-TSLP antibody or antigen-binding fragment thereof according to the invention can be administered in combination with an antibody to another cytokine or cytokine receptor, e.g., an anti-IgE antibody, an anti-IL31 antibody, an anti-IL31R antibody, an anti-IL13 antibody, an anti-endoglin antibody, an anti-IL1b antibody, another anti-TSLP antibody, or an anti-hTSLPR antibody, or a combination thereof.

[0101] Anti-TSLP antibodies or antigen-binding fragments thereof according to the invention can be administered in combination with the following: leukotriene antagonists, such as montelukast, zafirlukast, and pranlukast; PDE4 inhibitors, such as roflumilast; xanthenes; anti-IgE antibodies; IL-13 antagonists; IL-6 antagonists; and antagonists of IL-1, IL-33, IL-25, or TNF-α.

[0102] Anti-TSLP antibodies or antigen-binding fragments thereof according to the invention can be administered in combination with antihistamines or antitussives, such as cetirizine hydrochloride, acetaminophen, clemastine fumarate, promethazine, loratadine, desloratadine, diphenhydramine, and fexofenadine hydrochloride, activastine, astazole, azelastine, ebastine, epinastine, mizolastine, and tefenadine.

[0103] TSLP-binding antibodies or antigen-binding fragments thereof of the present invention may be administered in combination with other agents, including, but not limited to, immunosuppressants (e.g., corticosteroids, nonsteroidal glucocorticoid receptor agonists, leukotriene D4 antagonists, leukotriene B4 antagonists, A2A agonists, A2B antagonists, dopamine receptor agonists, pirfenidone, nintedanib, or avB6 antagonists), bronchodilators (e.g., beta2 adrenergic receptor agonists, muscarinic antagonists, short-acting beta2 receptor agonists, long-acting beta2 receptor agonists, short-acting anticholinergics, methylxanthine drugs, long-acting anticholinergics), other cytokines or or one or more other active agents selected from a cytokine receptor antagonist or antibody (e.g., an IL-13 antagonist, an IL-6 antagonist, an IL-1, IL-33, IL-25 or TNFalpha antagonist, an anti-IgE antibody, an anti-IL31 antibody, an anti-IL31R antibody, an anti-IL13 antibody, an anti-endoglin antibody, an anti-IL1b antibody, another anti-TSLP antibody or an anti-hTSLPR antibody), an antibiotic, radiation therapy, a leukotriene antagonist (e.g., montelukast, zafirlukast or pranlukast), a PDE4 inhibitor (e.g., roflumilast, xanthene), an antihistamine or an antitussive.

[0104] In certain embodiments, the pharmaceutical composition is administered simultaneously with other treatments, separately or sequentially, eg, before, simultaneously with, or after an additional pharmaceutically active agent.

[0105] In another aspect of the invention, antibodies or antigen-binding fragments thereof, nucleic acids, vectors, host cells, conjugates, or multispecific antibodies of the invention are provided for use in: (1) inhibiting or blocking TSLP binding to TSLPR / IL7Rα; (2) downregulating or ablating TSLP activity; (3) downregulating or blocking OX40L expression; (4) inhibiting or blocking TSLP-induced activation and / or proliferation of mast cells, DCs, or NKT cells; (5) inhibiting or blocking TSLP-induced osteoprotegerin (OPG) secretion; (6) inhibiting or blocking TSLP-induced secretion of Th2 cytokines, e.g., TARC, CCL22, IL-4, IL-13, or IL-5; and / or (7) preventing or treating an allergic disease, allergic response, or autoimmune disease.

[0106] In another aspect of the invention, (1) Inhibition or blocking of TSLP binding to TSLPR / IL7Rα, (2) downregulation or ablation of TSLP activity; (3) downregulation or blocking of OX40L expression; (4) inhibiting or blocking TSLP-induced activation and / or proliferation of mast cells, DCs, and NKT cells; (5) Inhibition or blocking of TSLP-induced osteoprotegerin (OPG) secretion, (6) inhibiting or blocking TSLP-induced secretion of Th2 cytokines, such as TARC, CCL22, IL-4, IL-13, or IL-5; and / or (7) Prevention or treatment of allergic diseases, allergic responses, or autoimmune diseases The present invention provides the use of an antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, conjugate, or multispecific antibody of the invention in the preparation of a medicament for:

[0107] In certain embodiments, when used for the preparation of a medicament, the host cell of the invention comprises an isolated nucleic acid molecule or vector as described above.

[0108] In certain embodiments, when the antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, multispecific antibody, or conjugate is used in the preparation of a medicament, the medicament is used to prevent and / or treat asthma, allergic inflammation, an allergic reaction, or an autoimmune disease in a subject (e.g., a human).

[0109] In certain embodiments, the subject is a mammal, including non-human mammals and humans, hi certain embodiments, the subject is a human.

[0110] In certain embodiments, the antibodies or antigen-binding fragments thereof, nucleic acids, vectors, host cells, multispecific antibodies, conjugates, or medicaments of the invention are used to prevent and / or treat allergic inflammatory diseases, including allergic and non-allergic diseases, such as asthma (including severe asthma), idiopathic pulmonary fibrosis, atopic dermatitis (AD), allergic conjunctivitis, allergic rhinitis (AR), Netherton syndrome (NS), eosinophilic esophagitis (EOE), food allergies, allergic diarrhea, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis (ABPA), allergic fungal sinusitis, rheumatoid arthritis, chronic obstructive pulmonary disease (COPD), and systemic sclerosis. , keloids, ulcerative colitis, chronic rhinosinusitis (CRS) and nasal polyps, chronic eosinophilic pneumonia, eosinophilic bronchitis; abdominal diseases, e.g., eosinophilic gastroenteritis, Churg-Strauss syndrome; eosinophil-associated gastrointestinal diseases, e.g., eosinophilia / eosinophilic granulomatosis with polyangiitis, eosinophilic esophagitis and inflammatory bowel disease; urticaria, systemic mastocytosis, cutaneous mastocytosis, and recurrent idiopathic angioedema.

[0111] In certain embodiments, the antibodies or antigen-binding fragments thereof, nucleic acids, vectors, host cells, multispecific antibodies, or conjugates of the invention are used to prevent and / or treat autoimmune-related diseases, including, but not limited to, hyperthyroidism, diabetes, myasthenia gravis, ulcerative colitis, gastritis, pemphigus, primary biliary cirrhosis, multiple sclerosis, lupus erythematosus, rheumatoid arthritis, etc.

[0112] In another aspect, the invention provides a method for at least one of (1) inhibiting or blocking TSLP binding to TSLPR / IL7Rα, (2) downregulating or ablating the activity of TSLP, (3) downregulating or blocking OX40L expression, (4) inhibiting or blocking TSLP-induced osteoprotegerin (OPG) secretion, (5) inhibiting or blocking TSLP-induced secretion of Th2-like cytokines, or (6) inhibiting or blocking TSLP-induced activation and / or proliferation of mast cells, DCs, or NKT cells, in vivo or in vitro, comprising the step of administering to a cell or a subject any one of the antibodies or antigen-binding fragments thereof, nucleic acids, vectors, host cells, multispecific antibodies, conjugates, or pharmaceutical compositions of the invention.

[0113] Optionally, the additional pharmaceutically active agent is administered simultaneously with, before or after administration of the antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, multispecific antibody, conjugate or pharmaceutical composition.

[0114] In certain embodiments, the subject is a mammal, including non-human mammals and humans; preferably, the subject is a human.

[0115] Accordingly, in another aspect, the present invention provides a method for preventing and / or treating asthma, an allergic reaction, allergic inflammation, or an autoimmune disease in a subject, comprising the step of administering to a subject in need thereof an effective amount of an antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, multispecific antibody, conjugate, or pharmaceutical composition of the invention.

[0116] The antibodies or antigen-binding fragments thereof, nucleic acids, vectors, host cells, multispecific antibodies, conjugates, or pharmaceutical compositions of the present invention can be formulated into any dosage form known in the pharmaceutical art, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated injection solutions), inhalants, sprays, etc. The preferred dosage form depends on the intended route of administration and therapeutic use. The pharmaceutical compositions of the present invention must be sterile and stable under production and storage conditions and can be prepared into injections.

[0117] In addition, the antibodies or antigen-binding fragments thereof according to the invention may be present in unit dosage form of a pharmaceutical composition for ease of administration.

[0118] Pharmaceutical compositions according to the present invention may comprise a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, multispecific antibody, or conjugate of the present invention. A "prophylactically effective amount" refers to an amount sufficient to prevent, arrest, or delay the onset of a disease. A "therapeutically effective amount" refers to an amount sufficient to cure or at least partially prevent a disease and its complications in a patient suffering from the disease, e.g., 0.1 mg / mL to 5000 mg / mL.

[0119] In the present invention, the subject may be a mammal (including non-human mammals and humans), for example, a human. Detection Method and Kit The antibodies or antigen-binding fragments thereof according to the invention are capable of binding to TSLP and are therefore useful in detecting the presence or level of TSLP in a sample.

[0120] Thus, in another aspect, the present invention provides a kit comprising an antibody or antigen-binding fragment thereof according to the present invention. In certain embodiments, the antibody or antigen-binding fragment thereof according to the present invention carries a detectable marker. In a preferred embodiment, the kit further comprises a secondary antibody that specifically recognizes the antibody or antigen-binding fragment thereof according to the present invention. Preferably, the secondary antibody further comprises a detectable marker.

[0121] In the present invention, a detectable marker can be any substance that can be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, light, or chemistry. It is particularly preferred that such a marker be suitable for immunoassays (e.g., enzyme-linked immunoassays, radioimmunoassays, fluorescence immunoassays, chemiluminescence immunoassays, etc.). Such markers are well known in the art and include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., H, I, S, C, or P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa750)), acridine ester compounds, magnetic beads (e.g., Dynabeads®), calorimetric markers, such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin used to bind to avidin (e.g., streptavidin) modified with the above markers. The use of such markers is taught in patents, including, but not limited to, U.S. Patent Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241, all of which are incorporated herein by reference. Markers encompassed by the present invention can be detected by methods known in the art. For example, radioactive markers may be detected using photographic film or scintillation counters, and fluorescent markers may be detected using a photodetector that detects emitted light. Enzymatic markers may generally be detected by providing a substrate for the enzyme and detecting the reaction product produced by the action of the enzyme on the substrate, and calorimetric markers may be detected using a single visual colorimetric marker.In some embodiments, the detectable markers described above may be attached to the recombinant proteins of the present invention via linkers of various lengths to reduce potential steric hindrance.

[0122] In another aspect, the invention provides a method for detecting the presence or level of TSLP in a sample, comprising utilizing an antibody or antigen-binding fragment thereof according to the invention.

[0123] In a preferred embodiment, the antibody or antigen-binding fragment thereof according to the invention carries a detectable marker.

[0124] In another preferred embodiment, the method further comprises the step of detecting an antibody or antigen-binding fragment thereof according to the invention using a reagent in conjunction with a detectable marker.

[0125] The methods may be used for diagnostic or non-diagnostic purposes (e.g., TSLP-TSLP / IL-7Ra pathway testing, drug screening, histochemical analysis, etc.) In certain embodiments, the sample for non-diagnostic purposes is a cell sample, e.g., a cell line or ex vivo cell culture.

[0126] In one embodiment, the invention provides a method for detecting the presence or level of TSLP in a sample, comprising contacting the sample with an antibody or antigen-binding fragment thereof according to the invention under conditions that allow the formation of a complex between the antibody or antigen-binding fragment thereof and TSLP, and detecting the formation of the complex.

