Anti-tslp antibodies and uses thereof

Abstract

Antibodies or antigen-binding fragments thereof against TSLP, nucleic acid molecules encoding them, methods of making them. The anti-TSLP antibodies or antigen-binding fragments thereof have high affinity for TSLP, are capable of effectively binding TSLP and blocking the proliferative effect of TSLP on Ba / F3-hTSLPR-hIL7Rα cells, and blocking the ability of TSLP to activate, secrete cytokines, PBMCs. Further relates to pharmaceutical compositions comprising the antibodies or antigen-binding fragments thereof, and their use in the preparation of drugs for the prevention and / or treatment of asthma, allergic inflammation, anaphylaxis or autoimmune diseases.

Description

Technical Field

[0001] This invention belongs to the field of therapeutic monoclonal antibodies, and more specifically, this invention relates to an antibody against TSLP, and also to the use of said antibody in the treatment of diseases. Technical Background

[0002] Thymic stromal lymphopoietin (TSLP) is an inflammatory cytokine similar to IL-7. TSLP responds to microorganisms, physical damage, or inflammatory cytokines (such as IL-1β and TNF) and is primarily secreted by epithelial cells of the skin, lungs, thymus, and gastrointestinal tract. Under pathological conditions such as inflammation, stromal cells, keratinocytes, dendritic cells (DCs), and mast cells can also secrete TSLP. TSLP plays an important role in the initial triggering of allergic and adaptive airway inflammation. Compared with healthy controls, TSLP is highly expressed in the airways of asthmatic patients, and its level is directly correlated with the expression of TH2 cytokines and chemokines, as well as the severity of the disease. TSLP can induce dendritic cell (DC) maturation and upregulate OX40L expression. The interaction between OX40 and OX40L participates in nascent T cell-induced TH2 cell polarization. After differentiation, these cells release cytokines such as IL-4, IL-5, and IL-13, causing mast cell and eosinophil infiltration and a series of allergic inflammatory responses, leading to pathological changes in the airways and thus triggering asthma attacks. TSLP can effectively activate mast cells and natural killer T (NKT) cells, producing TH2-type cytokines such as IL-13, exacerbating the occurrence and development of airway inflammation.

[0003] The TSLP receptor is a heterodimeric receptor complex composed of IL-7Rα and a unique TSLPR chain (CRFL2). Binding of the TSLP heterodimeric receptor induces STAT5 activation and cell proliferation. DC cells highly express TSLPR and IL-7Rα.

[0004] Therefore, it is urgent and necessary to develop anti-TSLP antibodies with high specificity and affinity, low toxicity and side effects, and excellent clinical efficacy, which will provide asthma patients with more medication options. Summary of the Invention

[0005] In this invention, the inventors first developed a chimeric antibody with excellent properties capable of binding to human TSLP. Based on this, the inventors further studied and modified the chimeric antibody, developing a fully human antibody derived from it. The fully human antibody of this invention possesses essentially the same (or even better) biological functions as the chimeric antibody. It not only has a strong affinity for TSLP but also effectively blocks the proliferative effect of TSLP on Ba / F3 cells, as well as the ability of TSLP to activate PBMCs and secrete cytokines. This invention further relates to pharmaceutical compositions comprising the antibody or its antigen-binding fragment, and their use in the preparation of medicaments for the prevention and / or treatment of asthma, allergic inflammation, allergic reactions, or autoimmune diseases.

[0006] The antibodies of this invention have a very high degree of humanization, even being fully human antibodies, thus they can be safely administered to human subjects without inducing immunogenic reactions. Therefore, the antibodies of this invention have significant clinical value.

[0007] The antibody of the present invention

[0008] In one aspect, the present invention provides an antibody or antigen-binding fragment thereof that binds to TSLP, said antibody or antigen-binding fragment comprising a complementarity-determining region (CDR):

[0009] (a) CDR-H1 contained in the heavy chain variable region (VH) shown in SEQ ID NO: 1, 17, 30, 40, 53 or 68

[0010] or variants thereof, CDR-H2 or variants thereof, and CDR-H3 or variants thereof; and / or

[0011] Or, the CDR-L1 or a variant thereof, CDR-L2 or a variant thereof, and CDR-L3 or a variant thereof contained in the variable region (VL) of the light chain shown in SEQ ID NO:2, 18, 31, 41, 54, or 69; or

[0012] (b) CDR-H1 or a variant thereof, CDR-H2 or a variant thereof, and CDR-H3 or a variant thereof contained in VH shown in SEQ ID NO:1; and / or CDR-L1 or a variant thereof, CDR-L2 or a variant thereof, and CDR-L3 or a variant thereof contained in VL shown in SEQ ID NO:2.

[0013] In some embodiments, the antibody or its antigen-binding fragment comprises CDR-H1 or a variant thereof, CDR-H2 or a variant thereof, and CDR-H3 or a variant thereof contained in VH as shown in SEQ ID NO:17; and / or CDR-L1 or a variant thereof, CDR-L2 or a variant thereof, and CDR-L3 or a variant thereof contained in VL as shown in SEQ ID NO:18.

[0014] In some embodiments, the antibody or its antigen-binding fragment comprises CDR-H1 or a variant thereof, CDR-H2 or a variant thereof, and CDR-H3 or a variant thereof contained in VH shown in SEQ ID NO:30; and / or CDR-L1 or a variant thereof, CDR-L2 or a variant thereof, and CDR-L3 or a variant thereof contained in VL shown in SEQ ID NO:31.

[0015] In some embodiments, the antibody or its antigen-binding fragment comprises CDR-H1 or a variant thereof, CDR-H2 or a variant thereof, and CDR-H3 or a variant thereof contained in VH shown in SEQ ID NO:40; and / or CDR-L1 or a variant thereof, CDR-L2 or a variant thereof, and CDR-L3 or a variant thereof contained in VL shown in SEQ ID NO:41.

[0016] In some embodiments, the antibody or its antigen-binding fragment comprises CDR-H1 or a variant thereof, CDR-H2 or a variant thereof, and CDR-H3 or a variant thereof contained in VH shown in SEQ ID NO:53; and / or CDR-L1 or a variant thereof, CDR-L2 or a variant thereof, and CDR-L3 or a variant thereof contained in VL shown in SEQ ID NO:54.

[0017] In some embodiments, the antibody or its antigen-binding fragment comprises CDR-H1 or a variant thereof, CDR-H2 or a variant thereof, and CDR-H3 or a variant thereof contained in VH as shown in SEQ ID NO:68; and / or CDR-L1 or a variant thereof, CDR-L2 or a variant thereof, and CDR-L3 or a variant thereof contained in VL as shown in SEQ ID NO:69.

[0018] In some preferred embodiments, the variant of the sequence is a CDR with one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids) compared to its source CDR.

[0019] In some preferred embodiments, the substitution is a conservative substitution.

[0020] Preferably, the CDR is defined according to the AbM, Chothia, Kabat or IMGT numbering system.

[0021] In some embodiments, the antibody or its antigen-binding fragment includes a framework region (FR) derived from human immunoglobulins in its VH and / or VL regions.

[0022] In some embodiments, the antibody or its antigen-binding fragment binds to human TSLP and / or monkey TSLP.

[0023] In one aspect, the present invention provides an antibody or antigen-binding fragment thereof capable of binding to TSLP, said antibody or antigen-binding fragment comprising a heavy chain variable region (VH) and / or a light chain variable region (VL).

[0024] In some embodiments, the antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDR is defined according to the IMGT numbering system:

[0025] (a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO: 3 or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); CDR-H2 with the sequence of SEQ ID NO: 4 or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); and CDR-H3 with the sequence of SEQ ID NO: 5 or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); and / or,

[0026] The light chain variable region (VL) contains the following three CDRs: CDR-L1, which has the sequence of SEQ ID NO: 6 or has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); CDR-L2, which has the sequence of SEQ ID NO: 7 or has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); and CDR-L3, which has the sequence of SEQ ID NO: 8 or has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids).

[0027] (b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO: 19 or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); CDR-H2 with the sequence of SEQ ID NO: 20 or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); and CDR-H3 with the sequence of SEQ ID NO: 21 or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); and / or,

[0028] The light chain variable region (VL) contains the following three CDRs: CDR-L1, which has the sequence of SEQ ID NO: 22 or has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); CDR-L2, which has the sequence of SEQ ID NO: 23 or has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); and CDR-L3, which has the sequence of SEQ ID NO: 24 or has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids).

[0029] (c) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO: 32 or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); CDR-H2 with the sequence of SEQ ID NO: 33 or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); and CDR-H3 with the sequence of SEQ ID NO: 34 or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); and / or,

[0030] The light chain variable region (VL) contains the following three CDRs: CDR-L1, which has the sequence of SEQ ID NO: 35 or has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); CDR-L2, which has the sequence of SEQ ID NO: 23 or has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); and CDR-L3, which has the sequence of SEQ ID NO: 24 or has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids).

[0031] (d) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO: 42 or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); CDR-H2 with the sequence of SEQ ID NO: 43 or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); and CDR-H3 with the sequence of SEQ ID NO: 44 or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); and / or,

[0032] The light chain variable region (VL) contains the following three CDRs: CDR-L1, which has the sequence of SEQ ID NO: 45 or has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); CDR-L2, which has the sequence of SEQ ID NO: 46 or has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); and CDR-L3, which has the sequence of SEQ ID NO: 47 or has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids).

[0033] or

[0034] (e) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO: 55 or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); CDR-H2 with the sequence of SEQ ID NO: 56 or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); and CDR-H3 with the sequence of SEQ ID NO: 57 or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); and / or,

[0035] The light chain variable region (VL) contains the following three CDRs: CDR-L1, which has the sequence of SEQ ID NO: 58 or has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); CDR-L2, which has the sequence of SEQ ID NO: 59 or has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); and CDR-L3, which has the sequence of SEQ ID NO: 60 or has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids).

[0036] In some embodiments, the antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDR is defined according to the IMGT numbering system:

[0037] (a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO: 3, CDR-H2 with sequence SEQ ID NO: 4, and CDR-H3 with sequence SEQ ID NO: 5; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO: 6, CDR-L2 with sequence SEQ ID NO: 7, and CDR-L3 with sequence SEQ ID NO: 8;

[0038] (b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 19, CDR-H2 of SEQ ID NO: 20, and CDR-H3 of SEQ ID NO: 21; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 22, CDR-L2 of SEQ ID NO: 23, and CDR-L3 of SEQ ID NO: 24;

[0039] (c) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO: 32, CDR-H2 with sequence SEQ ID NO: 33, and CDR-H3 with sequence SEQ ID NO: 34; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO: 35, CDR-L2 with sequence SEQ ID NO: 23, and CDR-L3 with sequence SEQ ID NO: 24;

[0040] (d) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO: 42, CDR-H2 with sequence SEQ ID NO: 43, and CDR-H3 with sequence SEQ ID NO: 44; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO: 45, CDR-L2 with sequence SEQ ID NO: 46, and CDR-L3 with sequence SEQ ID NO: 47;

[0041] or

[0042] (e) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 55, CDR-H2 of SEQ ID NO: 56, and CDR-H3 of SEQ ID NO: 57; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 58, CDR-L2 of SEQ ID NO: 59, and CDR-L3 of SEQ ID NO: 60.

[0043] In some embodiments, the antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDR is defined according to the AbM numbering system:

[0044] (a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO: 9 or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); CDR-H2 with the sequence of SEQ ID NO: 10 or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); and CDR-H3 with the sequence of SEQ ID NO: 11 or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); and / or,

[0045] The light chain variable region (VL) contains the following three CDRs: CDR-L1, which has the sequence of SEQ ID NO: 12 or has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); CDR-L2, which has the sequence of SEQ ID NO: 13 or has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); and CDR-L3, which has the sequence of SEQ ID NO: 8 or has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids).

[0046] (b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO: 25 or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); CDR-H2 with the sequence of SEQ ID NO: 26 or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); and CDR-H3 with the sequence of SEQ ID NO: 27 or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); and / or,

[0047] The light chain variable region (VL) contains the following three CDRs: CDR-L1, which has the sequence of SEQ ID NO: 28 or has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); CDR-L2, which has the sequence of SEQ ID NO: 29 or has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); and CDR-L3, which has the sequence of SEQ ID NO: 24 or has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids).

[0048] (c) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO: 36 or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); CDR-H2 with the sequence of SEQ ID NO: 37 or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); and CDR-H3 with the sequence of SEQ ID NO: 38 or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); and / or,

[0049] The light chain variable region (VL) contains the following three CDRs: CDR-L1, which has the sequence of SEQ ID NO: 39 or has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); CDR-L2, which has the sequence of SEQ ID NO: 29 or has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); and CDR-L3, which has the sequence of SEQ ID NO: 24 or has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids).

[0050] (d) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO: 48 or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); CDR-H2 with the sequence of SEQ ID NO: 49 or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); and CDR-H3 with the sequence of SEQ ID NO: 50 or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); and / or,

[0051] The light chain variable region (VL) contains the following three CDRs: CDR-L1, which has the sequence of SEQ ID NO: 51 or has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids); CDR-L2, which has the sequence of SEQ ID NO: 52 or has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids); and CDR-L3, which has the sequence of SEQ ID NO: 47 or has one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1, 2 or 3 amino acids).