[0127] In another aspect, The present invention relates to a method for treating TSLP comprising contacting a sample from a subject with an antibody or antigen-binding fragment thereof, a multispecific antibody, or a conjugate according to the present invention under conditions that allow for the formation of a complex between the antibody or antigen-binding fragment thereof and TSLP; detecting the formation of a complex; wherein elevated levels of TSLP compared to a healthy control are indicative of asthma, allergic inflammation, an allergic response, or an autoimmune disease.

[0128] Optionally, the subject is a mammal, including non-human mammals and humans. Preferably, the subject is a human.

[0129] Preferably, the allergic inflammation, allergic reaction or autoimmune disease is as described above.

[0130] In another aspect, the present invention provides the use of an antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, multispecific antibody, conjugate or pharmaceutical composition according to the invention in the preparation of a medicament or kit for diagnosing asthma, allergic inflammation, an allergic reaction, or an autoimmune disease.

[0131] In another aspect, there is provided use of an antibody or antigen-binding fragment thereof according to the invention in the preparation of a kit for detecting the presence or level of TSLP in a sample. In another aspect, the invention provides a diagnostic or therapeutic kit comprising one or more of an antibody or antigen-binding fragment thereof, a nucleic acid, a vector, a host cell, a multispecific antibody, a conjugate, or a pharmaceutical composition according to the invention. Optionally, the diagnostic or therapeutic kit also comprises instructions.

[0132] The antibodies or antigen-binding fragments thereof according to the present invention have high affinity and good specificity for binding to TSLP. Therefore, the antibodies or antigen-binding fragments thereof according to the present invention are suitable for the prevention and / or treatment of asthma, allergic inflammation, allergic reactions, or autoimmune diseases. The fully human antibodies of the present invention are highly human and can be safely administered to human subjects without eliciting an immunogenic response. Therefore, the antibodies or antigen-binding fragments thereof according to the present invention are of great clinical value. Definition of Terms Unless otherwise defined herein, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. In addition, cell culture, biochemistry, nucleic acid chemistry, immunology laboratory and other operation steps used herein are conventional steps widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of relevant terms are provided below.

[0133] As used herein and in the appended claims, the singular forms "a / an" and "the" include the plural reference unless the context clearly dictates otherwise. Thus, the singular word "a / an" includes the reference to "one or more."

[0134] As used herein, the term "antibody" refers to an immunoglobulin molecule typically consisting of two pairs of polypeptide chains, each of which has a light chain (LC) and a heavy chain (HC). Light chains are classified as either kappa (κ) or lambda (λ). Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes can be defined as IgM, IgD, IgG, IgA, and IgE, respectively. In light and heavy chains, the variable and constant regions are connected by a "J" segment of about 12 or more amino acids, and heavy chains also contain a "D" segment of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant domains are not directly involved in binding of antibodies to antigens but exhibit various effector functions, mediating the binding of immunoglobulins to host tissues or factors, including, for example, various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into hypervariable regions (called complementarity-determining regions (CDRs)) interspersed with relatively conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of each heavy / light chain pair (VH and VL) each form an antigen-binding site.

[0135] In the present invention, the CDRs contained in the antibody or antigen-binding fragment thereof according to the present invention can be determined according to various numbering systems known in the art. In a specific embodiment, the CDRs contained in the antibody or antigen-binding fragment thereof according to the present invention are preferably determined according to the Kabat, Chothia, AbM, or IMGT numbering system.

[0136] As used herein, the term "framework region" or "FR" residues refers to amino acid residues in an antibody variable region other than the CDR residues as defined above.

[0137] As used herein, the term "germline antibody gene" refers to an immunoglobulin-encoding gene expressed by non-lymphocytes that does not undergo the maturation process of gene rearrangement and maturation resulting in the expression of a specific immunoglobulin. Advantages provided by various embodiments of the present invention stem from the common recognition that the amino acid sequences encoded by germline antibody genes retain a more significant amino acid sequence structure characteristic of an individual animal species than the amino acid sequences encoded by mature antibody genes. Therefore, when applied therapeutically to that species, the amino acid sequences are less likely to be recognized as exogenous by that species.

[0138] The term "antibody" is not limited by any particular method for producing the antibody. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be of various isotypes, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0139] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide fragment of an antibody, e.g., a polypeptide fragment of a full-length antibody, that retains the ability to specifically bind to the same antigen as the full-length antibody and / or competes with the full-length antibody for specific binding to an antigen, also known as an "antigen-binding portion." See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd Edition, Raven Press, NY (1989)), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies, linear antibodies, nanobodies (e.g., by Ablynx technology), domain antibodies (e.g., by Domantis technology), and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide. The engineering of modified antibody variants is reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.

[0140] As used herein, the term "full-length antibody" refers to an antibody consisting of two "full-length heavy chains" and two "full-length light chains." Here, a "full-length heavy chain" refers to a polypeptide chain consisting of, from N-terminus to C-terminus, a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain; if the full-length antibody is an IgE isotype, it optionally further contains a heavy chain constant region CH4 domain. Preferably, a "full-length heavy chain" is a polypeptide chain consisting of, from N-terminus to C-terminus, a VH, CH1, HR, CH2, and CH3. A "full-length light chain" is a polypeptide chain consisting of, from N-terminus to C-terminus, a light chain variable region (VL) and a light chain constant region (CL). Two pairs of full-length antibody chains are bound by disulfide bridges between the CL and CH1 domains and between the HRs of the two full-length heavy chains. The full-length antibodies of the present invention may be derived from a single species, such as human, or may be chimeric or humanized. The full-length antibodies of the present invention comprise two antigen-binding sites, each formed by a VH and VL pair, which specifically recognize / bind to the same antigen.

[0141] As used herein, the term "Fd fragment" refers to an antibody fragment consisting of the VH and CH1 domains, the term "dAb fragment" refers to an antibody fragment consisting of the VH domain (Ward et al., Nature 341:544 546 (1989)), the term "Fab fragment" refers to an antibody fragment consisting of the VL, VH, CL and CH1 domains, the term "F(ab')2 fragment" refers to an antibody fragment comprising two Fab fragments linked by disulfide bridges in the hinge region, and the term "Fab' fragment" refers to a fragment obtained by reduction of the disulfide bridges linking the two heavy chain fragments in the F(ab')2 fragment, which consists of an intact light chain and an Fd fragment of the heavy chain (consisting of the VH and CH1 domains).

[0142] As used herein, the term "Fv fragment" refers to an antibody fragment consisting of the VL and VH domains of a single antibody arm. The Fv fragment is generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. The six CDRs are generally considered to confer antigen-binding specificity to the antibody. However, even a single variable region (e.g., an Fd fragment containing only three antigen-specific CDRs) can recognize and bind to an antigen, although possibly with lower affinity than the complete binding site.

[0143] As used herein, the term "Fc fragment" refers to an antibody fragment formed by linking the second and third constant regions of a first antibody heavy chain to the second and third constant regions of a second antibody heavy chain via disulfide bridges. The Fc fragment of an antibody has many different functions, but is not involved in antigen binding.

[0144] As used herein, the term "scFv" refers to a single-chain polypeptide comprising VL and VH domains, where the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Ed. Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Linkers preferred in the art consist of a repeated GGGGS amino acid sequence or variants thereof. For example, a linker with the amino acid sequence (GGGGS)4 or a variant thereof can be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in the present invention are described in Alfthan et al. (1995), Protein Eng. 8:725-731; Choi et al. (2001), Eur. J. Immunol. 31:94-106; Hu et al. (1996), Cancer Res. 56:3055-3061; Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bridge can also be present between the VH and VL of an scFv. As used herein, the term "di-scFv" refers to an antibody fragment formed by the binding of two scFvs.

[0145] As used herein, the term "diabody" refers to a single polypeptide chain in which VH and VL domains are expressed, but a linker is used that is too short to allow pairing between the two domains of that chain; therefore, the domains are forced to pair with complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) and Poljak RJ et al., Structure 2:1121-1123 (1994)).

[0146] As used herein, the term "antibody mimetic" refers to a substance that specifically binds to an antigen as an antibody but lacks the antibody structure. These are typically artificial peptides or proteins with a molar mass of approximately 3-20 kDa. Examples include designed ankyrin repeat proteins (DARPins) and phenomers. Designed ankyrin repeat proteins (DARPins) can be conjugated to IgG antibodies, scFv-Fc antibody fragments, or combinations thereof, as described in Chinese Patent Publication No. 104341529. Anti-IL-17a phenomers can be conjugated to anti-IL-6R antibodies, as described in International Publication No. 2015141862.

[0147] Each of the above antibody fragments retains the ability to specifically bind to the same antigen as bound by the full-length antibody and / or compete with the full-length antibody for specific binding to an antigen.

[0148] Antigen-binding fragments (e.g., the antibody fragments described above) may be obtained from a given antibody (e.g., an antibody provided by the present invention) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and antigen-binding fragments of the antibody are screened in detail in the same manner as for intact antibodies.

[0149] In the present invention, unless the context clearly dictates otherwise, reference to the term "antibody" includes not only intact antibodies but also antigen-binding fragments thereof.

[0150] As used herein, the terms "monoclonal antibody," "McAb," and "mAb" have the same meaning and are used interchangeably to refer to an antibody derived from a population of highly homologous antibody molecules, i.e., antibody molecules that are identical except for naturally occurring mutations that may occur naturally. Monoclonal antibodies have high specificity for a single epitope of an antigen. Compared to monoclonal antibodies, polyclonal antibodies usually contain at least two or more different antibodies, which usually recognize different epitopes of an antigen. Furthermore, the modifier "monoclonal" simply indicates that the characteristics of the antibody are obtained from a population of highly homologous antibodies, and is not understood to require any particular method of antibody preparation.

[0151] As used herein, the term "chimeric antibody" refers to an antibody in which one portion of its light or / and heavy chain is derived from one antibody (which may be from a particular species or belong to a particular antibody class or subclass) and the other portion of the light or / and heavy chain is derived from another antibody (which may be from the same or a different species or belong to the same or a different antibody class or subclass), which in either case retains binding activity for the target antigen (Cabilly et al., U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

[0152] In the present invention, the expected properties of the antibodies of the present invention include (1) inhibition or blocking of TSLP binding to TSLPR / IL7Rα, (2) downregulation or elimination of TSLP activity, (3) downregulation or blocking of OX40L expression, (4) inhibition or blocking of Th2-like cytokine secretion, and (5) prevention and / or treatment of allergic inflammatory diseases. The humanized antibodies of the present invention retain one or more of the above expected properties of the parent antibody (human antibody or mouse-human chimeric antibody).

[0153] In preparing humanized antibodies, murine CDR regions can be inserted into human framework sequences using methods known in the art (see Winter, U.S. Pat. Nos. 5,225,539; 5,530,101; 5,585,089; 5,693,762 and Queen et al., U.S. Pat. No. 6,180,370; and Lo, Benny, KC, editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004). Alternatively, transgenic animals can be utilized that are incapable of producing endogenous immunoglobulins after immunization, but are capable of producing intact human antibody libraries (see, e.g., Jakobovits et al., 1993, Proc. Natl. Acad. Sci. USA 90:2551; Jakobovits et al., 1993, Nature 362:255-258; Bruggermann et al., 1993, Year in Immunology 7:33; and Duchosal et al., 1992, Nature 355:258; Lonberg et al. (1994) Nature 368(6474):856-859; WO 02 / 43478). Other methods of antibody humanization include phage display technology (Hoogenboom et al., 1991, J. Mol. Biol. 227:381; Marks et al., J. Mol. Biol. 1991, 222:581-597; Vaughan et al., 1996, Nature Biotech 14:309).

[0154] As used herein, the term "degree of humanization" is an index for evaluating the number of non-human amino acid residues in a humanized antibody. The degree of humanization of a humanized antibody can be predicted based on the homology of the variable region sequence with a human V domain, for example, using DomainGapAlign on the IMGT website.

[0155] As used herein, the term "specifically binds" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its antigen. The strength or affinity of a specific binding interaction is determined by the equilibrium dissociation constant (K D In the present invention, the term "K D " refers to the dissociation equilibrium constant of a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the dissociation equilibrium constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen. In certain embodiments, an antibody that specifically binds to an antigen (or an antibody specific for an antigen) is one that binds to an antigen at a dissociation equilibrium constant of about 10 -8 Less than m, e.g., about 10 -8 M, 10 -9 M, 10 -10 M or 10 -11 K below M D In some embodiments, K D ≦10×10 -8 When M, the antibody or antigen-binding fragment thereof of the invention is considered to specifically bind to TSLP.

[0156] Specific binding between two molecules can be measured using methods well known in the art. One of these methods involves measuring the rates of formation and dissociation of the antigen-binding site / antigen complex. Both the "association rate constant" (ka or k) and the "dissociation rate constant" (k or k) can be calculated from the concentrations and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361:186-187). The ratio of k / k is the dissociation constant, K D (See Davies et al., Annual Rev Biochem, 1990;59:439-473). K DThe k, k, and k values ​​can be measured by any valid method. In certain embodiments, the dissociation constant can be measured by bioluminescence interferometry (e.g., ForteBio Octet assay). In addition, the dissociation constant can also be measured by surface plasmon resonance technology (e.g., Biacore) or Kinexa.

[0157] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. If a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is called an expression vector. A vector can be introduced into a host cell by transformation, transduction, or transfection, thereby allowing the genetic material elements carried therein to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); phages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). Vectors may contain elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, vectors may also contain an origin of replication.

[0158] As used herein, the term "host cell" refers to a cell into which a vector can be introduced, including, but not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as Drosophila S2 cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells.

[0159] As used herein, the term "identity" refers to the degree of sequence match between two polypeptides or two nucleic acids. If a position in two sequences for comparison is occupied by the same base or amino acid monomer subunit (e.g., if each position in two DNA molecules is occupied by adenine, or each position in two polypeptides is occupied by lysine), the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared, multiplied by 100. For example, if six of ten positions in two sequences are matched, the two sequences are 60% identical. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (three of six total positions are matched). Typically, two sequences are compared and aligned for maximum identity. Such alignments can be achieved, for example, by using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed using a computer program, such as the Align program (DNAstar, Inc.). In addition, the percentage identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) in the GAP program incorporated into the GCG software package (available at www.gcg.com), using either Blossum Matrix 62 or PAM250, gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6. The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Computer. App. Biosci., 4:11-17 (1988)).

[0160] As used herein, a sequence with a "percent identity" retains the important biological activity, e.g., antibody binding specificity, of the sequence from which it is aligned or derived. A sequence with one or more amino acid substitutions, deletions, or additions, or any combination thereof, retains the important biological activity, e.g., antibody binding specificity, of the sequence from which it is aligned or derived. Nucleotide sequences with a "percent identity" or differences of 3, 6, 15, 30, or 45 nucleotides or less may exhibit similar function to the nucleotide sequence from which it is aligned or derived, e.g., all of the expressed proteins may specifically bind to the same antigen or molecule.

[0161] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or change the expected properties of a protein / polypeptide containing the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include the replacement of an amino acid residue with an amino acid residue having a similar side chain, e.g., a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., has similar size, shape, charge, chemical properties including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with alkaline side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), polar uncharged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl. Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0162] The compilation of the 20 conventional amino acids involved in the present invention follows conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in the present invention, amino acids are generally represented by one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented as A or Ala, arginine as R or Arg, glycine as G or Gly, and glutamine as Q or Gln.

[0163] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and active ingredient, and is well known in the art (see, e.g., Remington's Pharmaceutical Sciences, edited by Gennaro AR, 19th ed., Pennsylvania: Mack Publishing Company, 1995). Examples of such carriers include, but are not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancing agents, diluents, osmolality maintaining agents, absorption delaying agents, and preservatives. For example, pH adjusting agents include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancing agents include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as, for example, p-hydroxybenzoic acid, trichloro-tert-butyl alcohol, phenol, sorbic acid, etc. Osmolality-maintaining agents include, but are not limited to, sugars, NaCl, etc. Absorption-delaying agents include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffer solutions (e.g., buffered saline), alcohols, and polyols (e.g., glycerin), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as, for example, thiomersalate, 2-phenoxyethanol, p-hydroxybenzoic acid, trichloro-tert-butyl alcohol, phenol, sorbic acid, etc.The stabilizer has the meaning commonly understood by those skilled in the art, which is capable of stabilizing the desired activity of the active ingredient in the drug, and includes, but is not limited to, sodium glutamate, gelatin, SPGA, sugars (e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin, or casein), or degradation products thereof (e.g., lactalbumin hydrolysate).

[0164] As used herein, the term "prevention" refers to a process for preventing or delaying the onset of a disease or symptom or sign (e.g., asthma, allergic inflammation, allergic reaction, or autoimmune disease) in a subject in vivo. As used herein, the term "treatment" refers to a process for achieving a beneficial or desired clinical result. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in the extent of disease, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, amelioration or alleviation of the disease state, and remission of symptoms (whether partial or complete), whether detectable or undetectable. In addition, "treatment" can also mean prolonging survival as compared to expected survival (in the absence of treatment).

[0165] As used herein, the term "subject" refers to a mammal, e.g., a primate, e.g., a non-human primate, or a human. In some embodiments, the subject (e.g., a human) has or is at risk of developing asthma, allergic inflammation, an allergic reaction, or an autoimmune disease.

[0166] As used herein, the term "effective amount" refers to an amount sufficient to achieve or at least partially achieve a desired effect. For example, an effective amount for the prevention of a disease (e.g., asthma, allergic inflammation, allergic reaction, or autoimmune disease) is an amount sufficient to prevent, stop, or delay the disease (e.g., asthma, allergic inflammation, allergic reaction, or autoimmune disease), and a therapeutically effective amount refers to an amount sufficient to cure or at least partially prevent the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is within the capabilities of one skilled in the art. For example, an amount effective for therapeutic use depends on the severity of the disease to be treated, the general state of the patient's own immune system, the patient's general condition, such as age, weight, and sex, the route of administration, and other treatments administered at the same time.

[0167] As used herein, the term "immune cells" includes cells that are derived from the hematopoietic system and play a role in the immune response, such as lymphocytes, e.g., B cells and T cells; natural killer cells; myeloid cells, e.g., monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0168] As used herein, the term "immune response" refers to the action of immune cells (e.g., lymphocytes, antigen-presenting cells, phagocytes, or granulocytes) and soluble macromolecules (including antibodies, cytokines, and complement) produced by immune cells or the liver, resulting in the selective damage, destruction, or removal from the body of invasive pathogens, pathogen-infected cells or tissues, cancer cells, or normal human cells or tissues in cases of autoimmune or pathological inflammation. As used herein, the term "antigen-specific T cell response" refers to an immune response generated by a T cell when the T cell is stimulated by an antigen specific for the T cell. Non-limiting examples of responses generated by T cells in response to antigen-specific stimulation include T cell proliferation and cytokine (e.g., IL-2) production.

[0169] As used herein, the term "effector functions" refers to the biological activities attributed to the Fc region of an antibody (a native sequence Fc region or amino acid sequence variant Fc region), which vary depending on the antibody isotype.

[0170] The term "pharmaceutically acceptable" means that a molecular entity, molecular fragment, or composition does not produce undesired, allergic, or other adverse reactions when administered appropriately to an animal or human. Specific examples of substances that can be used as pharmaceutically acceptable carriers or components thereof include sugars (e.g., lactose), starch, cellulose and its derivatives, vegetable oils, gelatin, polyols (e.g., polyethylene glycol), alginic acid, etc.

[0171] Beneficial Effects of the Invention Compared with the prior art, the technical solution of the present invention has the following beneficial effects: (1) The antibodies of the present invention specifically recognize / bind to TSLP with high affinity, inhibit or block TSLP binding to TSLPR / IL7RR, inhibit or block TSLP-induced proliferation of Ba / F3 cells in vitro / in vivo, and block TSLP-induced activation and cytokine secretion of PBMCs. Therefore, the antibodies of the present invention can inhibit or block TSLP-induced activation and proliferation of mast cells, DCs, and NKT cells, and inhibit or block TSLP-induced OX40L expression, osteoprotegerin (OPG) secretion, or TSLP-induced secretion of Th2 cytokines, such as TARC, CCL22, IL-4, IL-13, or IL-5. Therefore, the antibodies of the present invention have potential in the prevention and / or treatment of asthma, other allergic reactions, or autoimmune diseases. (2) The antibodies of the present invention have good thermal stability, hydrophilicity, isoelectric point, and affinity for FcRn. (3) Some of the antibodies of the present invention are fully human and can therefore be safely administered to subjects without eliciting an immunogenic response, making the antibodies of the present invention of great clinical value.

[0172] Abbreviation CDR Complementarity-Determining Region within the variable region of an immunoglobulin FR antibody framework region: amino acid residues in the variable region of an antibody other than CDR residues VH antibody heavy chain variable region VL antibody light chain variable region IgG immunoglobulin G AbM The AbM CDR definition comes from the work of Martin (Martin ACR, Cheetham JC, Rees AR (1989) Modeling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272), which incorporates parts of the Kabat and Chothia definitions. The immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutions of Health, Bethesda, Md., 1991). The immunoglobulin numbering system proposed by Chothia et al. is a classical rule for identifying the boundaries of CDR regions based on the location of the ring structure regions (see, e.g., Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883). IMGT: A numbering system based on the International Immunogenetics Information System® (IMGT) initiated by Lefranc et al. See Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003. mAb Monoclonal antibody EC 50 Concentration that produces 50% efficacy or binding I C 50 Concentration that produces 50% inhibition ELISA Enzyme-linked immunosorbent assay PCR polymerase chain reaction HRP horseradish peroxidase TSLP thymic stromal lymphopoietin TSLPR Thymic stromal lymphopoietin receptor IL7Rα Interleukin 7 receptor alpha subunit TARC: Thymus and activation-regulated chemokine hFc Fc segment of human IgG antibody KD dissociation equilibrium constant CDR-H1 Complementarity-determining region 1 in the heavy chain variable region of immunoglobulin CDR-H2 Complementarity-determining region 2 within the heavy chain variable region of immunoglobulin CDR-H3 Complementarity-determining region 3 within the heavy chain variable region of immunoglobulin CDR-L1 Complementarity-determining region 1 within the immunoglobulin light chain variable region CDR-L2 Complementarity-determining region 2 within the light chain variable region of immunoglobulin CDR-L3 Complementarity-determining region 3 within the light chain variable region of immunoglobulin [Brief explanation of the drawings]