[0052] or

[0053] (e) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with the sequence of SEQ ID NO: 61 or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); CDR-H2 with the sequence of SEQ ID NO: 62 or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); and CDR-H3 with the sequence of SEQ ID NO: 63 or having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); and / or,

[0054] The light chain variable region (VL) contains the following three CDRs: CDR-L1, which has the sequence of SEQ ID NO: 64 or has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); CDR-L2, which has the sequence of SEQ ID NO: 65 or has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids); and CDR-L3, which has the sequence of SEQ ID NO: 60 or has one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1, 2, or 3 amino acids).

[0055] In some embodiments, the antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the CDR is defined according to the AbM numbering system:

[0056] (a) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO: 9, CDR-H2 with sequence SEQ ID NO: 10, and CDR-H3 with sequence SEQ ID NO: 11; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO: 12, CDR-L2 with sequence SEQ ID NO: 13, and CDR-L3 with sequence SEQ ID NO: 8;

[0057] (b) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 25, CDR-H2 of SEQ ID NO: 26, and CDR-H3 of SEQ ID NO: 27; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 28, CDR-L2 of SEQ ID NO: 29, and CDR-L3 of SEQ ID NO: 24;

[0058] (c) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 of SEQ ID NO: 36, CDR-H2 of SEQ ID NO: 37, and CDR-H3 of SEQ ID NO: 38; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 of SEQ ID NO: 39, CDR-L2 of SEQ ID NO: 29, and CDR-L3 of SEQ ID NO: 24;

[0059] (d) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO: 48, CDR-H2 with sequence SEQ ID NO: 49, and CDR-H3 with sequence SEQ ID NO: 50; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO: 51, CDR-L2 with sequence SEQ ID NO: 52, and CDR-L3 with sequence SEQ ID NO: 47;

[0060] or

[0061] (e) A heavy chain variable region (VH) comprising the following three CDRs: CDR-H1 with sequence SEQ ID NO: 61, CDR-H2 with sequence SEQ ID NO: 62, and CDR-H3 with sequence SEQ ID NO: 63; and / or a light chain variable region (VL) comprising the following three CDRs: CDR-L1 with sequence SEQ ID NO: 64, CDR-L2 with sequence SEQ ID NO: 65, and CDR-L3 with sequence SEQ ID NO: 60.

[0062] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein at least one CDR in the heavy chain variable region (VH) and / or the light chain variable region (VL) contains a mutation compared to the CDR defined by the aforementioned IMGT or AbM, said mutation being a substitution, deletion or addition of one or more amino acids or any combination thereof (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids or any combination thereof).

[0063] Preferably, the substitution described in this invention is a conservative substitution.

[0064] In some embodiments, the VH of the antibody or antigen-binding fragment of the present invention comprises a framework region (FR) derived from the heavy chain variable region (VH) of human immunoglobulin, and / or the VL of the antibody or antigen-binding fragment of the present invention comprises a framework region (FR) derived from the light chain variable region (VL) of human immunoglobulin. Therefore, in some embodiments, the antibody or antigen-binding fragment of the present invention is humanized. In some embodiments, the antibody or antigen-binding fragment of the present invention is fully human.

[0065] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises:

[0066] (a) The heavy chain framework region of a human immunoglobulin or a variant thereof, said variant having up to 20 conserved substitutions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions) compared to the amino acid sequence encoded by the germline antibody gene from which it is derived; and / or

[0067] (b) The light chain framework region of a human immunoglobulin or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 20, up to 15, up to 10 or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions) compared to the amino acid sequence encoded by the germline antibody gene from which it is derived.

[0068] In some embodiments, the degree of humanization of the antibody or antigen-binding fragment of the present 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%.

[0069] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises:

[0070] (a) Heavy chain variable region (VH), which contains an amino acid sequence selected from the following:

[0071] (i) The sequence shown in SEQ ID NO: 1, 17, 30, 40, 53 or 68;

[0072] (ii) A sequence having one or more amino acid substitutions, deletions, or additions, or any combination thereof (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, or any combination thereof), compared to the sequence shown in SEQ ID NO: 1, 17, 30, 40, 53, or 68; or

[0073] (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 with the sequence shown in SEQ ID NO: 1, 17, 30, 40, 53, or 68;

[0074] and / or

[0075] (b) Light chain variable region (VL), which contains an amino acid sequence selected from the following:

[0076] (iv) The sequence shown in SEQ ID NO: 2, 18, 31, 41, 54 or 69;

[0077] (v) A sequence having one or more amino acid substitutions, deletions, or additions, or any combination thereof (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, or any combination thereof), compared to the sequence shown in SEQ ID NO: 2, 18, 31, 41, 54, or 69; or

[0078] (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 compared to the sequence shown in SEQ ID NO: 2, 18, 31, 41, 54, or 69.

[0079] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises VH as shown in SEQ ID NO: 1, and / or VL as shown in SEQ ID NO: 2.

[0080] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises VH as shown in SEQ ID NO: 17, and / or VL as shown in SEQ ID NO: 18.

[0081] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises VH as shown in SEQ ID NO: 30, and / or VL as shown in SEQ ID NO: 31.

[0082] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises VH as shown in SEQ ID NO: 40, and / or VL as shown in SEQ ID NO: 41.

[0083] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises VH as shown in SEQ ID NO: 53, and / or VL as shown in SEQ ID NO: 54.

[0084] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises VH as shown in SEQ ID NO: 68, and / or VL as shown in SEQ ID NO: 69.

[0085] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises:

[0086] (a) VH of the sequence shown in SEQ ID NO: 1 and VL of the sequence shown in SEQ ID NO: 2;

[0087] (b) VH of the sequence shown in SEQ ID NO: 17 and VL of the sequence shown in SEQ ID NO: 18;

[0088] (c) VH of the sequence shown in SEQ ID NO: 30 and VL of the sequence shown in SEQ ID NO: 31;

[0089] (d) VH of the sequence shown in SEQ ID NO: 40 and VL of the sequence shown in SEQ ID NO: 41;

[0090] (e) VH of the sequence shown in SEQ ID NO: 53 and VL of the sequence shown in SEQ ID NO: 54;

[0091] (f) VH of the sequence shown in SEQ ID NO: 68 and VL of the sequence shown in SEQ ID NO: 69;

[0092] (g) a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) and the light chain variable region (VL) 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, respectively, compared to the VH and VL described in any of (a) to (f); or

[0093] (h) Heavy chain variable region (VH) and light chain variable region (VL), wherein the heavy chain variable region (VH) and light chain variable region (VL) independently have, compared with the VH and VL described in any of (a) to (f), one or more amino acid substitutions, deletions, or additions, or any combination thereof (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, or any combination thereof). Preferably, the substitution is a conservative substitution.

[0094] In some embodiments, the heavy chain of the antibody or antigen-binding fragment of the present invention comprises the heavy chain constant region (CH) of a human immunoglobulin or a variant thereof, said variant having up to 50 conserved 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 substitutions; e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions) compared to its derived wild-type sequence. In some embodiments, the light chain of the antibody or antigen-binding fragment of the present invention comprises a light chain constant region (CL) of a human immunoglobulin or a variant thereof, said variant having up to 50 conserved 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 substitutions; e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions) compared to the wild-type sequence from which it is derived.

[0095] In some implementations, the constant region is altered, for example, mutated, to modify the properties of the anti-TSLP antibody molecule (e.g., altering one or more of the following properties: Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, or complement function). Functional changes can be produced by replacing at least one amino acid residue in the antibody's constant region with a different residue, for example, altering the antibody's affinity for effector ligands (such as FcR or complement C1q), thereby changing effector function (e.g., reducing it). The Fc region of an antibody mediates several important effector functions, such as ADCC, phagocytosis (ADCP), CDC, etc.

[0096] In some embodiments, the antibody or antigen-binding fragment of the present invention has a heavy chain constant region (Fc) selected from, for example, the heavy chain constant regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE; particularly selected from, for example, the heavy chain constant regions of IgG1, IgG2, IgG3, and IgG4, and more particularly selected from the heavy chain constant region of IgG1 (e.g., human IgG1). In some embodiments, the human IgG1 heavy chain constant region is as shown in SEQ ID NO: 14. In some embodiments, the antibody or antigen-binding fragment of the present invention has a light chain constant region selected from, for example, the κ or λ light chain constant region, preferably the κ light chain constant region (e.g., the human κ light chain constant region).

[0097] In some embodiments, the antibody or its antigen-binding fragment comprises a human IgG1 heavy chain constant region. In some preferred embodiments, the antibody or its antigen-binding fragment comprises the human IgG1 constant region shown in uniprot ID P01857 (SEQ ID NO: 74).

[0098] In some embodiments, the antibody or its antigen-binding fragment comprises a constant region of the human IgG1 heavy chain (e.g., SEQ ID NO: 74) or a variant thereof, wherein, according to the EU numbering system, the variant is mutated at at least one of sites 234, 235, 237, 265, 297, 331, 329, and 434. In some embodiments, the variant contains 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 contains at least one of the following mutations: L234A, L235A, G237A, and N434A. In some embodiments, the IgG1 heavy chain constant region variant comprises L234A, L235A, and G237A. In some embodiments, the IgG1 heavy chain constant region variant comprises mutant L234A, L235A, G237A, and N434A.

[0099] In some embodiments, the antibody or its antigen-binding fragment comprises CH as shown in SEQ ID NO: 14 or a variant thereof, the variant having at most 20 conserved substitutions (e.g., at most 20, 15, 10, or 5 amino acid substitutions; e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions) compared to SEQ ID NO: 14, 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 compared to SEQ ID NO: 14. According to the EU numbering system, the variant contains N297A and / or N434A. In some embodiments, the variant contains N434A.

[0100] In some embodiments, the antibody or its antigen-binding fragment comprises CH as shown in SEQ ID NO: 15 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid 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 compared to SEQ ID NO: 15.

[0101] In some embodiments, the antibody or its antigen-binding fragment comprises a constant region of the human IgG4 heavy chain (e.g., SEQ ID NO: 75) or a variant thereof, which, according to the EU numbering system, is mutated at at least one site at site 228 and / or 434.

[0102] In some embodiments, the mutant comprises S228P and / or N434A. In some embodiments, the human IgG4 heavy chain constant region variant comprises S228P and N434A.

[0103] In some embodiments, the antibody or its antigen-binding fragment comprises CH as shown in SEQ ID NO: 70 or a variant thereof, the variant having up to 20 conserved substitutions (e.g., up to 20, up to 15, up to 10, or up to 5 amino acid substitutions; e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions) compared to SEQ ID NO: 70, 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 compared to SEQ ID NO: 70.

[0104] In some embodiments, the antibody or its antigen-binding fragment comprises a light chain constant region or a variant thereof. In some embodiments, the light chain constant region comprises a κ light chain constant region. In some embodiments, the light chain constant region comprises the light chain constant region (CL) shown in SEQ ID NO: 16 or a variant thereof, the variant having at most 20 conserved substitutions (e.g., at most 20, 15, 10, or 5 amino acid substitutions; e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions) compared to SEQ ID NO: 16, 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 compared to SEQ ID NO: 16;

[0105] In some embodiments, the antibody or its antigen-binding fragment comprises the heavy chain constant region (CH) shown in SEQ ID NO: 14 and the light chain constant region (CL) shown in SEQ ID NO: 16.

[0106] In some embodiments, the antibody or its antigen-binding fragment comprises the heavy chain constant region (CH) shown in SEQ ID NO: 15 and the light chain constant region (CL) shown in SEQ ID NO: 16.

[0107] In some embodiments, the antibody or its antigen-binding fragment comprises the heavy chain constant region (CH) shown in SEQ ID NO: 70 and the light chain constant region (CL) shown in SEQ ID NO: 16.

[0108] In some embodiments, the above-mentioned site mutation causes the antibody or antigen-binding fragment to have no or reduced ADCP, ADCC, and / or CDC activity compared to the corresponding antibody or antigen-binding fragment containing human IgG4 or the constant region of the IgG4 heavy chain.

[0109] In some embodiments, the above-mentioned site mutation causes the antibody or antigen-binding fragment to have no or reduced ADCP, ADCC, and / or CDC activity compared to the corresponding antibody or antigen-binding fragment without the mutation or substitution.

[0110] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises:

[0111] (a) Heavy chain, which contains an amino acid sequence selected from the following:

[0112] (i) A sequence comprising the VH sequence shown in SEQ ID NO: 1 and the CH sequence shown in SEQ ID NO: 14, 15 or 70;

[0113] (ii) A sequence having one or more amino acid substitutions, deletions, or additions, or any combination thereof (e.g., substitutions, deletions, or additions of up to 50, 45, 35, 25, 15, 10, or 5 amino acids, or any combination thereof; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, or any combination thereof) compared to the sequence shown in (i); or

[0114] (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 with the sequence shown in (i); and

[0115] (b) A light chain comprising an amino acid sequence selected from the following:

[0116] (iv) A sequence comprising the VL sequence shown in SEQ ID NO: 2 and the CL sequence shown in SEQ ID NO: 16;

[0117] (v) A sequence having, compared to the sequence shown in (iv), one or more amino acid substitutions, deletions, or additions, or any combination thereof (e.g., substitutions, deletions, or additions of up to 50, 45, 35, 25, 15, 10, or 5 amino acids, or any combination thereof; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, or any combination thereof); or

[0118] (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 compared to the sequence shown in (iv).

[0119] In some implementations, the permutation described in (ii) or (v) is a conservative permutation.