[0173] [Figure 1] FIG. 1 shows the detection of a Ba / F3 cell line that highly expresses both the human TSLPR and IL7Rα genes. [Figure 2A] FIG. 1 shows the detection of the inhibitory activity of chimeric antibody 25A5C5 on the proliferation of Ba / F3-hTSLPR-hIL7Rα cells. [Figure 2B] FIG. 1 shows the detection of the inhibitory activity of chimeric antibodies 27C2B6 and 37C2D10 on the proliferation of Ba / F3-hTSLPR-hIL7Rα cells. [Figure 2C] FIG. 1 shows the detection of the inhibitory activity of chimeric antibodies 43B1A8 and 90H3H11 on the proliferation of Ba / F3-hTSLPR-hIL7Rα cells. [Figure 3A] FIG. 1 shows the detection of binding affinity of recombinant fully human antibodies 25A5C5-hIgG, 27C2B6-hIgG, and 37C2D10-hIgG to human TSLP protein. [Figure 3B]FIG. 1 shows the detection of binding affinity of recombinant fully human antibodies 43B1A8-hIgG and 90H3H11-hIgG to human TSLP protein. [Figure 4A] FIG. 1 shows the detection of the inhibitory activity of recombinant fully human antibodies 25A5C5-hIgG, 27C2B6-hIgG, and 37C2D10-hIgG on the proliferation of Ba / F3-hTSLPR-hIL7Rα cells. [Figure 4B] FIG. 1 shows the detection of the inhibitory activity of recombinant fully human antibody 43B1A8-hIgG on the proliferation of Ba / F3-hTSLPR-hIL7Rα cells. [Figure 4C] FIG. 1 shows the detection of the inhibitory activity of recombinant fully human antibody 90H3H11-hIgG on the proliferation of Ba / F3-hTSLPR-hIL7Rα cells. [Figure 5] FIG. 1 shows detection of the inhibitory activity of recombinant fully human antibodies 43B1A8-hIgG and 90H3H11-hIgG on the secretion of TARC by PBMC. [Figure 6] FIG. 1 shows the detection of thermal stability (Tm) of recombinant fully human antibodies 43B1A8-hIgG and 90H3H11-hIgG. [Figure 7A] FIG. 1 shows the detection of the inhibitory activity of recombinant fully human antibody 43B1-H2L2 on the proliferation of Ba / F3-hTSLPR-hIL7Rα cells. [Figure 7B] FIG. 1 shows the detection of the inhibitory activity of recombinant fully human antibody 43B1-H6L1 on the proliferation of Ba / F3-hTSLPR-hIL7Rα cells. [Figure 8] FIG. 1 shows the detection of the inhibitory activity of recombinant fully human antibody 43B1-H2L2 on the secretion of MDC cytokines by PBMC cells. [Figure 9] FIG. 1 shows pharmacokinetic analysis of recombinant fully human antibody 43B1-H2L2 molecule in cynomolgus monkeys. [Example]

[0174] Sequence information Sequence information relevant to the present invention is set forth in the table below. [Table 1] TIFF2026012690000002.tif226149

[0175] The invention will now be illustrated by reference to the following examples, which are intended to illustrate, but not limit, the invention.

[0176] Unless otherwise indicated, the molecular biology experiments and immunoassay processes used in the present invention are essentially carried out with reference to the processes described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, 1989; and F.M.Ausubel et al., Short Protocols in Molecular Biology, 3rd ed., John Wiley & Sons, Inc., 1995. Those skilled in the art will understand that the examples are intended to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0177] Example 1: Antigen preparation Human TSLP or monkey TSLP was expressed in Escherichia coli or mammalian cells. The amino acid sequence of human TSLP is located under NP_149024.1 in the NCBI protein database. The amino acid sequence of cynomolgus monkey (Macaca fascicularis) TSLP is located under XP_005557555.1 in the NCBI protein database. The antigen used in this application contains TSLP expressed in a modified form, such as a fusion of six consecutive histidines as a tag fused to the C-terminus of the TSLP sequence (TSLP-His). The above human and monkey TSLP sequences were codon-optimized by GenScript (Nanjing) Co., Ltd., synthesized into expression vectors (i.e., plasmids containing the complete coding sequence of human TSLP), and expressed and purified in Escherichia coli or mammalian cells HEK293F.

[0178] The native receptor for TSLP is a heterodimer composed of human TSLPR and human IL7R alpha (IL7Rα) subunits. The sequence of human TSLPR is found in Uniprot: Q9HC73.1, and the sequence of human IL7Rα subunit is found in GenBank: AAR08908.1. The human hIL7Rα-hTSLPR-hFc fusion protein is composed of the extracellular domain of human IL7Rα (E21-D239), the extracellular domain of human TSLPR receptor (Q23-K231), and a portion of the coding sequence for the human IgG1 Fc region (hinge-CH2-CH3) in tandem. This fusion protein was constructed in the expression vector pLVX after codon optimization. A stable HEK293F cell line expressing hIL7Rα-hTSLPR-hFc was established using the pLVX vector, and the hIL7Rα-hTSLPR-hFc protein was finally obtained by purification.

[0179] Example 2: Construction and identification of cell lines overexpressing human TSLPR / IL7Rα co-genes 2.1: Construction of a cell line overexpressing both human TSLPR and IL7Rα genes To verify the efficacy of human TSLP antibodies in blocking the binding of human TSLP to the human TSLPR / IL7Rα receptor, the complete amino acid coding sequence of human TSLPR (Gene ID: UniProtKB / Swiss-Prot: Q9HC73.1, synthesized by GenScript (Nanjing) Co., Ltd.) and the complete amino acid coding sequence of human IL7R alpha subunit (Gene ID: GenBank: AAR08908.1, synthesized by GenScript (Nanjing) Co., Ltd.) were codon-optimized by GenScript (Nanjing) Co., Ltd. and cloned into the lentiviral vectors pLVX-IRES-puro and pLVX-IRES-zeocin, respectively, and transfected into the mouse pro-B cell line Ba / F3 cells (purchased from COBIOER BIOSCIENCES CO., LTD.) as described in MohammadiZ et al., Mol Biotechnol. 2015. After infection with the virus obtained by the preparation method using the lentiviral packaging system described in Sep;57(9):793-800, the cells were selected with puromycin + bleomycin, and single clones were screened to obtain the stable monoclonal cell line Ba / F3-hTSLPR-hIL7Rα.

[0180] 2.2: Detection of Ba / F3 cell lines overexpressing both human TSLPR and IL7Rα genes The cell line was detected by flow cytometry (flow cytometry: Beckman, CytoFlex; detection antibodies: APC-anti-human TSLPR (Biolegend), APC-anti-human IL7Rα (Biolegend)) to determine whether the monoclonal cells properly expressed human TSLPR and human IL7Rα. As shown in Figure 1, the flow cytometry results indicated that Ba / F3-hTSLPR-hIL7Rα was a monoclonal cell line that expressed both genes with good uniformity (almost 100%), which could be used for subsequent experiments.

[0181] Example 3: Mouse immunization and hybridoma fusion 3.1: Immunization of mice Fully human transgenic mice, H2L2 (HarbourBioMed), were immunized multiple times with plasmids containing human TSLP (NP_149024.1) expressed in E. coli, human TSLP (NP_149024.1) expressed in mammalian cells, monkey TSLP (XP_005557555.1) expressed in mammalian cells, and the complete coding sequence of human TSLP. Booster immunizations were administered every two weeks for a total of five to six times. During immunization, serum titers of anti-human TSLP antibodies were detected every two weeks by ELISA (see Example 4.1). After multiple immunizations, mice with the best titers were selected for hybridoma fusion.

[0182] 3.2: Fusion method After mixing the single cell suspensions of spleen and lymph nodes, an equal volume of SP2 / 0 myeloma cells was added and mixed homogeneously. The cell mixture was washed and resuspended in electrofusion buffer. After electrofusion using a BTX-ECM2001 electrofusion device, the cell suspension was immediately transferred from the fusion chamber to complete fusion culture medium and incubated at 37°C for 1 hour. 2 × 10 cells were added per well. 4 Cells were seeded into 96-well plates at a density of 1000 x 1000. After 5 days of culture, the medium was replaced with complete fusion medium, and within 7 to 10 days, the supernatant was collected for hybridoma screening.

[0183] Example 4: Hybridoma screening 4.1: ELISA screening for human TSLP binding Soluble human TSLP-His protein was diluted to 1 μg / mL in 1×CBS coating buffer and then added to a 96-well plate and incubated overnight at 4°C. The 96-well plate was washed with PBST and blocked with blocking solution (PBS + 2% BSA) for 2 hours at 37°C. Hybridoma supernatant or the same volume of blocking solution was added to the plate and incubated in an incubator at 37°C for 2 hours. Goat anti-rat IgG-HRP was added to the 96-well plate and incubated in an incubator at 37°C for 1 hour, then washed and the OD was read at 450 nm.

[0184] 4.2: ELISA screening for monkey TSLP binding Monkey TSLP-His protein was diluted to 1 μg / mL in CBS coating buffer and then added to a 96-well plate and incubated overnight at 4°C. The 96-well plate was washed with PBST and blocked with blocking solution (PBS + 2% BSA) at 37°C for 2 hours. Hybridoma supernatant or the same volume of blocking solution was added to the plate and incubated in an incubator at 37°C for 2 hours. Goat anti-rat IgG-HRP was added to the 96-well plate and incubated in an incubator at 37°C for 1 hour, then washed and the OD was read at 450 nm.

[0185] 4.3: ELISA screening for blocking of human TSLP-His binding to the chimeric receptor IL7Rα-TSLPR-hFc Hybridoma supernatants or purified antibodies were quantified for blocking of human TSLP-His binding to the chimeric receptor IL7Rα-TSLPR-hFc according to the following scheme.

[0186] Soluble hIL7Rα-hTSLPR-hFc protein was diluted with 1×CBS coating buffer and then added to a 96-well plate and incubated overnight at 4°C. The 96-well plate was washed with PBST and blocked with blocking solution (PBS + 2% BSA) at 37°C for 2 hours. hTSLP-His protein (+ / -) recombinantly expressed by mammalian cells was added to the plate together with hybridoma supernatant or the same volume of blocking solution and incubated in an incubator at 37°C for 2 hours. Mouse anti-His-HRP was added to the 96-well plate and incubated in an incubator at 37°C for 1 hour, then washed and the OD was read at 450 nm. Hybridomas with strong inhibition rates were selected as candidate clones.

[0187] Positive clones were selected based on the activity quantified by affinity ELISA and the above-mentioned competitive ELISA, and subcloned by limiting dilution to obtain subclones. Finally, hybridoma subclones were obtained that not only bound to both human TSLP-His and monkey TSLP-His but also blocked the binding of human TSLP-His to the chimeric receptor hIL7Rα-hTSLPR-hFc. As shown in Table 1, five single clones potently bound both human TSLP (hTSLP) and monkey TSLP (cTSLP) and blocked the binding of human TSLP to its receptor hIL7Rα-hTSLPR-hFc by more than 60%. [Table 2]

[0188] Example 5: Preparation of anti-TSLP chimeric antibodies Expression of control antibody: The sequence of the control antibody was referenced in the chEMBL database (ID: CHEMBL3707229). The base sequences of the heavy and light chains of the control antibody were synthesized in a pTT5 expression vector and transiently transfected into CHO-S cells (purchased from Thermo), which were then affinity purified using Protein A (MabSelectSuRe, GE) to obtain the control antibody.

[0189] Single hybridoma clones were cultured in serum-free medium to obtain 50 mL of supernatant, which was then purified using Protein A (MabSelectSuRe, GE) to obtain chimeric hybridoma antibodies. The H2L2 mouse antibody constant region was genetically modified with a rat constant region, resulting in chimeric antibodies with a rat Fc possessing a fully human variable region. The purified antibodies were quantified spectrophotometrically to obtain chimeric antibodies 25A5C5, 27C2B6, 37C2D10, 43B1A8, and 90H3H11.

[0190] Example 6: ELISA detection of the affinity of anti-TSLP chimeric antibodies to TSLP The affinity of chimeric antibodies 25A5C5, 27C2B6, 37C2D10, 43B1A8, and 90H3H11 for human or monkey TSLP was quantified by ELISA. The specific method is summarized as follows: human or monkey TSLP-His antigen was coated onto a 96-well plate, incubated overnight at 4°C, and then various gradient dilutions of antibody were added. After 2 hours of incubation, goat anti-rat Fc-HRP secondary antibody was added. After 1 hour of incubation, the absorbance was read at 450 nm using a microplate reader.