[0120] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises:

[0121] (a) Heavy chain, which contains an amino acid sequence selected from the following:

[0122] (i) A sequence comprising the VH sequence shown in SEQ ID NO: 17 and the CH sequence shown in SEQ ID NO: 14, 15 or 70;

[0123] (ii) A sequence having one or more amino acid substitutions, deletions, or additions, or any combination thereof (e.g., substitutions, deletions, or additions of up to 50, 45, 35, 25, 15, 10, or 5 amino acids, or any combination thereof; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, or any combination thereof) compared to the sequence shown in (i); or

[0124] (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 with the sequence shown in (i); and

[0125] (b) A light chain comprising an amino acid sequence selected from the following:

[0126] (iv) A sequence comprising the VL sequence shown in SEQ ID NO: 18 and the CL sequence shown in SEQ ID NO: 16;

[0127] (v) A sequence having, compared to the sequence shown in (iv), one or more amino acid substitutions, deletions, or additions, or any combination thereof (e.g., substitutions, deletions, or additions of up to 50, 45, 35, 25, 15, 10, or 5 amino acids, or any combination thereof; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, or any combination thereof); or

[0128] (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 compared to the sequence shown in (iv).

[0129] In some implementations, the permutation described in (ii) or (v) is a conservative permutation.

[0130] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises:

[0131] (a) Heavy chain, which contains an amino acid sequence selected from the following:

[0132] (i) A sequence comprising the VH sequence shown in SEQ ID NO: 30 and the CH sequence shown in SEQ ID NO: 14, 15 or 70;

[0133] (ii) A sequence having one or more amino acid substitutions, deletions, or additions, or any combination thereof (e.g., substitutions, deletions, or additions of up to 50, 45, 35, 25, 15, 10, or 5 amino acids, or any combination thereof; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, or any combination thereof) compared to the sequence shown in (i); or

[0134] (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 with the sequence shown in (i); and

[0135] (b) A light chain comprising an amino acid sequence selected from the following:

[0136] (iv) A sequence comprising the VL sequence shown in SEQ ID NO: 31 and the CL sequence shown in SEQ ID NO: 16;

[0137] (v) A sequence having, compared to the sequence shown in (iv), one or more amino acid substitutions, deletions, or additions, or any combination thereof (e.g., substitutions, deletions, or additions of up to 50, 45, 35, 25, 15, 10, or 5 amino acids, or any combination thereof; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, or any combination thereof); or

[0138] (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 compared to the sequence shown in (iv).

[0139] In some implementations, the permutation described in (ii) or (v) is a conservative permutation.

[0140] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises:

[0141] (a) Heavy chain, which contains an amino acid sequence selected from the following:

[0142] (i) A sequence comprising the VH sequence shown in SEQ ID NO: 40 and the CH sequence shown in SEQ ID NO: 14, 15 or 70;

[0143] (ii) A sequence having one or more amino acid substitutions, deletions, or additions, or any combination thereof (e.g., substitutions, deletions, or additions of up to 50, 45, 35, 25, 15, 10, or 5 amino acids, or any combination thereof; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, or any combination thereof) compared to the sequence shown in (i); or

[0144] (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 with the sequence shown in (i); and

[0145] (b) A light chain comprising an amino acid sequence selected from the following:

[0146] (iv) A sequence comprising the VL sequence shown in SEQ ID NO: 41 and the CL sequence shown in SEQ ID NO: 16;

[0147] (v) A sequence having, compared to the sequence shown in (iv), one or more amino acid substitutions, deletions, or additions, or any combination thereof (e.g., substitutions, deletions, or additions of up to 50, 45, 35, 25, 15, 10, or 5 amino acids, or any combination thereof; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, or any combination thereof); or

[0148] (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 compared to the sequence shown in (iv).

[0149] In some implementations, the permutation described in (ii) or (v) is a conservative permutation.

[0150] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises:

[0151] (a) Heavy chain, which contains an amino acid sequence selected from the following:

[0152] (i) A sequence comprising the VH sequence shown in SEQ ID NO: 53 and the CH sequence shown in SEQ ID NO: 14, 15 or 70;

[0153] (ii) A sequence having one or more amino acid substitutions, deletions, or additions, or any combination thereof (e.g., substitutions, deletions, or additions of up to 50, 45, 35, 25, 15, 10, or 5 amino acids, or any combination thereof; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, or any combination thereof) compared to the sequence shown in (i); or

[0154] (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 with the sequence shown in (i); and

[0155] (b) A light chain comprising an amino acid sequence selected from the following:

[0156] (iv) A sequence comprising the VL sequence shown in SEQ ID NO: 54 and the CL sequence shown in SEQ ID NO: 16;

[0157] (v) A sequence having, compared to the sequence shown in (iv), one or more amino acid substitutions, deletions, or additions, or any combination thereof (e.g., substitutions, deletions, or additions of up to 50, 45, 35, 25, 15, 10, or 5 amino acids, or any combination thereof; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, or any combination thereof); or

[0158] (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 compared to the sequence shown in (iv).

[0159] In some implementations, the permutation described in (ii) or (v) is a conservative permutation.

[0160] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises:

[0161] (a) Heavy chain, which contains an amino acid sequence selected from the following:

[0162] (i) A sequence comprising the VH sequence shown in SEQ ID NO: 68 and the CH sequence shown in SEQ ID NO: 14, 15 or 70;

[0163] (ii) A sequence having one or more amino acid substitutions, deletions, or additions, or any combination thereof (e.g., substitutions, deletions, or additions of up to 50, 45, 35, 25, 15, 10, or 5 amino acids, or any combination thereof; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, or any combination thereof) compared to the sequence shown in (i); or

[0164] (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 with the sequence shown in (i); and

[0165] (b) A light chain comprising an amino acid sequence selected from the following:

[0166] (iv) A sequence comprising the VL sequence shown in SEQ ID NO: 69 and the CL sequence shown in SEQ ID NO: 16;

[0167] (v) A sequence having, compared to the sequence shown in (iv), one or more amino acid substitutions, deletions, or additions, or any combination thereof (e.g., substitutions, deletions, or additions of up to 50, 45, 35, 25, 15, 10, or 5 amino acids, or any combination thereof; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, or any combination thereof); or

[0168] (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 compared to the sequence shown in (iv).

[0169] In some implementations, the permutation described in (ii) or (v) is a conservative permutation.

[0170] In some embodiments, the antibody of the present invention comprises: a heavy chain including the VH shown in SEQ ID NO: 1 and the heavy chain constant region (CH) shown in SEQ ID NO: 14, 15 or 70, and a light chain including the VL shown in SEQ ID NO: 2 and the light chain constant region (CL) shown in SEQ ID NO: 16.

[0171] In some embodiments, the antibody of the present invention comprises: a heavy chain including the VH shown in SEQ ID NO: 17 and the heavy chain constant region (CH) shown in SEQ ID NO: 14, 15 or 70, and a light chain including the VL shown in SEQ ID NO: 18 and the light chain constant region (CL) shown in SEQ ID NO: 16.

[0172] In some embodiments, the antibody of the present invention comprises: a heavy chain including the VH shown in SEQ ID NO: 30 and the heavy chain constant region (CH) shown in SEQ ID NO: 14, 15 or 70, and a light chain including the VL shown in SEQ ID NO: 31 and the light chain constant region (CL) shown in SEQ ID NO: 16.

[0173] In some embodiments, the antibody of the present invention comprises: a heavy chain including the VH shown in SEQ ID NO: 40 and the heavy chain constant region (CH) shown in SEQ ID NO: 14, 15 or 70, and a light chain including the VL shown in SEQ ID NO: 41 and the light chain constant region (CL) shown in SEQ ID NO: 16.

[0174] In some embodiments, the antibody of the present invention comprises: a heavy chain including the VH shown in SEQ ID NO: 53 and the heavy chain constant region (CH) shown in SEQ ID NO: 14, 15 or 70, and a light chain including the VL shown in SEQ ID NO: 54 and the light chain constant region (CL) shown in SEQ ID NO: 16.

[0175] In some embodiments, the antibody of the present invention comprises: a heavy chain including the VH region shown in SEQ ID NO: 68 and the heavy chain constant region (CH) shown in SEQ ID NO: 14, 15 or 70, and a light chain including the VL region shown in SEQ ID NO: 69 and the light chain constant region (CL) shown in SEQ ID NO: 16.

[0176] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises a heavy chain and a light chain.

[0177] The heavy chain includes:

[0178] (i) The sequence shown in SEQ ID NO: 66 or 73;

[0179] (ii) A sequence having one or more amino acid substitutions, deletions, or additions, or any combination thereof (e.g., substitutions, deletions, or additions of up to 50, 45, 35, 25, 15, 10, or 5 amino acids, or any combination thereof; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, or any combination thereof) compared to the sequence shown in (i); or

[0180] (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 with the sequence shown in (i); and

[0181] The light chain comprises:

[0182] (iv) The sequence shown in SEQ ID NO: 67;

[0183] (v) A sequence having, compared to the sequence shown in (iv), one or more amino acid substitutions, deletions, or additions, or any combination thereof (e.g., substitutions, deletions, or additions of up to 50, 45, 35, 25, 15, 10, or 5 amino acids, or any combination thereof; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, or any combination thereof); or

[0184] (vi) Sequences showing 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;

[0185] Preferably, the substitution described in (ii) or (v) is a conservative substitution.

[0186] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises a heavy chain and a light chain.

[0187] The heavy chain includes:

[0188] (i) The sequence shown in SEQ ID NO: 71;

[0189] (ii) A sequence having one or more amino acid substitutions, deletions, or additions, or any combination thereof (e.g., substitutions, deletions, or additions of up to 50, 45, 35, 25, 15, 10, or 5 amino acids, or any combination thereof; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, or any combination thereof) compared to the sequence shown in (i); or

[0190] (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 with the sequence shown in (i); and

[0191] The light chain comprises:

[0192] (iv) The sequence shown in SEQ ID NO: 72;

[0193] (v) A sequence having, compared to the sequence shown in (iv), one or more amino acid substitutions, deletions, or additions, or any combination thereof (e.g., substitutions, deletions, or additions of up to 50, 45, 35, 25, 15, 10, or 5 amino acids, or any combination thereof; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, or any combination thereof); or

[0194] (vi) Sequences showing 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;

[0195] Preferably, the substitution described in (ii) or (v) is a conservative substitution.

[0196] In some embodiments, the antibody of the present invention is a chimeric antibody, a humanized antibody, or a fully human antibody. In some embodiments, the antibody of the present invention or its antigen-binding fragment is selected from scFv, Fab, Fab', F(ab')2, Fv fragment, disulfide-linked Fv (dsFv), and diabody.

[0197] In some embodiments, the antibody molecule or its antigen-binding fragment of the present invention may exhibit at least one of the following properties:

[0198] (a) Combining TSLP (e.g., human TSLP) with a KD of less than about 50 nM, such as less than about 40 nM, 30 nM, 20 nM, 10 nM, 1 nM, 0.1 nM, 1 pM, 0.1 pM or lower; said KD can be measured by techniques known in the art, such as by Fortebio or ELISA;

[0199] (b) TSLP (e.g., human TSLP) is bound with an EC50 of less than about 50 nM, such as less than 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 smaller; said EC50 can be measured by techniques known in the art, such as flow cytometry, ELISA such as affinity ELISA or cell competition ELISA.

[0200] (c) Inhibit the binding of TSLP to IL7Rα / TSLPR with an IC50 of less than about 50 nM, for example, 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 smaller; said IC50 is determined by ELISA.

[0201] (d) Inhibit or block TSLP-induced OX40L expression;

[0202] (e) Inhibit or block TSLP-induced activation and / or proliferation of mast cells, DCs, and NKT cells;

[0203] (f) Inhibit or block TSLP-induced osteoprotegerin (OPG) secretion;

[0204] (g) Inhibit or block the secretion of Th2 cytokines such as TARC, CCL22, IL-4, IL-13 or IL-5;

[0205] (h) Good affinity for binding with FcRn;

[0206] (i) The isoelectric point (PI) is about 6.5 to about 8.5, such as 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.

[0207] Furthermore, the antibody of the present invention exhibits good affinity for FcRn. In some embodiments, the KD(M) value of the affinity for FcRn is 10. -9The antibodies of this invention have a longer in vivo half-life. The antibodies of this invention also exhibit good hydrophilicity. In some embodiments, the hydrophobicity time of the antibodies of this invention, as detected by column chromatography, is between 8 and 14 minutes.

[0208] Antibody derivatives

[0209] The antibodies or antigen-binding fragments of the present invention can be derivatized, for example, by being linked to another molecule (e.g., another polypeptide or protein). Generally, derivatization (e.g., labeling) of the antibody or antigen-binding fragment does not adversely affect its binding to TSLP (particularly human TSLP). Therefore, the antibodies or antigen-binding fragments of the present invention are also intended to include such derivatized forms. For example, the antibodies or antigen-binding fragments of the present invention can be linked (by chemical coupling, gene fusion, non-covalent linkage, or other means) to one or more other molecular groups, such as another antibody (e.g., forming a bispecific antibody), a detection reagent, a pharmaceutical reagent, and / or a protein or polypeptide (e.g., an avidin or a multihistidine tag) capable of mediating the binding of the antibody or antigen-binding fragment to another molecule.

[0210] One type of derivatized antibody (e.g., a bispecific antibody) is produced by cross-linking two or more antibodies (of the same or different types). Methods for obtaining bispecific antibodies are well known in the art, and examples include, but are not limited to, chemical cross-linking, cell engineering (hybridoma method), or genetic engineering.

[0211] Another type of derivatized antibody is a labeled antibody. For example, the antibody of the present invention or its antigen-binding fragment can be linked to a detectable label. The detectable label described in the present invention can be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical, or chemical means. Such labels are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), 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, Alexa 750)), acrid esters, magnetic beads (e.g., ), calorimetric markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding avidin (e.g., streptavidin) modified with the aforementioned markers. Patents teaching the use of such markers include, but are not limited to, U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241 (all incorporated herein by reference). Detectable markers as described above can be detected by methods known in the art. For example, radioactive markers can be detected using photographic film or a scintillation calculator, and fluorescent markers can be detected using a photodetector to detect emitted light. Enzyme markers are generally detected by providing an enzyme with a substrate and detecting the reaction product produced by the enzyme's action on the substrate, and calorimetric markers are detected by simple, visually appealing colored markers. In some embodiments, such markers can be used for immunological assays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In some embodiments, the detectable markers described above can be linked to the antibodies or antigen-binding fragments of the present invention via linkers of varying lengths to reduce potential steric hindrance.

[0212] Furthermore, the antibodies or antigen-binding fragments of the present invention can also be derivatized with chemical groups, such as polyethylene glycol (PEG), methyl or ethyl groups, or glycosyl groups. These groups can be used to improve the biological properties of the antibodies, such as increasing serum half-life.

[0213] Therefore, one aspect of the present invention provides a conjugate comprising the monoclonal antibody or antigen-binding fragment thereof of the present invention and a conjugation portion, said conjugation portion being a detectable marker as described above, such as a radioactive isotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme. The conjugation portion may also be a therapeutic agent.

[0214] As a derivative of antibodies, this invention provides a multispecific antibody comprising a first antibody or a fragment thereof, and a further antibody or a fragment thereof, or an antibody analog, wherein the first antibody or a fragment thereof, the further antibody or a fragment thereof, or the antibody analog retains its original binding specificity. The first antibody or a fragment thereof is any TSLP-binding (monoclonal) antibody or antigen-binding fragment of this invention. As used herein, "antibody mimetic" refers to an antibody that binds to an antigen with the same specificity as an antibody but does not possess an antibody structure. These are typically artificial peptides or proteins with a molar mass of approximately 3 to 20 kDa, such as DARPin and fynomer. Designed DARPin can be linked to IgG antibodies, scFv-Fc antibody fragments, or combinations thereof, as described in CN104341529A. The fusion of anti-IL-17a fynomer with an anti-IL-6R antibody produces a bispecific fusion polypeptide, as described in WO2015141862A1.

[0215] In some embodiments, the multispecific antibody is formed by coupling a first antibody or its antigen-binding fragment with other antibodies or their antigen-binding fragments or antibody analogs, wherein each antibody or its antigen-binding fragment or antibody analog retains its original binding specificity, and the first antibody or its antigen-binding fragment is the antibody or its antigen-binding fragment described in this invention. In some embodiments, the multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

[0216] Antibody preparation

[0217] The antibodies of the present invention can be prepared by various methods known in the art, such as through genetic engineering recombination 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.

[0218] The antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). Alternatively, these antigen-binding fragments can also be directly produced from recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000)). For example, the Fab' fragment can be obtained directly from host cells; the Fab' fragment can be chemically coupled to form the F(ab')2 fragment (Carter et al., Bio / Technology, 10:163-167 (1992)). Furthermore, the Fv, Fab, or F(ab')2 fragments can also be directly isolated from the recombinant host cell culture medium. Other techniques for preparing these antigen-binding fragments are fully known to those skilled in the art.

[0219] 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, or a heavy chain variable region and / or a light chain variable region thereof, or one or more CDRs thereof. Based on codon degeneracy known in the art, in some embodiments, the nucleotide sequence may be substituted according to codon degeneracy. In some embodiments, the nucleotide sequence is codon-optimized.

[0220] In some embodiments, the isolated nucleic acid molecule of the present invention comprises: (i) a first nucleic acid and a second nucleic acid encoding the heavy chain variable region and the light chain variable region of the antibody or antigen-binding fragment of the present invention, respectively; or (ii) a first nucleic acid encoding the heavy chain variable region and the heavy chain constant region of the antibody or antigen-binding fragment of the present invention, and a second nucleic acid encoding the light chain variable region and the light chain constant region, respectively; or (iii) a first nucleic acid and a second nucleic acid encoding the heavy chain and the light chain of the antibody or antigen-binding fragment of the present invention, respectively. In some embodiments, the first nucleic acid and the second nucleic acid comprise a nucleic acid having a degenerate sequence or substantially identical sequence to any of the first nucleic acids and second nucleic acids in (i)-(iii) above. In some embodiments, the degenerate sequence or substantially identical sequence refers to a sequence having at least about 85%, 90%, 95%, 99% or higher sequence identity compared to the nucleic acid molecules described in (i)-(iii), or a sequence having one or more nucleotide substitutions, or a sequence differing by no more than 3, 6, 15, 30 or 45 nucleotides.

[0221] On the other hand, a vector (e.g., a cloning vector or an expression vector) is provided, which contains the isolated nucleic acid molecules of the present invention. In some embodiments, the vector of the present invention is, for example, a plasmid, a granulocyte, a bacteriophage, a lentivirus, etc. In some embodiments, the vector is capable of expressing the antibody of the present invention or its antigen-binding fragment in a subject (e.g., a mammal, such as a human).

[0222] On the other hand, a host cell is provided that contains the isolated nucleic acid molecules of the present invention or the vector of the present invention. The host cell can be a eukaryotic cell (e.g., mammalian cells, insect cells, yeast cells) or a prokaryotic cell (e.g., *E. 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 some embodiments, the host cell of the present invention is a mammalian cell, such as CHO (e.g., CHO-K1, CHO-S, CHO DXB11, CHO DG44).

[0223] On the other hand, a method for preparing the antibody or antigen-binding fragment thereof of the present invention is provided, comprising culturing the host cell of the present 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.

[0224] Uses, treatments and pharmaceutical compositions

[0225] In another aspect, the present invention provides pharmaceutical compositions comprising the antibody or antigen-binding fragment thereof of the present invention, nucleic acid, carrier, host cell, multispecific antibody, and / or conjugate, and pharmaceutically acceptable carrier and / or excipient.

[0226] In some embodiments, the pharmaceutical compositions of the present invention comprise the antibody or antigen-binding fragment of the present invention, and a pharmaceutically acceptable carrier and / or excipient.

[0227] In some embodiments, the pharmaceutical composition of the present invention comprises the host cell of the present invention, and a pharmaceutically acceptable carrier and / or excipient, wherein the host cell comprises isolated nucleic acid molecules or carriers as described above.

[0228] In some embodiments, the pharmaceutical compositions of the present invention comprise the multispecific antibody of the present invention, as well as a pharmaceutically acceptable carrier and / or excipient.

[0229] In some embodiments, the pharmaceutical compositions of the present invention comprise the conjugates of the present invention as well as pharmaceutically acceptable carriers and / or excipients.

[0230] On the other hand, the antibodies or antigen-binding fragments thereof, nucleic acids, carriers, host cells, multispecific antibodies, or conjugates in the pharmaceutical compositions of the present invention are used to produce at least one of the following biological activities in a subject:

[0231] (1) Inhibit or block the binding of TSLP to TSLPR / IL7Rα;

[0232] (2) Downregulate or eliminate the activity of TSLP;

[0233] (3) Downregulate or block the expression of OX40L;

[0234] (4) Inhibit or block TSLP-induced osteoprotegerin (OPG) secretion;

[0235] (5) Inhibit or block the secretion of Th2 cytokines such as TARC, CCL22, IL-4, IL-13 or IL-5;

[0236] (6) Inhibit or block TSLP-induced activation and / or proliferation of mast cells, DCs, and NKT cells.

[0237] The antibodies or antigen-binding fragments thereof, nucleic acids, carriers, 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. The binding of TSLP to TSLPR / IL7Rα can cause many allergic inflammatory diseases, including allergic and non-allergic diseases. These diseases include, but are not limited to, 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, COPD, systemic sclerosis, keloids, ulcerative colitis, chronic sinusitis (CRS) and nasal polyps, chronic eosinophilic pneumonia, eosinophilic bronchitis; abdominal diseases such as eosinophilic gastroenteritis, Churg-Strauss syndrome; eosinophil-related gastrointestinal diseases such as eosinophilia / eosinophilic granuloma with polyangiitis, eosinophilic esophagitis and inflammatory bowel disease; urticaria, systemic mastocytosis, cutaneous mastocytosis, and recurrent idiopathic angioedema. Therefore, the antibodies or antigen-binding fragments, nucleic acids, vectors, host cells, multispecific antibodies or conjugates in the compositions of the present invention can prevent or treat the aforementioned diseases.

[0238] TSLP binding to TSLPR / IL7Rα is also associated with autoimmune diseases. Therefore, the antibodies or their antigen-binding fragments, nucleic acids, vectors, host cells, multispecific antibodies, or conjugates in the 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, rheumatoid arthritis, psoriasis, and thyroid diseases.

[0239] In some embodiments, in the pharmaceutical composition, the antibody or antigen-binding fragment of the present invention, along with the additional pharmaceutically active agent, is provided as a separate component or as a component of the same composition. Therefore, the antibody or antigen-binding fragment of the present invention, along with the additional pharmaceutically active agent, can be administered in combination or separately, simultaneously or sequentially.

[0240] In some embodiments, the pharmaceutical composition may also contain additional pharmaceutically active agents.

[0241] The anti-TSLP antibody or its antigen-binding fragment of the present invention can be administered alone or in combination with other active agents. The anti-TSLP antibody or its antigen-binding fragment and one or more other active agents can be administered separately, simultaneously, or sequentially.

[0242] The anti-TSLP antibody or its antigen-binding fragment of the present invention can be administered in combination with any suitable immunosuppressant, including but not limited to anti-inflammatory drugs, particularly inhaled, intranasal, or parenteral corticosteroids such as budesonide, beclamethasone dipropionate, fludioxonide propionate, cicosonelide, voltadone furoate, fluticasone furoate, fluticasone propionate, budesonide, cicosonelide, beclomethasone dipropionate, mometasone furoate, triamcinolone acetonide, and prednisolone. In one embodiment, the anti-TSLP antibody or its antigen-binding fragment of the present 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, the anti-TSLP antibody or its antigen-binding fragment of the present invention may be administered in combination with a nonsteroidal glucocorticoid receptor agonist; an LTD4 antagonist or LTB4 antagonist, including montelukast, pramexazol, zafirlukast, acetaminophen, etc.; an A2A agonist; an A2B antagonist; a dopamine receptor agonist; or a PDE4 inhibitor. In one embodiment, the anti-TSLP antibody or its antigen-binding fragment of the present invention may be administered in combination with pirfenidone or nintedanib or an aVB6 antagonist.

[0243] The anti-TSLP antibody or its antigen-binding fragment of the present invention can be administered in combination with bronchodilators, such as β-2-adrenergic receptor agonists and / or muscarinic antagonists. Suitable β-2-adrenergic receptor agonists include vilanterol, salmeterol, salbutamol, formoterol, samethamol, fenoterol, carmoterol, etantenoterol, nalmenterol, clenbuterol, pibuterol, flubuterol, reproterol, bambuterol, indacaterol, terbutaline, and their salts. Suitable muscarinic antagonists include uracilium bromide, tiotropium bromide, glycopyrronium bromide, ipratropium bromide, and their salts such as uracilium bromide hydrobromide. In one embodiment, the anti-TSLP antibody or its antigen-binding fragment of the present invention can be administered in a fixed-dose combination with a β-2 adrenergic receptor agonist and / or a muscarinic antagonist, such as a fixed-dose combination with vilanterol triphenylacetate, urodimethyl bromide, or a dual combination with vilanterol triphenylacetate and urodimethyl bromide.

[0244] The anti-TSLP antibody or its antigen-binding fragment of the present invention can be administered in combination with one or more bronchodilators and an inhaled steroid. Such combinations may include dual-drug regimens such as fluticasone furoate and vilanterol triphenylacetate, fluticasone furoate and urodesulfuron-methyl, fluticasone propionate and salmeterol, budesonide and formoterol, mometasone and formoterol, and triple therapy such as fluticasone furoate, vilanterol triphenylacetate and urodesulfuron-methyl. In one embodiment, the anti-TSLP antibody or its antigen-binding fragment of the present invention can be administered in a fixed-dose combination with an inhaled corticosteroid and one or more bronchodilators, such as with fluticasone furoate and vilanterol triphenylacetate, or fluticasone propionate and salmeterol, or fluticasone furoate and urodesulfuron-methyl, or a fixed-dose combination of fluticasone furoate, vilanterol triphenylacetate and urodesulfuron-methyl.

[0245] In one embodiment, the anti-TSLP antibody or its antigen-binding fragment of the present invention can be administered in combination with antagonists of cytokine receptors, such as antagonists of CCR-1, CCR-3, CCR-4, CCR-5, CCR-6, CCR-7, CCR-8, CCR-9 and CCR10, CXCR1, CXCR2, CXCR3, CXCR4, and CXCR5. In one embodiment, the anti-TSLP antibody or its antigen-binding fragment of the present invention can be administered in combination with antibodies against other cytokines or cytokine receptors, such as anti-IgE antibodies, anti-IL31 antibodies, anti-IL31R antibodies, anti-IL13 antibodies, anti-endothelial glycoprotein antibodies, anti-IL1b antibodies, another anti-TSLP antibody, or an anti-hTSLPR antibody, or combinations thereof.