[0191] The results are shown in Table 2. Here, chimeric antibodies 25A5C5 and 27C2B6 each had affinity for human TSLP substantially comparable to that of the control antibody, while 43B1A8 and 90H3H11 each had affinity for human TSLP higher than that of the control antibody. Antibodies 25A5C5 and 90H3H11 did not bind to monkey TSLP. On the other hand, 37C2D10, 27C2B6, and 43B1A8 each strongly bound to monkey TSLP. [Table 3]

[0192] Example 7: Detection of the activity of anti-TSLP chimeric antibodies to block TSLP / hIL7Rα-hTSLPR-hFc binding by competitive ELISA Human TSLP binds to the human hIL7Rα-hTSLPR-hFc heterodimeric receptor and activates downstream signaling pathways. A competitive ELISA was applied to quantify the activity of chimeric antibodies in blocking the binding of the chimeric receptor hIL7Rα-hTSLPR-hFc to antigen. The specific steps are described in Example 4.3. Gradient-diluted chimeric antibodies were added to the plate. The results are shown in Table 3. All five chimeric antibodies effectively blocked the binding of human TSLP to the human hIL7Rα-hTSLPR-hFc heterodimeric receptor. [Table 4]

[0193] Example 8: Inhibition of proliferation of Ba / F3-hTSLPR-hIL7Rα cells by anti-TSLP chimeric antibodies The activity of anti-TSLP chimeric antibodies was detected by inhibiting Ba / F3-hTSLPR-hIL7Rα cell proliferation. The receptor proteins hTSLPR and hIL7Rα were expressed on the cell surface of Ba / F3-hTSLPR-hIL7Rα. Here, a dimer of the extracellular domains of these two receptor proteins can bind to human TSLP, and their intracellular domains can further transduce signals to activate intracellular STAT5 phosphorylation and promote proliferation of Ba / F3-hTSLPR-hIL7Rα cells.

[0194] The specific steps were as follows: an appropriate amount of Ba / F3-hTSLPR-hIL7Rα stable cell line was harvested and centrifuged, and washed twice with 1640 + 10% FBS culture medium to remove recombinant mouse IL3 in the culture medium; hTSLP-His(+ / -) was incubated with purified anti-TSLP chimeric antibody or control anti-TSLP antibody (control antibody) in each well at room temperature for 30 minutes; and 1.5 × 10 Ba / F3-hTSLPR-hIL7Rα cells were added per well. 4 CCK8 (RHINO BIO, QDY-003-D) is added to each well and the OD is read at 450 nm, the data is exported and the inhibition of the chimeric antibody on cell proliferation is analyzed by using PrismGraphpad software.

[0195] As shown in Figures 2A to 2C and Table 4, all five chimeric antibodies clearly inhibited the proliferation of Ba / F3-hTSLPR-hIL7Rα cells. [Table 5]

[0196] Example 9: Amplification of the variable regions of anti-TSLP chimeric antibodies Hybridoma cells were grown in approximately 2 × 10 6The cells were cultured individually and lysed using TRIzol Reagent (Thermo Fisher Sci. Cat# 15596026) to extract RNA. Then, a cDNA reverse transcription kit (Thermo Fisher Sci. Cat# 18080-200) was used to synthesize first-strand cDNA. Referring to the methods of IMGT and AbM and sequence analysis of all mouse antibodies, multiple pairs of upstream primers for the variable regions were designed by selecting regions with high homology, and downstream primers were designed using the CH1 homologous sequence. The light and heavy chain variable regions of the antibodies were obtained by PCR amplification using a primer pool. The PCR products were purified using a DNA purification kit (Qiagen, Cat# 28104) and cloned into the pTT-5 vector. Approximately 10 clones were selected and sequenced for each ligation reaction to obtain the variable region sequences. These were further analyzed using the IMGT and AbM databases. [Table 6]

[0197] Example 10: Expression, purification, and binding affinity detection of recombinant antibodies The amino acid sequences of the light chain variable regions of 25A5C5, 27C2B6, 37C2D10, 43B1A8, and 90H3H11 were fused to the amino acid sequence of the light chain kappa constant region (SEQ ID NO: 16), and the amino acid sequences of these heavy chain variable regions were fused to the amino acid sequence of the heavy chain constant region of IgG1 (SEQ ID NO: 14). Nucleotide sequences corresponding to these sequences were constructed in the pTT5 vector. The pTT5 vectors corresponding to the heavy and light chains of each recombinant fully human antibody were co-transfected into CHO-S (purchased from Thermo). The supernatants were purified using Protein A (MabSelectSuRe, GE). The purified recombinant fully human antibodies were designated 25A5C5-hIgG, 27C2B6-hIgG, 37C2D10-hIgG, 43B1A8-hIgG, and 90H3H11-hIgG, respectively, and their protein concentrations were quantified spectrophotometrically.

[0198] Detection of affinity of anti-TSLP recombinant fully human antibodies to mammalian-expressed recombinant human or monkey TSLP-His. The specific experimental steps are summarized as follows: human TSLP-His or monkey TSLP-His antigen is coated into a 96-well plate, incubated overnight at 4°C, and then serially diluted antibodies are added. After 2 hours of incubation, HRP-labeled goat-human Fc secondary antibody is added, and after 1 hour of incubation, reading is performed at a wavelength of 450 nm.

[0199] The results are shown in Figures 3A and 3B. All five candidate antibodies were able to bind to human TSLP. Furthermore, as shown in Table 6, 37C2D10-hIgG, 43B1A8-hIgG, and 90H3H11-hIgG each had higher affinity for hTSLP than the control antibody. 25A5C5-hIgG and 27C2B6-hIgG each had affinity for hTSLP comparable to that of the control antibody. 27C2B6-hIgG, 37C2D10-hIgG, and 43B1A8-hIgG each had affinity for monkey TSLP comparable to that of the control antibody. [Table 7]

[0200] Example 11: Detection of the dynamic affinity of anti-TSLP fully human antibodies to TSLP The dynamic affinity of anti-TSLP fully human chimeric antibody to TSLP was detected by Fortibio, a commonly used dynamic affinity detector. The method is briefly described as follows: serial dilutions of human or monkey TSLP were carried out using PBST to obtain 100 nM, 50 nM, 25 nM, 12.5 μM, 6.25 nM, 3.125 nM, 1.5625 nM, and 0 nM; ProA biosensor (Pall Life Sciences) was pre-wetted with PBST buffer before use; recombinant fully human antibody was diluted to 5 μg / mL with PBST and immobilized on the ProA sensor; the antibody-immobilized sensor was then placed in PBST buffer and equilibrated for 60 seconds to obtain a baseline, transferred to antigen dilution for 60 seconds to allow binding, and then dissociated in PBST for 180 seconds; after the analysis cycle, the sensor was regenerated with 10 mM Gly (pH 1.5). Date analysis 11.0 version (Pall) was used to determine the association rate constant (Ka) and dissociation rate constant (Kd), which were then used to calculate the dissociation equilibrium constant (KD).

[0201] As shown in Table 7, the KD values ​​of 25A5C5-hIgG, 43B1A8-hIgG, and 90H3H11-hIgG binding to human TSLP were lower than that of the control antibody, indicating strong affinity. This was consistent with the affinity ELISA results. Regarding binding to monkey TSLP, 25A5C5-hIgG had no affinity, while 37C2D10-hIgG, 90H3H11-hIgG, and 43B1A8-hIgG each showed good binding, with 10 -8 M~10 -9 It had M-order affinity. [Table 8]

[0202] Example 12: Inhibition of recombinant fully human antibodies on the proliferation of Ba / F3-hTSLPR-hIL7Rα cells The activity of the recombinant fully human antibody was quantified by the growth inhibition method of Ba / F3-hTSLPR-hIL7Rα cells (see Example 8 above).

[0203] The results are shown in Table 8. Here, as shown in Figures 4A to 4C, 27C2B6-hIgG and 37C2D10-hIgG were able to inhibit human TSLP-induced proliferation of Ba / F3-hTSLPR-hIL7Rα cells, 25A5C5-hIgG and 90H3H11-hIgG were substantially comparable to the control antibody in inhibiting human TSLP-induced proliferation of Ba / F3-hTSLPR-hIL7Rα cells, while 43B1A8-hIgG had significantly more potent inhibitory activity than the control antibody (approximately 0.25-fold). [Table 9]

[0204] Example 13: Detection of the inhibitory activity of recombinant fully human antibodies against human TSLP-induced PBMC secretion of thymus and activation-regulated chemokine (TARC) The functional activity of fully human anti-TSLP antibodies in primary cells was assessed by inhibiting human TSLP-induced secretion of TARC (a Th2-like cytokine) by human PBMC cells. PBMCs are CD11 + DC cells, and studies have also shown that TSLP binds to the TSLPR / IL7Rα receptor and mediates CD11 + Activates DC cells and upregulates OX40L to CD11 + It has been shown that TSLP can stimulate DC cell secretion of Th2-like cytokines (e.g., TARC and CCL22). Anti-TSLP antibodies block the binding of TSLP to TSLPR / IL7Rα on the surface of DC cells, thereby blocking DC cell activation and secretion of Th2-like cytokines.

[0205] The method was briefly described as follows: Human peripheral blood PBMCs were isolated using Ficoll separation solution (GE), and hTSLP-His(+ / -) was incubated with recombinant fully human antibody or control antibody for 30 minutes at room temperature, and 2 × 10 PBMC cells were plated per well. 5The cells were incubated for 48 hours after addition of the ELISA kit. The supernatants were collected and analyzed for human TARC by ELISA to quantify the inhibition of TARC secretion by the hybridoma supernatants or purified antibodies. TARC levels in the supernatants were detected using a TARC ELISA kit (Sino Biological).

[0206] The results are shown in Table 9 and Figure 5. Here, the EC50 values ​​of the fully human antibodies 43B1A8-hIgG and 90H3H11-hIgG were lower than that of the control antibody, indicating that their activity in inhibiting TSLP-induced secretion of TARC by PBMCs was greater than that of the control antibody. [Table 10]

[0207] Example 14: Assay of Tm values ​​of recombinant fully human antibodies The Tm value of the anti-TSLP antibody was measured by differential fluorescence scanning (DSF). The specific experimental steps were as follows: 12.5 μL of 40× SYPRO Orange dye (Life Technologies Co., Ltd., Cat. No. 56651), 5 μL of 1 mg / mL fully human anti-TSLP antibody (diluted with PBS), and 7.5 μL of sterile water were mixed in an EP tube, and the sample mixture was added to a Q-PCR system (AB Applied Biosystems ABI, 7500) for reaction. Q-PCR parameters were set as follows: target (ROX), program (25°C, 3 minutes; 1% ratio, 95°C; 95°C, 2 minutes). The results were input into GraphPrism software to calculate the V50 value. As shown in Figure 6 and Table 10, the Tm values ​​of 43B1A8-hIgG (74.72°C) and 90H3H11-hIgG (72.58°C) were higher than that of the control antibody (66.47°C), clearly indicating that the fully human antibodies prepared according to the present invention had better thermal stability. [Table 11]

[0208] Example 15: Hydrophobicity assay of recombinant fully human antibodies Hydrophobicity comparison was performed using an Agilent 1260 HPLC column with a TOSOH TskgelButy-NPR (2.5) chromatography column. To compare the hydrophobicity differences among the three antibodies, these three antibodies were directly loaded and analyzed. Mobile phase A: 1.5 M (NH4)2SO4; Mobile phase B: 25 mM Na2HPO4 (pH 7.0) + 25% IPA. The retention times of the three antibodies are shown in Table 11. Here, the longer the retention time, the stronger the antibody's hydrophobicity. 90H3H11-hIgG has hydrophilicity comparable to that of the control antibody, while 43B1A8-hIgG has better hydrophilicity than the control antibody, indicating that process development and antibody aggregation are superior to those of the control antibody. [Table 12]

[0209] Example 16: Preparation of recombinant modified fully human antibodies 43B1-H6L1 and 43B1-H2L2 The in vivo half-life of antibodies is closely related to their isoelectric point, affinity for FcRn, glycosylation modification, and immunogenicity. To extend the half-life of the 43B1A8-hIgG antibody, the affinity for FcRn was increased by modifying the amino acid sequences of the heavy and light chains of the antibody.

[0210] Modification scheme 1: hIgG1Fc (SEQ ID NO: 14) was substituted with IgG4 (SEQ ID NO: 70) containing two amino acid mutations: on the one hand, N to A at amino acid position 434 (EU numbering system) of IgG4, and on the other hand, S to P at amino acid position 228 (EU numbering system).

[0211] Modification scheme 2: N was substituted with A at amino acid position 434 (EU numbering system) of hIgG1Fc (SEQ ID NO: 14) to obtain SEQ ID NO: 15, R was mutated to G at position 16 (Chothia numbering system) of FR1 of the heavy chain of 43B1A8-hIgG, and R was mutated to Q at position 79 (Chothia numbering system) of FR3 of the light chain of 43B1A8-hIgG.

[0212] The antibodies obtained by the above modification schemes 1 and 2 were designated 43B1-H6L1 and 43B1-H2L2, respectively.

[0213] The heavy chain variable region sequence of 43B1-H2L2 is SEQ ID NO: 68, its heavy chain constant region sequence is SEQ ID NO: 15, its light chain variable region sequence is SEQ ID NO: 69, and its light chain constant region sequence is SEQ ID NO: 16.

[0214] The heavy chain variable region sequence of 43B1-H6L1 is SEQ ID NO: 40, its heavy chain constant region sequence is SEQ ID NO: 70, its light chain variable region sequence is SEQ ID NO: 41, and its light chain constant region sequence is SEQ ID NO: 16. [Table 13]

[0215] The heavy and light chains of the two modified molecules 43B1-H2L2 and 43B1-H6L1 were constructed in pTT5 expression vectors, respectively. After extracting the plasmids, they were transfected into CHO-S cells (purchased from Thermo). After culturing for approximately 10 days, the cell supernatant was purified using Protein A (MabSelectSuRe, GE). The purified recombinant fully human antibody was quantified for protein content by spectrophotometry.

[0216] Example 17: Dynamic affinity assay of recombinant fully human antibodies 43B1-H6L1 and 43B1-H2L2 against TSLP The dynamic affinity of anti-TSLP fully human antibodies to TSLP was detected using Fortibio. The method is described in Example 11 above. The results are shown in Table 13. Here, the 43B1-H6L1 and 43B1-H2L2 human antibody molecules each had a dynamic affinity for hTSLP that was comparable to that of the control antibody. [Table 14]

[0217] Example 18: Assay of the isoelectric point (PI) of recombinant fully human antibodies 43B1-H6L1 and 43B1-H2L2 The isoelectric points of the 43B1-H6L1, 43B1-H2L2, and 43B1A8-hIgG antibodies were detected by isoelectric focusing, as briefly described below: A Maurice isoelectric focusing system (ProteinSimple) was used in conjunction with a capillary cartridge for analysis. To compare the differences in the isoelectric points of the three antibodies, the three antibodies were diluted with water and a pH gradient was formed by using ampholytes 3-10 (GE) at a final concentration of 4% in the detection system. The results are shown in Table 14. Here, the isoelectric points of 43B1-H6L1 and 43B1-H2L2 were 1.3 and 0.4 lower than that of 43B1A8-hIgG, respectively, indicating that the modification scheme was successful. [Table 15]

[0218] Example 19: Dynamic affinity assay of recombinant fully human antibodies 43B1-H6L1 and 43B1-H2L2 for FcRn The dynamic affinity of the modified anti-TSLP fully human antibody for FcRn was detected using Fortibio. The method is briefly described as follows: serial dilutions of the anti-TSLP antibody were performed using PBST (pH 6.0) to obtain 200 nM, 100 nM, 50 nM, 25 nM, 12.5 μM, 6.25 nM, 3.125 nM, 1.5625 nM, and 0 nM; SA biosensors (Pall Life Sciences) were pre-wetted with PBST (pH 6.0) buffer before use; biotin-labeled FcRn was diluted to 2.3 μg / mL and immobilized on the SA sensor; the FcRn-immobilized sensor was then placed in PBST (pH 6.0) buffer and equilibrated for 60 seconds to obtain a baseline, transferred to the antibody diluent for 60 seconds to allow binding, and then dissociated in PBST (pH 6.0) for 60 seconds; after the analysis cycle, the sensor was regenerated with PBST (pH 7.4). Date analysis version 11.0 (Pall) using a 1:1 model was used to determine the association rate constant (Ka) and dissociation rate constant (Kd), which were then used to calculate the dissociation equilibrium constant (KD).

[0219] As shown in Table 15, 43B1-H6L1 and 43B1-H2L2 each had a lower dynamic affinity (KD) for FcRn than that of the control antibody, and exhibited an affinity for FcRn that was approximately twice that of the control antibody. [Table 16]

[0220] Example 20: Assay of the inhibitory activity of recombinant fully human antibodies 43B1-H6L1 and 43B1-H2L2 on the proliferation of Ba / F3-hTSLPR-hIL7Rα cells The activity of recombinant fully human antibodies 43B1-H6L1 and 43B1-H2L2 was detected by the Ba / F3 cell proliferation inhibition method. The method is as described in Example 8 above, and the antibody activity EC50 values ​​were analyzed based on the data. The results are shown in Table 16 and Figures 7A and 7B. Here, 43B1-H2L2 and 43B1-H6L1 were able to inhibit human TSLP-induced proliferation of Ba / F3-hTSLPR-hIL7Rα cells, and these EC50 values ​​were comparable to those of the control antibody, indicating that the fully human antibodies 43B1-H2L2 and 43B1-H6L1 each had comparable abilities to inhibit the cellular activity of TSLP. [Table 17]

[0221] Example 21: Detection of the inhibitory activity of recombinant fully human antibodies against human TSLP-induced PBMC secretion of macrophage-derived chemokine (MDC) The functional activity of anti-TSLP fully human antibodies in primary cells was assessed by inhibiting human TSLP-induced secretion of MDC by human PBMC cells. PBMCs contain DC cells. Studies have shown that TSLP can activate DC cells by binding to the TSLPR / IL7Rα receptor on the cell surface and upregulate OX40L, promoting DC secretion of Th2-like cytokines (e.g., TARC and MDC). Anti-TSLP antibodies can block the binding of TSLP to TSLPR / IL7Rα on the surface of DC cells, thereby blocking DC activation and secretion of Th2-like cytokines by DC cells.

[0222] The method was briefly described as follows: Human peripheral blood PBMCs were isolated using Ficoll separation solution (GE), and hTSLP-His(+ / -) was incubated with recombinant fully human antibody or control antibody for 30 minutes at room temperature, and 2 × 10 PBMC cells were plated per well. 5The cells were incubated for 120 hours after addition of 1000 μg of TARC. The supernatants were collected and analyzed for human MDC production by ELISA to measure antibody inhibition of MDC secretion. TARC levels in the supernatants were detected using an MDC ELISA kit (Raybiotech).

[0223] The results are shown in Table 17 and Figure 8, where 43B1-H2L2 and the control antibody were able to significantly inhibit TSLP-induced production of MDC, with EC50 values ​​of 7.84 pM and 5.63 pM, respectively. [Table 18]

[0224] Example 22: In vivo pharmacokinetic analysis of recombinant fully human antibodies in cynomolgus monkeys The sequence of the candidate molecule 43B1-H2L2 of the present application contains the N434A mutation, which affects its affinity for FcRn and thereby extends its in vivo drug metabolic half-life. Therefore, in this example, the pharmacokinetics of the 43B1-H2L2 molecule in cynomolgus monkeys in vivo was tested by subcutaneous injection and compared with that of a control antibody. The method and results were as follows: Four cynomolgus monkeys (Macaca fascicularis, available from Hainan Jingang Biotech Co., Ltd.) were selected and divided into two groups, one male and one female per group. The subcutaneous injection dose was 5 mg / kg. Blood samples were collected at 0, 5 minutes, 30 minutes, 2 hours, 4 hours, 8 hours, 1 day, 2 days, 3 days, 4 days, 7 days, 10 days, 14 days, 21 days, 28 days, 35 days, 42 days, 49 days, and 56 days after administration, allowed to clot at room temperature for 1 hour, and centrifuged to obtain serum samples, which were then frozen at -80°C and tested. Antibody concentrations in serum were determined by ELISA, and the results were analyzed as follows: Pharmacokinetic parameters and curves for a single subcutaneous administration are shown in Table 18 and Figure 9. These results indicated that the 43B1-H2L2 antibody had a longer half-life and a high AUC of blood concentration, approximately 1-fold higher than that of the control antibody. [Table 19]

[0225] Although specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes may be made to the details in accordance with all published teachings, which teachings also fall within the scope of the present invention, the scope of protection of which is defined by the appended claims and any equivalents thereof.

Claims

1. An antibody or antigen-binding fragment thereof that binds to thymic stromal lymphopoietin (TSLP), comprising the following complementarity-determining regions (CDRs): (a) CDR-H1 or a sequence variant thereof, CDR-H2 or a sequence variant thereof, and CDR-H3 or a sequence variant thereof, which are contained in the heavy chain variable region (VH) set forth in SEQ ID NO: 1, 17, 30, 40, 53, or 68; and / or (b) CDR-L1 or a sequence variant thereof, CDR-L2 or a sequence variant thereof, and CDR-L3 or a sequence variant thereof, which are contained in the light chain variable region (VL) of SEQ ID NO: 2, 18, 31, 41, 54, or 69. Including, Preferably, the sequence variant is a CDR having one or more amino acid substitutions, deletions or additions (e.g., 1, 2 or 3 amino acid substitutions, deletions or additions) compared to the CDR from which it is derived, and preferably the substitutions are conservative substitutions.