[0246] The anti-TSLP antibody or its antigen-binding fragment of the present invention can be administered in combination with the following: anti-leukotriene antagonists such as montelukast, zafirlukast, and pramlukast; PDE4 inhibitors such as roflumilast; xanthan; anti-IgE antibody; IL-13 antagonist; IL-6 antagonist; and antagonists of IL-1, IL-33, IL-25, or TNF-α.

[0247] The anti-TSLP antibody or its antigen-binding fragment of the present invention can be administered in combination with antihistamines or antitussive drugs, such as cetirizine hydrochloride, acetaminophen, chlormastine fumarate, promethazine, loratadine, desloratadine, chlorpheniramine, fexofenadine hydrochloride, activastine, astemizole, azelastine, ebastine, epinastine, mizolastine, and tefenadine.

[0248] The TSLP-binding antibody or its antigen-binding fragment described above may be administered in combination with one or more other active agents, which may be selected from, but are 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., β-2 adrenergic receptor agonists, muscarinic antagonists, short-acting β2 receptor agonists, long-acting β2 receptor agonists, short-acting anticholinergic drugs, methylxanthines). Antibiotics, long-acting anticholinergic drugs, antagonists or antibodies against other cytokines or cytokine receptors (e.g., IL-13 antagonists, IL-6 antagonists, IL-1, IL-33, IL-25 or TNF-α antagonists, anti-IgE antibodies, anti-IL31 antibodies, anti-IL31R antibodies, anti-IL13 antibodies, anti-endothelial glycoprotein antibodies, anti-IL1b antibodies, another anti-TSLP antibody or anti-hTSLPR antibody), antibiotics, radiation therapy, leukotriene antagonists (such as montelukast, zafirlukast or premexazol), PDE4 inhibitors (such as roflumilast, xanthan), antihistamines or antitussives.

[0249] In some embodiments, the pharmaceutical composition is administered simultaneously, separately, or sequentially with other treatments, such as before, simultaneously with, or after another pharmaceutically active agent.

[0250] In another aspect, the present invention provides antibodies or antigen-binding fragments thereof, nucleic acids, vectors, host cells, conjugates or multispecific antibodies for (1) inhibiting or blocking the binding of TSLP to TSLPR / IL7Rα, (2) downregulating or eliminating the activity of TSLP, (3) downregulating or blocking the expression of OX40L, (4) inhibiting or blocking the activation and / or proliferation of mast cells, DCs, NKT cells induced by TSLP, (5) inhibiting or blocking the secretion of osteoprotegerin (OPG) induced by TSLP, (6) inhibiting or blocking the secretion of Th2 cytokines such as TARC, CCL22, IL-4, IL-13 or IL-5 induced by TSLP, and / or (7) preventing or treating allergic diseases, hypersensitivity reactions or autoimmune diseases.

[0251] In another aspect, the present invention provides the use of the antibody or its antigen-binding fragment, nucleic acid, vector, host cell, conjugate, or multispecific antibody of the present invention in the preparation of a medicament, said medicament being used for:

[0252] (1) Inhibit or block the binding of TSLP to TSLPR / IL7Rα.

[0253] (2) Downregulate or eliminate the activity of TSLP;

[0254] (3) Downregulate or block the expression of OX40L;

[0255] (4) Inhibit or block TSLP-induced activation and / or proliferation of mast cells, DCs, and NKT cells.

[0256] (5) Inhibit or block TSLP-induced osteoprotegerin (OPG) secretion,

[0257] (6) Inhibit or block TSLP-induced secretion of Th2 cytokines such as TARC, CCL22, IL-4, IL-13, or IL-5; and / or

[0258] (7) Prevention or treatment of allergic diseases, hypersensitivity or autoimmune diseases.

[0259] In some embodiments, when the host cell of the present invention is used to prepare a drug, the host cell contains isolated nucleic acid molecules or carriers as described above.

[0260] In some embodiments, when the antibody of the present invention or its antigen-binding fragment, nucleic acid, vector, host cell, multispecific antibody or conjugate is used to prepare a medicament, the medicament is used in a subject (e.g., a human) to prevent and / or treat asthma, allergic inflammation, allergic reactions or autoimmune diseases.

[0261] In some embodiments, the subject is a mammal, including non-human mammals and humans. In some embodiments, the subject is a human.

[0262] In some embodiments, the antibodies or antigen-binding fragments thereof, nucleic acids, vectors, host cells, multispecific antibodies, conjugates, or drugs described in this invention are used to prevent and / or treat allergic inflammatory diseases, including allergic and non-allergic diseases. These diseases include, but are not limited to, 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), systemic sclerosis, keloids, ulcerative colitis, chronic sinusitis (CRS) and nasal polyps, chronic eosinophilic pneumonia, eosinophilic bronchitis; abdominal diseases such as eosinophilic gastroenteritis, Churg-Strauss syndrome; eosinophil-related gastrointestinal diseases such as eosinophilia / eosinophilic granuloma with polyangiitis, eosinophilic esophagitis and inflammatory bowel disease; urticaria, systemic mastocytosis, cutaneous mastocytosis, and recurrent idiopathic angioedema.

[0263] In some embodiments, the antibodies or antigen-binding fragments, nucleic acids, vectors, host cells, multispecific antibodies, or conjugates described in this invention are used for the prevention and / or treatment of autoimmune-related diseases. In some embodiments, the diseases include, but are not limited to: hyperthyroidism, diabetes, myasthenia gravis, ulcerative colitis, gastritis, pemphigus, primary biliary cirrhosis, multiple sclerosis, lupus erythematosus, rheumatoid arthritis, etc.

[0264] In another aspect, the present invention provides a method for (1) inhibiting or blocking the binding of TSLP to TSLPR / IL7Rα, (2) downregulating or eliminating the activity of TSLP, (3) downregulating or blocking the expression of OX40L, (4) inhibiting or blocking the secretion of osteoprotegerin (OPG) induced by TSLP, (5) inhibiting or blocking the secretion of Th2 cytokines, and (6) inhibiting or blocking the activation and / or proliferation of mast cells, DCs, and NKT cells induced by TSLP, comprising: administering to cells or subjects any of the antibodies or antigen-binding fragments thereof described in the present invention, nucleic acids, vectors, host cells, multispecific antibodies, conjugates, or pharmaceutical compositions thereof.

[0265] Optionally, additional pharmaceutically active agents may be applied simultaneously, before, or after the application of the antibody or its antigen-binding fragment, nucleic acid, vector, host cell, multispecific antibody, conjugate, or pharmaceutical composition.

[0266] In some implementations, the subject is a mammal, including non-human mammals and humans; preferably, the subject is a human.

[0267] Therefore, in another aspect, the present invention provides a method for preventing and / or treating asthma, allergic reactions, allergic inflammation, or autoimmune diseases in a subject, the method comprising administering to the subject in need an effective amount of the antibody or antigen-binding fragment thereof described in the present invention, nucleic acid, vector, host cell, multispecific antibody, conjugate, or pharmaceutical composition thereof.

[0268] The antibodies or antigen-binding fragments thereof, nucleic acids, carriers, host cells, multispecific antibodies, conjugates, and pharmaceutical compositions of the present invention can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalers, sprays, etc. Preferred dosage forms depend on the intended route of administration and therapeutic use. The pharmaceutical compositions of the present invention should be sterile and stable under the conditions of manufacture and storage, and can be prepared as injections.

[0269] Furthermore, the antibody or its antigen-binding fragment of the present invention may be present in the pharmaceutical composition in unit dose form for ease of administration.

[0270] The pharmaceutical compositions of the present invention may include, in a “therapeutic effective amount” or a “preventive effective amount”, an antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, multispecific antibody, or conjugate of the present invention. A “preventive effective amount” refers to an amount sufficient to prevent, stop, or delay the onset of a disease. A “therapeutic effective amount” refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease, for example, from 0.1 mg / ml to 5000 mg / ml.

[0271] In this invention, the subject can be a mammal (including non-human mammals and humans), such as a human.

[0272] Detection methods and kits

[0273] The antibody or its antigen-binding fragment of the present invention can bind to TSLP, thereby enabling it to be used to detect the presence or level of TSLP in a sample.

[0274] Therefore, in another aspect, the present invention provides a kit comprising the antibody of the present invention or its antigen-binding fragment. In some embodiments, the antibody of the present invention or its antigen-binding fragment is labeled with a detectable marker. In a preferred embodiment, the kit further comprises a second antibody that specifically recognizes the antibody of the present invention or its antigen-binding fragment. Preferably, the second antibody further comprises a detectable marker.

[0275] In this invention, the detectable label can be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical, or chemical means. Particularly preferred is that such labels are suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). Such labels are well known in the art and include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), 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, Alexa 750)), acridine esters, and magnetic beads (e.g., The invention includes, but is not limited to, calorimetric markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding avidin (e.g., streptavidin) modified with the aforementioned markers. Patents teaching the use of these markers include, but are not limited to, U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241 (all incorporated herein by reference). The markers covered in this invention can be detected by methods known in the art. For example, radioactive markers can be detected using photographic film or a scintillation calculator, and fluorescent markers can be detected using a photodetector to detect emitted light. Enzyme markers are generally detected by providing an enzyme with a substrate and detecting the reaction product produced by the enzyme's action on the substrate, and calorimetric markers are detected by simple, visually appealing colored markers. In some embodiments, the detectable markers described above can be attached to the recombinant protein of the present invention via connectors of different lengths to reduce potential steric hindrance.

[0276] In another aspect, the present invention provides a method for detecting the presence or level of TSLP in a sample, comprising the step of using an antibody or an antigen-binding fragment thereof of the present invention.

[0277] In a preferred embodiment, the antibody or antigen-binding fragment of the present invention is further labeled with a detectable tag.

[0278] In another preferred embodiment, the method further includes using a reagent with a detectable label to detect the antibody or antigen-binding fragment of the present invention.

[0279] The method can be used for diagnostic purposes or non-diagnostic purposes (e.g., for TSLP-TSLP / IL-7Ra pathway studies, drug screening, histochemical analysis, etc.). In some embodiments, the sample used for non-diagnostic purposes is a cell sample, such as a cell line or ex vivo cell culture.

[0280] In one embodiment, the present invention provides a method for detecting the presence or level of TSLP in a sample, the method comprising contacting the sample with the antibody or antigen-binding fragment thereof, under conditions allowing the formation of a complex between the antibody or antigen-binding fragment thereof and TSLP, and detecting the formation of the complex.

[0281] In another aspect, the present invention provides a method for diagnosing asthma, allergic inflammation, allergic reactions, or autoimmune diseases in a subject, including...

[0282] - The antibody or its antigen-binding fragment, multispecific antibody, or conjugate described in this invention is contacted with a sample from a subject under conditions that allow the antibody or its antigen-binding fragment to form a complex with the TSLP.

[0283] - Detect the formation of the complex.

[0284] Compared with healthy controls, elevated TSLP levels indicate the presence of asthma, allergic inflammation, allergic reactions, or autoimmune diseases.

[0285] Optionally, the subject is a mammal, including non-human mammals and humans. Preferably, the subject is a human.

[0286] Preferably, the allergic inflammation, allergic reaction, or autoimmune disease is as described above.

[0287] In another aspect, the present invention provides the use of the antibodies or antigen-binding fragments thereof, nucleic acids, vectors, host cells, multispecific antibodies, conjugates or pharmaceutical compositions described herein in the preparation of medicaments or kits for the diagnosis of asthma, allergic inflammation, allergic reactions or autoimmune diseases.

[0288] In another aspect, the invention provides the use of the antibody or antigen-binding fragment thereof of the present 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 the following substances: the antibody or antigen-binding fragment thereof described in the invention, nucleic acid, vector, host cell, multispecific antibody, conjugate, or pharmaceutical composition. Optionally, the diagnostic or therapeutic kit further includes instructions for use.

[0289] The antibody or antigen-binding fragments of the present invention exhibit high affinity and good specificity for TSLP. Therefore, the antibody or antigen-binding fragments of 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 retain a high degree of humanization, thus allowing safe administration to human subjects without inducing immunogenic reactions. Therefore, the antibody or antigen-binding fragments of the present invention have significant clinical value.

[0290] Terminology Definition

[0291] In this document, unless otherwise stated, scientific and technical terms used have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, biochemistry, nucleic acid chemistry, and immunology laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0292] The singular forms “a” and “the” used in this invention and in the appended claims include the plural meaning unless the context clearly indicates otherwise. Thus, the singular word “a” includes “one or more”.

[0293] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of one light chain (LC) and one heavy chain (HC)). Antibody light chains can be classified as κ (kappa) and λ (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 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. Constant domains do not directly participate in antibody-antigen binding but exhibit various effector functions, such as mediating the binding of immunoglobulins to host tissues or factors, including 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 highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy / light chain pair form the antigen-binding sites.

[0294] In this document, the CDR contained in the antibody or antigen-binding fragment of the present invention can be determined according to various numbering systems known in the art. In some embodiments, the CDR contained in the antibody or antigen-binding fragment of the present invention is preferably determined by the Kabat, Chothia, AbM, or IMGT numbering systems.

[0295] As used herein, the term “framework region” or “FR” residues refer to those amino acid residues in the antibody variable region other than the CDR residues as defined above.