2. (1) VH and / or VL as defined by the IMGT numbering system, (1a) the VH comprises CDR-H1 having the sequence of SEQ ID NO: 3, CDR-H2 having the sequence of SEQ ID NO: 4, and CDR-H3 having the sequence of SEQ ID NO: 5; and / or the VL comprises a CDR-L1 having the sequence of SEQ ID NO: 6, a CDR-L2 having the sequence of SEQ ID NO: 7, and a CDR-L3 having the sequence of SEQ ID NO: 8; (1b) the VH comprises CDR-H1 having the sequence of SEQ ID NO: 19, CDR-H2 having the sequence of SEQ ID NO: 20, and CDR-H3 having the sequence of SEQ ID NO: 21; and / or the VL comprises a CDR-L1 having the sequence of SEQ ID NO: 22, a CDR-L2 having the sequence of SEQ ID NO: 23, and a CDR-L3 having the sequence of SEQ ID NO: 24; (1c) the VH comprises CDR-H1 having the sequence of SEQ ID NO: 32, CDR-H2 having the sequence of SEQ ID NO: 33, and CDR-H3 having the sequence of SEQ ID NO: 34; and / or the VL comprises a CDR-L1 having the sequence of SEQ ID NO: 35, a CDR-L2 having the sequence of SEQ ID NO: 23, and a CDR-L3 having the sequence of SEQ ID NO: 24; (1d) the VH comprises CDR-H1 having the sequence of SEQ ID NO: 42, CDR-H2 having the sequence of SEQ ID NO: 43, and CDR-H3 having the sequence of SEQ ID NO: 44; and / or the VL comprises a CDR-L1 having the sequence of SEQ ID NO: 45, a CDR-L2 having the sequence of SEQ ID NO: 46, and a CDR-L3 having the sequence of SEQ ID NO: 47; or (1e) the VH comprises CDR-H1 having the sequence of SEQ ID NO: 55, CDR-H2 having the sequence of SEQ ID NO: 56, and CDR-H3 having the sequence of SEQ ID NO: 57; and / or VH and / or VL, wherein the VL comprises CDR-L1 having the sequence of SEQ ID NO: 58, CDR-L2 having the sequence of SEQ ID NO: 59, and CDR-L3 having the sequence of SEQ ID NO: 60; (2) VH and / or VL, as defined by the AbM numbering system: (2a) the VH comprises CDR-H1 having the sequence of SEQ ID NO: 9, CDR-H2 having the sequence of SEQ ID NO: 10, and CDR-H3 having the sequence of SEQ ID NO: 11; and / or the VL comprises a CDR-L1 having the sequence of SEQ ID NO: 12, a CDR-L2 having the sequence of SEQ ID NO: 13, and a CDR-L3 having the sequence of SEQ ID NO: 8; (2b) the VH comprises CDR-H1 having the sequence of SEQ ID NO: 25, CDR-H2 having the sequence of SEQ ID NO: 26, and CDR-H3 having the sequence of SEQ ID NO: 27; and / or the VL comprises a CDR-L1 having the sequence of SEQ ID NO: 28, a CDR-L2 having the sequence of SEQ ID NO: 29, and a CDR-L3 having the sequence of SEQ ID NO: 24; (2c) the VH comprises CDR-H1 having the sequence of SEQ ID NO: 36, CDR-H2 having the sequence of SEQ ID NO: 37, and CDR-H3 having the sequence of SEQ ID NO: 38; and / or the VL comprises a CDR-L1 having the sequence of SEQ ID NO: 39, a CDR-L2 having the sequence of SEQ ID NO: 29, and a CDR-L3 having the sequence of SEQ ID NO: 24; (2d) the VH comprises CDR-H1 having the sequence of SEQ ID NO: 48, CDR-H2 having the sequence of SEQ ID NO: 49, and CDR-H3 having the sequence of SEQ ID NO: 50; and / or the VL comprises a CDR-L1 having the sequence of SEQ ID NO: 51, a CDR-L2 having the sequence of SEQ ID NO: 52, and a CDR-L3 having the sequence of SEQ ID NO: 47; or (2e) the VH comprises CDR-H1 having the sequence of SEQ ID NO: 61, CDR-H2 having the sequence of SEQ ID NO: 62, and CDR-H3 having the sequence of SEQ ID NO: 63; and / or a VH and / or VL, wherein the VL comprises a CDR-L1 having the sequence of SEQ ID NO: 64, a CDR-L2 having the sequence of SEQ ID NO: 65, and a CDR-L3 having the sequence of SEQ ID NO: 60; and / or (3) A VH and / or VL in which at least one CDR comprises a mutation when compared to the VH and / or VL of any one of (1a), (1b), (1c), (1d), (1e), or (2a), (2b), (2c), (2d), (2e), wherein the mutation is a substitution, deletion, or addition of one or more amino acids, or any combination thereof (e.g., a substitution, deletion, or addition of one, two, or three amino acids, or any combination thereof), preferably a conservative substitution; The antibody or antigen-binding fragment thereof according to claim 1, which preferably binds to human TSLP and / or monkey TSLP.

3. (a) a VH set forth in SEQ ID NO: 1, 17, 30, 40, 53, or 68, and / or a VL set forth in any of SEQ ID NO: 2, 18, 31, 41, 54, or 69 (b) a VH having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any VH of (a), and / or a VL having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any VL of (a); or (c) a VH having one or more amino acid substitutions, deletions, or additions, or any combination thereof (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, or additions, or any combination thereof) compared to any VH in (a), and / or a VL having one or more amino acid substitutions, deletions, or additions, or any combination thereof (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, or additions, or any combination thereof) compared to any VL in (a), wherein preferably the substitutions are conservative substitutions.

4. (a) VH set forth in SEQ ID NO: 1 and VL set forth in SEQ ID NO: 2; (b) VH set forth in SEQ ID NO: 17 and VL set forth in SEQ ID NO: 18; (c) VH set forth in SEQ ID NO: 30 and VL set forth in SEQ ID NO: 31; (d) VH set forth in SEQ ID NO: 40 and VL set forth in SEQ ID NO: 41; (e) VH set forth in SEQ ID NO: 53 and VL set forth in SEQ ID NO: 54; (f) VH set forth in SEQ ID NO: 68 and VL set forth in SEQ ID NO: 69; (g) a VH and VL, wherein the VH has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, and / or the VL has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, to the VH and VL in any one of (a) to (f); or (h) VH and VL in any one of (a) to (f), wherein VH has one or more amino acid substitutions, deletions, or additions, or any combination thereof (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, or additions, or any combination thereof), and / or VL has one or more amino acid substitutions, deletions, or additions, or any combination thereof (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, or additions, or any combination thereof), and preferably the substitutions are conservative substitutions. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, comprising:

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which is a chimeric antibody, a humanized antibody, or a fully human antibody.

6. (a) a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof, and / or (b) a human immunoglobulin light chain constant region (CL) or a variant thereof wherein the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the wild-type sequence from which it is derived; or wherein the variant has one or more amino acid substitutions, deletions or additions, or any combination thereof (e.g. up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions, or any combination thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions, or any combination thereof) compared to the wild-type sequence from which it is derived, and preferably the substitutions are conservative substitutions; Preferably, the heavy chain constant region is an IgG heavy chain constant region, e.g., an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region; Preferably, it comprises the heavy chain constant region of human IgG1, The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the light chain constant region is preferably a kappa or lambda light chain constant region, more preferably a human kappa light chain constant region.

7. The heavy chain constant region or variant thereof is (1) The variant is mutated at at least one of positions 234, 235, 237, 265, 297, 331, 329, and 434 according to the EU numbering system, and preferably the variant is mutated at any of the following positions: L234A, L235A, D265A, N297A, L234F, L235E, P331S, P329G, N434A, N434Y, N434F, N434W, N434S, N434G, N434H, and N434 Q, more preferably the variant comprises at least one of the following mutations: L234A, L235A, G237A and N434A, more preferably the IgG1 variant comprises the mutations L234A, L235A and G237A, more preferably the IgG1 variant comprises the mutations L234A, L235A, G237A and N434A, or (2) CH according to SEQ ID NO: 14 or a variant thereof, wherein the variant comprises up to 20 amino acid conservative substitutions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid conservative substitutions, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid conservative substitutions) compared to SEQ ID NO: 14, or has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 14, wherein the variant comprises N297A and / or N434A according to the EU numbering system, preferably wherein the variant comprises N434A; (3) A CH as set forth in SEQ ID NO: 15 or a variant thereof, comprising up to 20 amino acid conservative substitutions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid conservative substitutions, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid conservative substitutions) compared to SEQ ID NO: 15, or having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 15; or (4) A heavy chain constant region of human IgG4 or a variant thereof, wherein the variant is mutated at at least one of positions 228 and / or 434 according to the EU numbering system, preferably the variant includes S228P and / or N434A, preferably the variant includes S228P and N434A; or (5) The variant comprises a CH as set forth in SEQ ID NO: 70 or a variant thereof, which comprises up to 20 amino acid conservative substitutions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid conservative substitutions, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid conservative substitutions) compared to SEQ ID NO: 70, or has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 70; and / or The light chain constant region or variant thereof is (6) a kappa light chain constant region, or (7) The variant comprises a light chain constant region (CL) set forth in SEQ ID NO: 16 or a variant thereof, which comprises up to 20 amino acid conservative substitutions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid conservative substitutions, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid conservative substitutions) compared to SEQ ID NO: 16, or has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 16; Preferably, it comprises a heavy chain constant region (CH) set forth in SEQ ID NO: 14 and a light chain constant region (CL) set forth in SEQ ID NO: 16, Preferably, it comprises a heavy chain constant region (CH) set forth in SEQ ID NO: 15 and a light chain constant region (CL) set forth in SEQ ID NO: 16, Preferably, it comprises a heavy chain constant region (CH) set forth in SEQ ID NO: 70 and a light chain constant region (CL) set forth in SEQ ID NO: 16, Preferably, the antibody or antigen-binding fragment thereof described in claim 6 has no or reduced ADCP, ADCC and / or CDC activity due to the mutation or substitution compared to a corresponding antibody or antigen-binding fragment thereof that does not contain the mutation or substitution.

8. The antibody, (a) a heavy chain comprising a VH set forth in SEQ ID NO: 1 and a CH set forth in SEQ ID NO: 14, 15 or 70, and a light chain comprising a VL set forth in SEQ ID NO: 2 and a CL set forth in SEQ ID NO: 16; (b) a heavy chain comprising a VH set forth in SEQ ID NO: 17 and a CH set forth in SEQ ID NO: 14, 15 or 70, and a light chain comprising a VL set forth in SEQ ID NO: 18 and a CL set forth in SEQ ID NO: 16; (c) a heavy chain comprising a VH set forth in SEQ ID NO: 30 and a CH set forth in SEQ ID NO: 14, 15 or 70, and a light chain comprising a VL set forth in SEQ ID NO: 31 and a CL set forth in SEQ ID NO: 16; (d) a heavy chain comprising a VH set forth in SEQ ID NO: 40 and a CH set forth in SEQ ID NO: 14, 15 or 70, and a light chain comprising a VL set forth in SEQ ID NO: 41 and a CL set forth in SEQ ID NO: 16; (e) a heavy chain comprising a VH set forth in SEQ ID NO: 53 and a CH set forth in SEQ ID NO: 14, 15 or 70, and a light chain comprising a VL set forth in SEQ ID NO: 54 and a CL set forth in SEQ ID NO: 16; (f) a heavy chain comprising a VH set forth in SEQ ID NO: 68 and a CH set forth in SEQ ID NO: 14, 15, or 70, and a light chain comprising a VL set forth in SEQ ID NO: 69 and a CL set forth in SEQ ID NO: 16; or (g) a heavy chain and a light chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, and / or a light chain having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, to the heavy chain and the light chain in any one of (a) to (f). The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, comprising:

9. (a) the heavy chain is (i) a sequence set forth in SEQ ID NO: 66 or 73; (ii) a sequence having one or more amino acid substitutions, deletions or additions, or any combination thereof, compared to the sequence of (i) (e.g., up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions, or any combination thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions, or any combination thereof); or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of (i); The light chain (iv) the sequence set forth in SEQ ID NO: 67; (v) a sequence having one or more amino acid substitutions, deletions or additions, or any combination thereof, compared to the sequence of (iv) (e.g., up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions, or any combination thereof, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions, or any combination thereof); or (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of (iv); Preferably, the substitutions in (ii) or (v) are conservative substitutions, or (b) the heavy chain is (i) the sequence set forth in SEQ ID NO: 71; (ii) a sequence having one or more amino acid substitutions, deletions or additions, or any combination thereof, compared to the sequence of (i) (e.g., up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions, or any combination thereof, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions, or any combination thereof); or (iii) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of (i); The light chain (iv) the sequence set forth in SEQ ID NO: 72; (v) a sequence having one or more amino acid substitutions, deletions or additions, or any combination thereof, compared to the sequence of (iv) (e.g., up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10 or up to 5 amino acid substitutions, deletions or additions, or any combination thereof, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, deletions or additions, or any combination thereof); or (vi) a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of (iv); The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, comprising a heavy chain and a light chain, wherein the substitution (ii) or (v) is preferably a conservative substitution.