[0296] As used herein, the term "germline antibody gene" refers to a gene encoding an immunoglobulin not expressed by lymphocytes that has not undergone the genetic rearrangement and maturation process that leads to the expression of a specific immunoglobulin. One advantage offered by the various embodiments of the present invention stems from the understanding that the amino acid sequence encoded by the germline antibody gene retains more of the characteristic amino acid sequence structure of the individual animal species than the amino acid sequence encoded by the mature antibody gene. Therefore, when therapeutically applied to the species, it is less likely to be recognized as a foreign substance by the species.

[0297] The term "antibody" is not limited to any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0298] As used herein, the term “antigen-binding fragment” of an antibody refers to a polypeptide fragment of an antibody, such as a polypeptide fragment of a full-length antibody, which retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen; this fragment is also referred to as the “antigen-binding moiety.” See also Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology 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, diabody, linear antibody, nanobody (e.g., technology from Ablynx), domain antibody (e.g., technology from Domantis), and peptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the peptide. Engineered antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23:1126-1136.

[0299] As used herein, the term "full-length antibody" refers to an antibody composed of two "full-length heavy chains" and two "full-length light chains." A "full-length heavy chain" is a polypeptide chain consisting of 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 in the N-terminal to C-terminal direction; and, optionally, a heavy chain constant region CH4 domain is also included when the full-length antibody is an IgE isotype. Preferably, the "full-length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2, and CH3 in the N-terminal to C-terminal direction. A "full-length light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction. The two pairs of full-length antibody chains are linked together by disulfide bonds between CL and CH1 and between the HRs of the two full-length heavy chains. The full-length antibody of this invention can be derived from a single species, such as humans; it can also be a chimeric antibody or a humanized antibody. The full-length antibody of this invention comprises two antigen-binding sites formed by VH and VL pairs, respectively, which specifically recognize / bind to the same antigen.

[0300] As used herein, the term “Fd fragment” refers to an antibody fragment consisting of VH and CH1 domains; the term “dAb fragment” refers to an antibody fragment consisting of VH domains (Ward et al., Nature 341:544 546 (1989)); the term “Fab fragment” refers to an antibody fragment consisting of VL, VH, CL and CH1 domains; the term “F(ab')2 fragment” refers to an antibody fragment containing two Fab fragments connected by disulfide bridges on the hinge region; the term “Fab' fragment” refers to the fragment obtained by reducing the disulfide bonds connecting the two heavy chain fragments in the F(ab')2 fragment, consisting of a complete light chain and heavy chain Fd fragment (consisting of VH and CH1 domains).

[0301] As used herein, the term "Fv fragment" refers to an antibody fragment consisting of the VL and VH domains of a single arm of the antibody. Fv fragments are generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that six CDRs confer antigen-binding specificity to the antibody. However, even a variable region (such as the Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind to the antigen, although its affinity may be lower than that of a complete binding site.

[0302] As used herein, the term "Fc fragment" refers to an antibody fragment formed by the disulfide bonds between the second and third constant regions of the first heavy chain and the second and third constant regions of the second heavy chain. The Fc fragment of an antibody has various functions but does not participate in antigen binding.

[0303] As used herein, the term “scFv” refers to a single polypeptide chain containing VL and VH domains linked by a linker (see, for example, 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, edited by Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a repeating GGGGS amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in this invention are described by 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 bond may also exist between VH and VL of scFv. As used in this article, the term "di-scFv" refers to an antibody fragment formed by the linking of two scFvs.

[0304] As used herein, the term “biantibody” means that its VH and VL domains are expressed on a single polypeptide chain, but the linker is too short to allow pairing between the two domains on the same chain, thus forcing the domain to pair with the complementary domain of another chain and creating two antigen-binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak RJ et al., Structure 2:1121-1123 (1994)).

[0305] As used in this article, "antibody mimetic" refers to an antibody that binds to an antigen specifically like an antibody, but without the antibody structure. They are typically artificial peptides or proteins with a molar mass of approximately 3 to 20 kDa. Examples include ankyrin repeat protein (DARPin) and fynomer. Designed ankyrin repeat protein (DARPin) can be linked to IgG antibodies, scFv-Fc antibody fragments, or combinations thereof, such as CN104341529A. Anti-IL-17a fynomer binds to anti-IL-6R antibodies, such as WO2015141862A1.

[0306] Each of the above antibody fragments retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen.

[0307] Antigen-binding fragments (e.g., the antibody fragments described above) of a given antibody (e.g., the antibody provided in this invention) can be obtained using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of the antibody can be specifically screened in the same manner as those used for intact antibodies.

[0308] In this article, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.

[0309] As used herein, the terms “monoclonal antibody,” “monoclonal antibody,” and “mAb” have the same meaning and are used interchangeably, referring to an antibody derived from a group of highly homologous antibody molecules (i.e., a group of identical antibody molecules, except for the possibility of spontaneous natural mutations). Monoclonal antibodies exhibit high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies that typically recognize different epitopes on an antigen. Furthermore, the modifier “monoclonal” only indicates that the antibody is derived from a highly homologous group of antibodies and should not be construed as requiring preparation by any specific method.

[0310] As used herein, the term "chimeric antibody" refers to an antibody whose light chain and / or heavy chain is derived from a portion of an antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another portion of the light chain and / or heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but which retains binding activity to the target antigen in any case (Cabilly et al., U.S. Patent 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)).

[0311] In this invention, the intended properties of the antibody include: (1) inhibiting or blocking the binding of TSLP to TSLPR / IL7Rα; (2) downregulating or eliminating the activity of TSLP; (3) downregulating or blocking the expression of OX40L; (4) inhibiting the secretion of Th2 cytokines; and (5) preventing or treating allergic inflammation. The humanized antibody of this invention retains one or more of the above-mentioned intended properties of the parent antibody (human antibody or mouse-human chimeric antibody).

[0312] To prepare humanized antibodies, mouse CDR regions can be inserted into human frame sequences using methods known in the art (see Winter’s U.S. Patent No. 5,225,539; Queen et al.’s U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,762 and 6,180,370; and Lo, Benny, KC, editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004). Alternatively, transgenic animals that do not produce endogenous immunoglobulins after immunization and can generate a complete human antibody library can be used (see, for example, 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; WO02 / 43478). Other methods for humanizing antibodies 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).

[0313] As used herein, the term "degree of humanization" is an indicator used to evaluate the number of non-human amino acid residues in a humanized antibody. The degree of humanization of a humanized antibody can be predicted, for example, by using the DomainGapAlign website on the IMGT platform to predict the homology between the variable region sequence and the human V domain.

[0314] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. The strength or affinity of a specific binding interaction can be expressed by the dissociation equilibrium constant (KD) of the interaction. Here, the term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. The smaller the dissociation equilibrium constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. In some embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific to an antigen) means that the antibody binds to an antigen at a concentration of less than about 10... -8M, for example, less than about 10 -8 M, 10 -9 M, 10 -10 M or 10 -11 M or smaller KD binds to the antigen. In some embodiments, when KD ≤ 10 × 10 -8 When M is present, the antibody or its antigen-binding fragment of the present invention is considered to specifically bind to TSLP.

[0315] The specific binding properties between two molecules can be determined using methods known in the art. One method involves measuring the rate of formation and dissociation of the antigen binding site / antigen complex. Both the “binding rate constant” (ka or kon) and the “dissociation rate constant” (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361:186-187). The ratio of kdis / kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59:439-473). The values ​​of KD, kon, and kdis can be measured using any effective method. In some embodiments, the dissociation constant can be measured using bioluminescent interferometry (e.g., the ForteBio Octet method). Alternatively, surface plasmon resonance techniques (e.g., Biacore) or Kinexa can be used to measure the dissociation constant.

[0316] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0317] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, 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 S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0318] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are identical at a position when the same base or amino acid monomeric subunit occupies the same location (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage 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 × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be made using methods readily available, for example, computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. In addition, the Needleman and Wunsch (J MoI Biol. 48:444-453 (1970)) algorithm, which is integrated into the GCG software package (available at www.gcg.com), can be used to determine the percentage identity between two amino acid sequences using a Blossum62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6. The % identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)).

[0319] As used herein, sequences with “% identity” retain important biological activities, such as antibody binding specificity, of their comparative or source sequences. Sequences with one or more amino acid substitutions, deletions, or additions, or any combination thereof, retain important biological activities, such as antibody binding specificity, of their comparative or source sequences. Nucleotide sequences with “% identity” or differing by no more than 3, 6, 15, 30, or 45 nucleotides can achieve functions similar to their comparative or source sequences, such as the expressed proteins specifically binding to the same antigen or molecule.

[0320] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, 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).

[0321] The twenty common amino acids referred to in this article are written in accordance with conventional usage. See, for example, Immunology-ASynthesis (2nd Edition, E.S. Golub and D.G. Ren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this article, the terms “polypeptide” and “protein” have the same meaning and are used interchangeably. And in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala; arginine by R or Arg; glycine by G or Gly; and glutamine by Q or Gln.

[0322] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintainers, absorption delayers, and preservatives. For example, pH adjusters 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 enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Osmotic pressure maintainers include, but are not limited to, sugars, NaCl, and their analogues. Absorption delayers include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art for stabilizing the desired activity of the active ingredient in a pharmaceutical product, including, but not limited to, monosodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate), etc.

[0323] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease or condition or symptom (e.g., asthma, allergic inflammation, anaphylactic reaction, or autoimmune disease) in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the extent of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the progression of the disease, improving or alleviating the state of the disease, and relieving symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also refer to prolonged survival compared to the expected survival (if no treatment was received).

[0324] As used herein, the term "subject" refers to a mammal, such as a primate, a non-human primate, or a human. In some embodiments, the subject (e.g., a human) suffers from asthma, allergic inflammation, allergic reactions, or an autoimmune disease, or is at risk of suffering from such diseases.

[0325] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for preventing disease (e.g., asthma, allergic inflammation, anaphylaxis, or autoimmune disease) means an amount sufficient to prevent, stop, or delay the onset of the disease (e.g., asthma, allergic inflammation, anaphylaxis, or autoimmune disease); an effective amount for treating disease means an amount sufficient to cure or at least partially stop the disease and its complications in a patient who already has the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the manner of administration of the drug, and other concurrent treatments, etc.

[0326] As used herein, the term “immune cells” includes cells that have a hematopoietic origin and play a role in the immune response, such as lymphocytes, such as B cells and T cells; natural killer cells; and myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0327] 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 selective damage, destruction, or clearance from the body of invasive pathogens, pathogen-infected cells or tissues, cancer cells, or normal human cells or tissues in cases of autoimmunity or pathological inflammation. In this document, the term "antigen-specific T-cell response" refers to an immune response generated by T cells in response to stimulation of the T cell by an antigen specific to that T cell. Non-limiting examples of responses generated by T cells in response to antigen-specific stimulation include T-cell proliferation and the production of cytokines (e.g., IL-2).

[0328] As used herein, the term "effector function" refers to the biological activity attributable to the antibody's Fc region (either the native Fc region or the Fc region of an amino acid sequence variant), which varies with antibody isotype.

[0329] The term "pharmaceutically acceptable" means that when the molecular bulk, molecular fragment, or composition is properly administered to an animal or human, it will not produce adverse, allergic, or other adverse reactions. Specific examples of substances that can serve as pharmaceutically acceptable carriers or components include sugars (such as lactose), starch, cellulose and its derivatives, vegetable oils, gelatin, polyols (such as propylene glycol), alginic acid, etc.

[0330] Beneficial effects of the invention

[0331] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0332] (1) The antibody of the present invention can specifically and with high affinity recognize / bind to TSLP, inhibit or block the binding of TSLP to TSLPPR / IL7RR, and can inhibit or block the proliferative effect of TSLP on Ba / F3 cells in vitro / in vivo, and block the ability of TSLP to activate PBMCs and secrete cytokines. Therefore, the antibody of the present invention can inhibit or block the activation and / or proliferation of mast cells, DCs, and NKT cells induced by TSLP, inhibit or block the expression of OX40L induced by TSLP, the secretion of osteoprotegerin (OPG), or the secretion of Th2 cytokines such as TARC, CCL22, IL-4, IL-13, or IL-5 induced by TSLP. Therefore, the antibody of the present invention has the potential to be used for the prevention and / or treatment of asthma, other allergic reactions, or autoimmune diseases.

[0333] (2) It has good thermal stability, hydrophilicity, isoelectric point and affinity for FcRn.

[0334] (3) Some of the antibodies of the present invention are fully human antibodies that can be safely administered to subjects without causing immunogenic reactions. Therefore, the antibodies of the present invention have significant clinical value.

[0335] abbreviations

[0336] Complementation-determining region in the CDR immunoglobulin variable region

[0337] FR antibody framework region: amino acid residues in the antibody variable region other than CDR residues.

[0338] VH antibody heavy chain variable region

[0339] VL antibody light chain variable region

[0340] IgG immunoglobulin G

[0341] The definition of AbMAbM CDR is derived from Martin's related research (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. ProcNatl Acad Sci USA 86:9268–9272), and this definition method integrates some of the definitions from Kabat and Chothia.

[0342] Kabat is an immunoglobulin matching and numbering system proposed by Elvin A. Kabat (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

[0343] The Chothia immunoglobulin numbering system, proposed by Chothia et al., is a classic rule for identifying the boundaries of CDR regions based on the location of structural loop regions (see, for example, Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883).

[0344] IMGT is based on the international Immunogenetics information system initiated by Lefranc et al. For the numbering system of (IMGT), see Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003.

[0345] mAb monoclonal antibody

[0346] EC50 produces 50% efficacy or a combined concentration

[0347] IC50 produces a concentration that inhibits 50% of the concentration.

[0348] ELISA (Enzyme-Linked Immunosorbent Assay)

[0349] PCR polymerase chain reaction

[0350] HRP (Hydrogen peroxidase)

[0351] TSLP thymic stromal lymphopoietin

[0352] TSLPR thymic stromal lymphopoietin receptor

[0353] IL7Rα interleukin-7 receptor alpha subunit

[0354] TARC thymus activation regulates chemokines

[0355] hFc human IgG antibody Fc fragment

[0356] KD dissociation equilibrium constant

[0357] Complementation-determining region 1 in the variable region of CDR-H1 immunoglobulin heavy chain

[0358] Complementation-determining region 2 in the variable region of CDR-H2 immunoglobulin heavy chain

[0359] Complementation-determining region 3 in the variable region of CDR-H3 immunoglobulin heavy chain

[0360] Complementation-determining region 1 in the variable region of CDR-L1 immunoglobulin light chain

[0361] Complementation-determining region 2 in the variable region of CDR-L2 immunoglobulin light chain

[0362] Complementation-determining region 3 in the variable region of CDR-L3 immunoglobulin light chain Attached Figure Description

[0363] Figure 1 Detection of human TSLPR / IL7Rα dual gene overexpression Ba / F3 cell line

[0364] Figure 2A Detection of the inhibitory activity of chimeric antibody 25A5C5 on the proliferation of Ba / F3-hTSLPR-hIL7Rα cells

[0365] Figure 2B Detection of the inhibitory activity of chimeric antibodies 27C2B6 and 37C2D10 on the proliferation of Ba / F3-hTSLPR-hIL7Rα cells

[0366] Figure 2C Detection of the inhibitory activity of chimeric antibodies 43B1A8 and 90H3H11 on the proliferation of Ba / F3-hTSLPR-hIL7Rα cells

[0367] Figure 3A Affinity assay of recombinant fully human antibodies 25A5C5-hIgG, 27C2B6-hIgG, and 37C2D10-hIgG binding to human TSLP protein.

[0368] Figure 3B Affinity assay of recombinant fully human antibodies 43B1A8-hIgG and 90H3H11-hIgG binding to human TSLP protein

[0369] Figure 4A 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.

[0370] Figure 4B Detection of the inhibitory activity of recombinant fully human antibody 43B1A8-hIgG on the proliferation of Ba / F3-hTSLPR-hIL7Rα cells.

[0371] Figure 4C Detection of the inhibitory activity of recombinant fully human antibody 90H3H11-hIgG on the proliferation of Ba / F3-hTSLPR-hIL7Rα cells.

[0372] Figure 5 Detection of the activity of recombinant fully human antibodies 43B1A8-hIgG and 90H3H11-hIgG in inhibiting TARC secretion by PBMCs

[0373] Figure 6 Thermostability (Tm) of recombinant fully human antibodies 43B1A8-hIgG and 90H3H11-hIgG

[0374] Figure 7A Detection of the inhibitory activity of recombinant fully human antibody 43B1-H2L2 on the proliferation of Ba / F3-hTSLPR-hIL7Rα cells.

[0375] Figure 7B Detection of the inhibitory activity of recombinant fully human antibody 43B1-H6L1 on the proliferation of Ba / F3-hTSLPR-hIL7Rα cells.

[0376] Figure 8 Detection of the activity of recombinant fully human antibody 43B1-H2L2 in inhibiting the secretion of MDC cytokines by PBMC cells.

[0377] Figure 9 Pharmacokinetic analysis of recombinant fully human antibody 43B1-H2L2 in cynomolgus monkeys

[0378] Sequence information

[0379] Information about the sequences involved in this invention is described in the table below.

[0380]

[0381]

[0382] Detailed Implementation

[0383] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).

[0384] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in this invention are substantially based on those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Ausubel et al., A Concise Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995. Those skilled in the art will appreciate that the examples illustrate the invention by way of illustration and are not intended to limit the scope of the invention as claimed.

[0385] Example 1: Preparation of Antigen

[0386] Human TSLP or monkey TSLP was expressed in *E. coli* or mammalian cells. The amino acid sequence of human TSLP is referenced from NP_149024.1 in the NCBI protein database. The amino acid sequence of cynomolgus monkey (Macaca fascicularis) TSLP is referenced from XP_005557555.1 in the NCBI protein database. The antigens used in this application include TSLP expressed in a modified form, such as by fusing six consecutive histidine residues as a tag (TSLP-His) to the C-terminus of the TSLP sequence. The above-mentioned human or monkey TSLP sequences were codon-optimized by GenScript, synthesized in expression vectors (i.e., plasmids containing the complete coding sequence of human TSLP), and expressed and purified in *E. coli* or mammalian HEK293F cells.

[0387] The natural receptor for TSLP is a heterodimer composed of human TSLPR and the human IL7R alpha (IL7Rα) subunit. The sequence references for human TSLPR are (Uniprot: Q9HC73.1) and human IL7R alpha subunit (GenBank: AAR08908.1). The human hIL7Rα-hTSLPR-hFc fusion protein is composed of a portion of the coding sequence (Hinge-CH2-CH3) of the extracellular region of human IL7Rα (E21-D239), the extracellular region of the human TSLPR receptor (Q23-K231), and the human IgG1 Fc region. After codon optimization, it was constructed on the pLVX expression vector. A stable expression cell line of HEK293F was established using the pLVX vector, and the hIL7Rα-hTSLPR-hFc fusion protein was finally purified.

[0388] Example 2: Construction and identification of human TSLPR / IL7Rα dual-gene overexpression cell lines

[0389] 2.1 Construction of human TSLPR / IL7Rα dual-gene overexpression cell lines

[0390] To verify the function of human TSLP antibody in blocking the binding of human TSLP to its receptor human TSLPR / IL7Rα, the complete amino acid sequence encoding human TSLPR (gene ID: UniProtKB / Swiss-Prot: Q9HC73.1, synthesized by Nanjing GenScript Biotech Co., Ltd.) and the complete amino acid sequence encoding the human IL7R alpha subunit (gene ID: GenBank: AAR08908.1, synthesized by Nanjing GenScript Biotech Co., Ltd.) were given to GenScript for codon optimization and cloned into the lentiviral vectors pLVX-IRES-puro and pLVX-IRES-zeocin, respectively. The results were then analyzed according to the literature (Mohammadi Z et al., Mol...). Biotechnol.2015Sep;57(9):793-800) Method for preparing viruses using lentiviral packaging system. The obtained virus was used to infect mouse original B cell line Ba / F3 cells (purchased from Nanjing Kebai). Through puromycin + bleomycin screening and monoclonal selection, a stable monoclonal Ba / F3-hTSLPR-hIL7Rα cell line was obtained.

[0391] 2.2 Detection of human TSLPR / IL7Rα dual gene overexpression Ba / F3 cell line

[0392] Cell lines were identified using flow cytometry (Beckman, CytoFlex; detection antibodies APC-anti-human TSLPR (Biolegend) and APC-anti-human IL7Rα (Biolegend)) to confirm that monoclonal cells correctly expressed human TSLPR and human IL7Rα. Figure 1 As shown, the flow cytometry results indicate that Ba / F3-hTSLPR-hIL7Rα is a monoclonal cell line expressing both genes (nearly 100%) with good homogeneity, and can be used for subsequent experiments.

[0393] Example 3: Fusion of mouse immune system with hybridoma

[0394] 3.1 Mouse Immunization

[0395] Human transgenic mice (H2L2, Harbour BioMed) were immunized multiple times using human TSLP expressed in *E. coli* (NP_149024.1), human TSLP expressed in mammalian cells (NP_149024.1), monkey TSLP expressed in mammalian cells (XP_005557555.1), and plasmids containing the complete coding sequence of human TSLP. Booster immunizations were performed every two weeks for a total of 5-6 times. During the immunization period, serum titers of anti-human TSLP antibodies were measured every two weeks using ELISA (see Example 4.1). Mice with the best titers after multiple rounds of immunization were selected for hybridoma generation using the following protocol.

[0396] 3.2 Fusion Method

[0397] Mouse spleen and lymph node single-cell suspensions were mixed, and the same number of SP2 / 0 myeloma cells were added and mixed thoroughly. The cell mixture was washed and resuspended with electrofusion buffer, and fused using a BTX-ECM2001 electrofusion instrument. Immediately after fusion, the cell suspension was transferred from the fusion chamber to complete fusion medium and incubated at 37°C for 1 hour. The mixture was then incubated at 2 × 10⁻⁶ cells / day. 4 Cells were seeded at a density of 100 cells per well in 96-well plates. After 5 days of culture, the medium was changed using complete fusion medium. The supernatant was collected for hybridoma screening at 7-10 days.

[0398] Example 4: Hybridoma Screening

[0399] 4.1 Individual TSLP combined with ELISA screening

[0400] Soluble human TSLP-His protein was diluted to 1 μg / ml in 1xCBS coating buffer and added to 96-well plates, then incubated overnight at 4°C. The 96-well plates were washed with PBST and blocked with blocking buffer (PBS + 2% BSA) at 37°C for 2 hours. Hybridoma supernatant or an equal volume of blocking buffer was added to the plates, and the plates were incubated at 37°C for 2 hours. Goat anti-rat IgG-HRP was added to the 96-well plates, and the plates were incubated at 37°C for 1 hour. The plates were then washed, and OD was read at 450 nm.

[0401] 4.2 Monkey TSLP combined with ELISA screening

[0402] Monkey TSLP-His protein was diluted to 1 μg / ml in CBS coating buffer and added to 96-well plates, then incubated overnight at 4°C. The 96-well plates were washed with PBST and blocked with blocking buffer PBS + 2% BSA at 37°C for 2 hours. Hybridoma supernatant or an equal volume of blocking buffer was added to the plates, and the plates were incubated at 37°C for 2 hours. Goat anti-rat IgG-HRP was added to the 96-well plates, and the plates were incubated at 37°C for 1 hour, washed, and OD was read at 450 nm.

[0403] 4.3 ELISA screening for blocking the binding of human TSLP-His to chimeric receptor IL7Rα-TSLPR-hFc

[0404] The following protocol was used to determine the binding of human TSLP-His to chimeric receptor IL7Rα-TSLPR-hFc by measuring hybridoma supernatant or purified antibody.

[0405] Soluble hIL7Rα-hTSLPR-hFc protein was diluted in 1xCBS coating buffer and added to 96-well plates, then incubated overnight at 4°C. The 96-well plates were washed with PBST and blocked at 37°C for 2 hours with blocking buffer PBS + 2% BSA. Recombinant mammalian cell-expressed hTSLP-His protein (+ / -) was added to the plates, along with hybridoma supernatant or an equal volume of blocking buffer, and the plates were incubated at 37°C for 2 hours. Mouse anti-His-HRP was added to the 96-well plates, and the plates were incubated at 37°C for 1 hour, washed, and OD read at 450 nm. Hybridomas with strong inhibition rates were selected as candidate clones.

[0406] Based on the activities of affinity ELISA and competition ELISA, positive clones were selected and subclonal sorted using a limiting dilution method to obtain subclones. Ultimately, hybridoma subclones capable of binding to human TSLP-His and monkey TSLP-His, and simultaneously blocking the binding of human TSLP-His to the chimeric receptor hIL7Rα-hTSLPR-hFc, were obtained. As shown in Table 1, all five monoclonal samples exhibited strong binding affinity to human TSLP (hTSLP) and monkey TSLP (cTSLP), and all were able to block the binding of human TSLP to its receptor hIL7Rα-hTSLPR-hFc, with inhibition rates exceeding 60%.

[0407] Table 1. Subclonal Screening for Anti-TSLP Hybridoma

[0408]

[0409]

[0410] Example 5: Preparation of anti-TSLP chimeric antibody

[0411] Control antibody expression: The control antibody sequence was referenced from the chEMBL database (ID: CHEMBL3707229). The heavy and light chain base sequences of the control antibody were synthesized in the pTT5 expression vector and transiently transfected and expressed in CHO-S cells (purchased from Thermo). The control antibody was then purified by affinity using Protein A (MabSelect SuRe, GE).

[0412] Hybridoma monoclonal antibodies were cultured in serum-free medium, yielding 50 mL of supernatant. The supernatant was then purified using Protein A (MabSelectSuRe, GE) to obtain chimeric hybridoma antibodies. Because the H2L2 mouse antibody constant region was genetically modified to resemble the rat constant region, the purified antibodies were chimeric antibodies carrying the rat Fc in the fully human variable region. The purified antibodies were quantified spectrophotometrically, yielding chimeric antibodies 25A5C5, 27C2B6, 37C2D10, 43B1A8, and 90H3H11.

[0413] Example 6: ELISA detection of anti-TSLP chimeric antibody and TSLP affinity

[0414] The affinity of chimeric antibodies 25A5C5, 27C2B6, 37C2D10, 43B1A8, and 90H3H11 for human or monkey TSLP was detected using ELISA. The specific method is briefly described below: Human TSLP-His or monkey TSLP-His antigen was coated onto 96-well plates and incubated overnight at 4°C. Then, serially diluted antibodies of different concentrations were added and incubated for 2 hours. Next, goat anti-rat Fc-HRP secondary antibody was added and incubated for 1 hour. The absorbance at 450 nm was then measured using a microplate reader.