10. scFv, Fab, Fab', F(ab') ２ 10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, which is selected from the group consisting of an Fv fragment, a disulfide-linked Fv (dsFv), and a diabody.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, which carries a marker, preferably a detectable marker, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance) or biotin.

12. The following characteristics: (a) a K of less than about 50 nM, e.g., about 20 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, 1 pM, 0.1 pM or less Ｄ Binding to TSLP (e.g., human TSLP) by K Ｄ is measured by Fortibio or ELISA. (b) binding to TSLP (e.g., human TSLP) with an EC50 of less than about 50 nM, e.g., about 20 nM, 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.01 nM, 1 pM, 0.1 pM or less, wherein the EC50 is measured by flow cytometry or ELISA; (c) inhibiting the binding of TSLP to IL7Rα / TSLPR with an IC50 of about 50 nM, e.g., about 50 nM, 20 nM, 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.01 nM, 1 pM, 0.1 pM or less, wherein the IC50 is measured by ELISA. (d) inhibiting or blocking TSLP-induced activity and / or proliferation of mast cells, DCs, NKT cells; (e) inhibiting or blocking TSLP-induced OX40L expression; (f) inhibiting or blocking TSLP-induced osteoprotegerin (OPG) secretion; (g) inhibiting or blocking TSLP-induced secretion of Th2 cytokines, e.g., TARC, CCL22, IL-4, IL-13, or IL-5; (h) having good affinity for binding to FcRn; (i) having an isoelectric point (PI) of about 6.5 to about 8.5, e.g., about 6.5, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.7, about 7.9, about 8.0, about 8.2, or about 8.5; The antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, wherein the antibody or antigen-binding fragment exhibits at least one of the following:

13. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 12.

14. A vector comprising the isolated nucleic acid molecule of claim 13, preferably a cloning vector or an expression vector.

15. 15. A host cell comprising the isolated nucleic acid molecule of claim 13 or the vector of claim 14.

16. 16. A method for preparing the antibody or antigen-binding fragment thereof of any one of claims 1 to 12, comprising culturing the host cell of claim 15 under conditions that allow expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

17. The antibody or antigen-binding fragment thereof that binds to TSLP according to any one of claims 1 to 12, and an additional antibody or fragment thereof or antibody mimetic, A multispecific antibody, preferably a bispecific antibody or a trispecific antibody or a tetraspecific antibody.

18. 13. A conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 12 and a coupling moiety, wherein the coupling moiety is a detectable marker, such as a radioisotope, a fluorescent substance, a luminescent substance, a chromogenic substance, or an enzyme, or the coupling moiety is a therapeutic agent.

19. 19. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 12, the nucleic acid of claim 13, the vector of claim 14, the host cell of claim 15, the multispecific antibody of claim 17, and / or the conjugate of claim 18, and a pharmaceutically acceptable carrier and / or excipient.

20. The following biological activities in a subject: (a) inhibiting or blocking the binding of TSLP to IL7Rα-TSLPR; (b) downregulating or eliminating the activity of TSLP; (c) downregulation or blocking of OX40L expression; (d) inhibiting or blocking TSLP-induced activation and / or proliferation of mast cells, DCs, NKT cells; (e) inhibiting or blocking TSLP-induced osteoprotegerin (OPG) secretion; (f) inhibiting or blocking TSLP-induced secretion of Th2 cytokines, e.g., TARC, CCL22, IL-4, IL-13, or IL-5. The pharmaceutical composition according to claim 19, wherein the pharmaceutical composition is used in at least one of the following conditions:

21. 21. A kit comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, and / or a nucleic acid according to claim 13, and / or a vector according to claim 14, and / or a host cell according to claim 15, a multispecific antibody according to claim 17, and / or a conjugate according to claim 18, and / or a pharmaceutical composition according to claim 19 or 20, and optionally instructions for use.

22. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, and / or a nucleic acid according to claim 13, and / or a vector according to claim 14, and / or a host cell according to claim 15, a multispecific antibody according to claim 17, and / or a conjugate according to claim 18, and / or a pharmaceutical composition according to claim 19 or 20 in the preparation of a medicament for the prevention and / or treatment of allergic inflammation or an autoimmune disease, Optionally, the allergic inflammation is selected from at least one of the group consisting of asthma, idiopathic pulmonary fibrosis, atopic dermatitis (AD), allergic conjunctivitis, allergic rhinitis (AR), Netherton syndrome, eosinophilic esophagitis (EOE), food allergy, allergic diarrhea, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis (ABPA), allergic fungal sinusitis, rheumatoid arthritis, chronic obstructive pulmonary disease (COPD), systemic sclerosis, keloids, ulcerative colitis, chronic rhinosinusitis (CRS) and nasal polyps, chronic eosinophilic pneumonia, eosinophilic bronchitis; Churg-Strauss syndrome, eosinophilia, eosinophilic granulomatosis with polyangiitis, inflammatory bowel disease, urticaria, systemic mastocytosis, cutaneous mastocytosis, and recurrent idiopathic angioedema; Optionally, the autoimmune disease is selected from the group consisting of diabetes, myasthenia gravis, gastritis, pemphigus, primary biliary cirrhosis, multiple sclerosis, lupus, colitis, rheumatic disease, psoriasis, and thyroid disease; Optionally, but not limited to, immunosuppressants (e.g., corticosteroids, nonsteroidal glucocorticoid receptor agonists, leukotriene D4 antagonists, leukotriene B4 antagonists, A2A agonists, A2B antagonists, dopamine receptor agonists, pirfenidone, nintedanib, or avB6 antagonists), bronchodilators (e.g., beta-2 adrenergic receptor agonists, muscarinic antagonists, short-acting beta-2 receptor agonists, long-acting beta-2 receptor agonists, short-acting anticholinergics, methylxanthines, long-acting anticholinergics), other cytokines or cytokine receptor antagonists the administration of said medicament in combination with one or more additional therapeutic agents selected from an IL-13 antagonist, an IL-6 antagonist, an IL-1, IL-33, IL-25 or TNFalpha antagonist, an anti-IgE antibody, an anti-IL31 antibody, an anti-IL31R antibody, an anti-IL13 antibody, an anti-endoglin antibody, an anti-IL1b antibody, another anti-TSLP antibody or an anti-hTSLPR antibody; an antibiotic; radiation therapy; a leukotriene antagonist (e.g., montelukast, zafirlukast or pranlukast); a PDE4 inhibitor (e.g., roflumilast, xanthene); an antihistamine; or an antitussive; Optionally, the antibody or antigen-binding fragment thereof is administered sequentially or simultaneously with an additional therapeutic agent.

23. 20. A method for at least one of (a) inhibiting or blocking binding of TSLP to IL7Rα-TSLPR, (b) downregulating or ablating the activity of TSLP, (c) downregulating or blocking OX40L expression, (d) inhibiting or blocking TSLP-induced activation and / or proliferation of mast cells, DCs, NKT cells, (e) inhibiting or blocking TSLP-induced osteoprotegerin (OPG) secretion, (f) inhibiting or blocking TSLP-induced secretion of Th2 cytokines, such as TARC, CCL22, IL-4, IL-13 or IL-5, in vivo / in vitro, comprising the step of administering an effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, the nucleic acid according to claim 13, the vector according to claim 14, the host cell according to claim 15, the multispecific antibody according to claim 17, the conjugate according to claim 18, or the pharmaceutical composition according to claim 19 or 20, Optionally, an additional therapeutic agent is administered simultaneously with, before, or after administration of the antibody or antigen-binding fragment thereof, the nucleic acid, the vector, the host cell, the multispecific antibody, the conjugate, or the pharmaceutical composition.

24. 21. A method for preventing and / or treating allergic inflammation or an autoimmune disease in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 19 or 20, wherein the subject is a mammal, preferably the subject is a human; Optionally, an additional therapeutic agent is administered simultaneously with, before, or after administration of said pharmaceutical composition; Optionally, the allergic inflammation is selected from at least one of the group consisting of asthma, idiopathic pulmonary fibrosis, atopic dermatitis (AD), allergic conjunctivitis, allergic rhinitis (AR), Netherton syndrome, eosinophilic esophagitis (EOE), food allergy, allergic diarrhea, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis (ABPA), allergic fungal sinusitis, rheumatoid arthritis, COPD, systemic sclerosis, keloids, ulcerative colitis, chronic rhinosinusitis (CRS) and nasal polyps, chronic eosinophilic pneumonia, eosinophilic bronchitis; Churg-Strauss syndrome, eosinophilia, eosinophilic granulomatosis with polyangiitis, inflammatory bowel disease, urticaria, systemic mastocytosis, cutaneous mastocytosis, and recurrent idiopathic angioedema; Optionally, the autoimmune disease is selected from the group consisting of diabetes, myasthenia gravis, gastritis, pemphigus, primary biliary cirrhosis, multiple sclerosis, lupus, colitis, rheumatic disease, psoriasis, and thyroid disease; Optionally, the additional therapeutic agent includes, but is not limited to, an immunosuppressant (e.g., a corticosteroid, a nonsteroidal glucocorticoid receptor agonist, a leukotriene D4 antagonist, a leukotriene B4 antagonist, an A2A agonist, an A2B antagonist, a dopamine receptor agonist, pirfenidone, nintedanib, or an avB6 antagonist), a bronchodilator (e.g., a beta-2 adrenergic receptor agonist, a muscarinic antagonist, a short-acting beta-2 receptor agonist, a long-acting beta-2 receptor agonist, a short-acting anticholinergic, a methylxanthine, a long-acting anticholinergic), etc. a cytokine or cytokine receptor antagonist or antibody (e.g., an IL-13 antagonist, an IL-6 antagonist, an IL-1, IL-33, IL-25 or TNFalpha antagonist, an anti-IgE antibody, an anti-IL31 antibody, an anti-IL31R antibody, an anti-IL13 antibody, an anti-endoglin antibody, an anti-IL1b antibody, another anti-TSLP antibody or an anti-hTSLPR antibody), an antibiotic, radiation therapy, a leukotriene antagonist (e.g., montelukast, zafirlukast or pranlukast), a PDE4 inhibitor (e.g., roflumilast, xanthene), an antihistamine or an antitussive.

25. 13. A method for detecting the presence or level of TSLP in a sample, comprising the steps of contacting the sample with the antibody or antigen-binding fragment thereof of any one of claims 1 to 12 under conditions that allow the formation of a complex between the antibody or antigen-binding fragment thereof and TSLP, and detecting the formation of said complex.

26. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, or the nucleic acid according to claim 13, or the vector according to claim 14, or the host cell according to claim 15, or the multispecific antibody according to claim 17, or the conjugate according to claim 18, or the pharmaceutical composition according to claim 19 or 20, in the preparation of a medicament or kit for diagnosing allergic inflammation or an autoimmune disease, Optionally, the allergic inflammation is selected from at least one of the group consisting of asthma, idiopathic pulmonary fibrosis, atopic dermatitis (AD), allergic conjunctivitis, allergic rhinitis (AR), Netherton syndrome, eosinophilic esophagitis (EOE), food allergy, allergic diarrhea, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis (ABPA), allergic fungal sinusitis, rheumatoid arthritis, COPD, systemic sclerosis, keloids, ulcerative colitis, chronic rhinosinusitis (CRS) and nasal polyps, chronic eosinophilic pneumonia, eosinophilic bronchitis; Churg-Strauss syndrome, eosinophilia, eosinophilic granulomatosis with polyangiitis, inflammatory bowel disease, urticaria, systemic mastocytosis, cutaneous mastocytosis, and recurrent idiopathic angioedema; Optionally, the autoimmune disease is selected from the group consisting of diabetes, myasthenia gravis, gastritis, pemphigus, primary biliary cirrhosis, multiple sclerosis, lupus, colitis, rheumatic diseases, psoriasis, and thyroid diseases.

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