[0415] The test results are shown in Table 2. The affinity of human TSLP was as follows: the chimeric antibodies 25A5C5 and 27C2B6 were roughly equivalent to the control antibody, while the antibodies 43B1A8 and 90H3H11 showed higher affinity than the control antibody. The antibodies 25A5C5 and 90H3H11 showed almost no binding to monkey TSLP; however, the antibodies 37C2D10, 27C2B6, and 43B1A8 all showed strong binding to monkey TSLP.

[0416] Table 2. Affinity of anti-TSLP chimeric antibodies to TSLP

[0417]

[0418] Example 7: Competitive ELISA detection of anti-TSLP chimeric antibody blocking TSLP / hIL7Rα-hTSLPR-hFc binding activity

[0419] Human TSLP activates downstream signaling pathways by binding to the human hIL7Rα-hTSLPR-hFc heterodimeric receptor. The activity of chimeric antibodies in blocking the binding of the chimeric receptor hIL7Rα-hTSLPR-hFc to the antigen was detected using a competitive ELISA method. The specific steps are described in Example 4.3, where serially diluted chimeric antibodies were added to the wells of 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.

[0420] Table 3: Competitive ELISA Activity of Anti-TSLP Chimeric Antibodies

[0421] Antibody 25A5C5 27C2B6 37C2D10 43B1A8 90H3H11 control antibody IC50(nM) 0.89 0.77 0.51 1.33 1.65 0.93

[0422] Example 8: Detection of Ba / F3-hTSLPR-hIL7Rα cell proliferation inhibition by anti-TSLP chimeric antibody

[0423] The activity of the anti-TSLP chimeric antibody was detected using a Ba / F3-hTSLPR-hIL7Rα cell proliferation inhibition method. Ba / F3-hTSLPR-hIL7Rα cells express hTSLPR and hIL7Rα receptor proteins on their surface. The extracellular dimer of these two receptor proteins can bind to human TSLP, while the intracellular region can further transduce signals, activating intracellular STAT5 phosphorylation and promoting the proliferation of Ba / F3-hTSLPR-hIL7Rα cells.

[0424] Specifically: Centrifuge an appropriate amount of the Ba / F3-hTSLPR-hIL7Rα stable cell line and wash twice with 1640+10% FBS medium to remove recombinant mouse IL3 used in the medium. Incubate hTSLP-His (+ / -) with purified anti-TSLP chimeric antibody / or control anti-TSLP antibody (control antibody) in each well at room temperature for 30 minutes. Add 1.5×10⁻⁶ cells / well. 4 Ba / F3-hTSLPR-hIL7Rα cells / well were cultured for 3 days. CCK8 (RHINO BIO, QDY-003-D) was added to each well, and OD was read at 450 nm. Data were exported and Prism Graphpad software was used to analyze the inhibitory response of the chimeric antibody to cell proliferation.

[0425] like Figure 2A-2C As shown in Table 4, all five chimeric antibodies significantly inhibited the proliferation of Ba / F3-hTSLPR-hIL7Rα cells.

[0426] Table 4: Detection of the activity of chimeric antibodies in blocking the proliferation of human TSLP-induced Ba / F3-hTSLPR-hIL7Rα cells

[0427] Antibody 25A5C5 27C2B6 37C2D10 43B1A8 90H3H11 control antibody EC50(nM) 0.11 1.22 0.28 0.10 0.25 0.05

[0428] Example 9: Amplification of the variable region of anti-TSLP chimeric antibody

[0429] Hybridoma cells cultured to 2×10 6Approximately 10 hybridoma cells were lysed using TRIzol reagent (Thermo Fisher Sci. Cat#15596026) to extract RNA, and first-strand cDNA was synthesized using a cDNA reverse transcription kit (Thermo Fisher Sci. Cat#18080-200). Following the methods of IMGT and AbM, and based on the analysis of all murine antibody sequences, multiple pairs of upstream primers were designed for the variable region, using CH1 homologous sequences as downstream primers. The light and heavy chain variable regions of the antibody were amplified by PCR using a primer pooling method. The PCR products were purified using a DNA purification kit (Qiagen, Cat#28104) and cloned into the pTT-5 vector. Approximately 10 clones from each ligation reaction were sequenced to obtain the variable region sequence, which was then further analyzed using the IMGT and AbM databases.

[0430] Table 5: Amino acid sequences and their numbers of the variable region and CDR of anti-human TSLP chimeric antibodies

[0431]

[0432]

[0433] Example 10: Expression, purification, and affinity binding activity assay of recombinant antibodies

[0434] The amino acid sequences of the variable regions of the light chains 25A5C5, 27C2B6, 37C2D10, 43B1A8, and 90H3H11 were linked to the amino acid sequences of the constant region of the light chain κ (SEQ ID NO: 16), and the amino acid sequences of the variable regions of the heavy chains were linked to the amino acid sequences of the constant region of the IgG1 heavy chain (SEQ ID NO: 14). The corresponding nucleotide sequences were then constructed into the pTT5 vector. The pTT5 vectors corresponding to the heavy and light chains of each recombinant fully human antibody were simultaneously transfected into CHO-S (purchased from Thermo). The supernatant was purified using Protein A (MabSelect SuRe, GE). The purified recombinant fully human antibodies were named 25A5C5-hIgG, 27C2B6-hIgG, 37C2D10-hIgG, 43B1A8-hIgG, and 90H3H11-hIgG, respectively, and their protein content was quantified spectrophotometrically.

[0435] The affinity of recombinant fully human anti-TSLP antibodies for recombinant human or monkey TSLP-His expressed in mammals was determined. The specific experimental procedures are briefly described below: Human TSLP-His or monkey TSLP-His antigen was coated into 96-well plates and incubated overnight at 4°C. Then, serially diluted antibodies of different concentrations were added and incubated for 2 hours. HRP-labeled goat anti-human Fc secondary antibody was added, and after incubation for 1 hour, the antibody was detected at an A450 nm wavelength.

[0436] The results are as follows Figures 3A-3B As shown in Table 6, all five candidate antibodies were able to bind to human TSLP. Furthermore, the hTSLP affinity of 37C2D10-hIgG, 43B1A8-hIgG, and 90H3H11-hIgG was stronger than that of the control antibody. The hTSLP affinity of 25A5C5-hIgG and 27C2B6-hIgG was comparable to that of the control antibody. The hTSLP affinity of 27C2B6-hIgG, 37C2D10-hIgG, and 43B1A8-hIgG was essentially equivalent to that of monkey TSLP.

[0437] Table 6. Binding affinity of recombinant fully human antibodies to human and monkey TSLP.

[0438]

[0439] Example 11: Detection of dynamic affinity between anti-TSLP fully human antibody and TSLP

[0440] Fortebio is a commonly used dynamic affinity assay device, used to detect the dynamic affinity of the anti-TSLP fully human antibody to TSLP. The method is briefly described below: Human or monkey TSLP antigens were serially diluted with PBST to obtain 100 nM, 50 nM, 25 nM, 12.5 μM, 6.25 nM, 3.125 nM, 1.5625 nM, and 0 nM. The ProA biosensor (Pall Life Sciences) was pre-wetted with PBST buffer before use. The recombinant fully human antibody was diluted to 5 μg / mL with PBST and immobilized on the ProA sensor. The antibody-immobilized sensor was then equilibrated in PBST buffer for 60 s to obtain a baseline, then transferred to antigen dilution buffer for binding for 60 s, and then dissociated in PBST for 180 s. After one analytical cycle, the sensor was regenerated with 10 mM Gly (pH 1.5). Using Date Analysis version 11.0 (Pall), a 1:1 model analysis was performed to determine the binding (Ka) and dissociation (Kd) rate constants and to calculate the dissociation equilibrium constant (KD).

[0441] As shown in Table 7, the KD values ​​of 25A5C5-hIgG, 43B1A8-hIgG, and 90H3H11-hIgG binding to human TSLP were lower than those of the control antibody, indicating stronger affinity, consistent with the affinity ELISA results. Regarding monkey TSLP binding, 25A5C5-hIgG showed no affinity; while 37C2D10-hIgG, 90H3H11-hIgG, and 43B1A8-hIgG showed good binding, with affinity around 10. -8 M to 10 -9 M level.

[0442] Table 7: Dynamic affinity analysis of fully human antibodies

[0443]

[0444] Example 12: Inhibition of Ba / F3-hTSLPR-hIL7a cell proliferation by recombinant fully human antibody

[0445] The activity of the recombinant fully human antibody was detected using the Ba / F3-hTSLPR-hIL7a cell proliferation inhibition method. The method was the same as described in Example 8 above.

[0446] The results are shown in Table 8. 27C2B6-hIgG and 37C2D10-hIgG inhibited the proliferation of Ba / F3-hTSLPR-hIL7Rα cells induced by human TSLP. The inhibitory activity of 25A5C5-hIgG and 90H3H11-hIgG on the proliferation of Ba / F3-hTSLPR-hIL7Rα cells induced by human TSLP was roughly equivalent to that of the control antibody. However, the inhibitory activity of 43B1A8-hIgG was significantly stronger than that of the control antibody, approximately 0.25 times greater. Figure 4A -C is shown.

[0447] Table 8: Inhibitory activity of recombinant fully human antibody against Ba / F3-hTSLPR-hIL7Rα cell proliferation

[0448]

[0449] Example 13: Detection of the activity of recombinant fully human antibody in inhibiting the secretion of thymus activation regulator factor (TARC) by human TSLP-induced PBMCs

[0450] The functional activity of a fully human TSLP antibody in primary cells was assessed by inhibiting the secretion of TARC (a Th2 cytokine) by human TSLP-induced TARC in human PBMCs. PBMCs contain CD11. + Studies have shown that TSLP can bind to the receptor TSLPR / IL7Rα in dendritic cells (DCs) and activate CD11. +DC cells upregulate OX40L and promote CD11 + DC cells secrete Th2 cytokines (such as TARC and CCL22). Anti-TSLP antibodies can block the binding of TSLP to TSLPR / IL7Rα on the surface of DC cells, thereby blocking the activation of DC cells and the secretion of Th2 cytokines by DC cells.

[0451] The method is briefly described as follows: Human peripheral blood PBMCs were separated using Ficoll separation buffer (GE). hTSLP-His (+ / -) was incubated with recombinant fully human antibody or control antibody for 30 minutes at room temperature, and 2×10⁻⁶ antibodies were added. 5 PBMC cells / well were cultured for 48 hours. The supernatant was harvested and human TARC production was analyzed by ELISA. The inhibition of TARC secretion by hybridoma supernatant or purified antibody was also determined. TARC levels in the supernatant were detected using a TARC ELISA kit (Sinosure).

[0452] The results are shown in Table 9 and Figure 5 The results showed that the EC50 values ​​of the fully human antibodies 43B1A8-hIgG and 90H3H11-hIgG were both lower than those of the control antibody, indicating that their activity in inhibiting TSLP-induced TARC secretion in PBMC cells was higher than that of the control antibody molecule.

[0453] Table 9: Recombinant fully human antibody inhibits TARC secretion induced by human TSLP in PBMCs

[0454] Antibody 43B1A8-hIgG 90H3H11-hIgG control antibody EC50(nM) 0.44 0.42 0.51

[0455] Example 14: Determination of Tm value of recombinant fully human antibody.

[0456] The Tm value of anti-TSLP antibody was determined using the DSF (Differential Fluorescence Sampling) method. The specific experimental procedure is as follows: 12.5 μl of 40×SYPRO Orange dye (purchased from Life Technologies, catalog number: 56651), 5 μl of 1 mg / ml fully human TSLP antibody (diluted in PBS), and 7.5 μl of sterile water were mixed in an EP tube. This sample mixture was then added to a Q-PCR system (AB Applied Biosystems ABI, 7500) for reaction. The Q-PCR parameters were set as follows: Target (ROX), program (25℃, 3 min; 1% rate, 95℃; 95℃, 2 min). The results were imported into Graph Prism software to calculate the V50 value. Figure 6As shown in Table 10, the Tm values ​​of 43B1A8-hIgG (74.72℃) and 90H3H11-hIgG (72.58℃) are higher than the Tm of the control antibody (66.47℃), which clearly indicates that the fully human antibody prepared in this invention has better thermal stability.

[0457] Table 10: Tm values ​​of recombinant fully human TSLP antibodies

[0458] Antibody control antibody 43B1A8-hIgG 90H3H11-hIgG Tm (°C) 66.47 74.72 72.58

[0459] Example 15: Determination of the hydrophobicity of recombinant fully human antibodies

[0460] Hydrophobicity comparison was performed using an Agilent 1260 HPLC system with a TOSOH Tskgel Buty-NPR (2.5) column. To compare the differences in hydrophobicity among the three antibodies, they were directly injected for analysis. Mobile phase A: 1.5M (NH4)2SO4; Mobile phase B: 25mM Na2HPO4 (pH 7.0) + 25% IPA. The retention times of the three antibodies are shown in Table 11. Longer retention times indicate stronger hydrophobicity. 90H3H11-hIgG showed hydrophilicity comparable to the control antibody; 43B1A8-hIgG showed better hydrophilicity than the control antibody, indicating that the process development and antibody aggregation were superior to the control antibody.

[0461] Table 11: Hydrophobicity detection of recombinant fully human antibodies

[0462] Antibody control antibody 43B1A8-hIgG 90H3H11-hIgG Retention time (minutes) 13.93 12.2 13.61

[0463] Example 16: Obtaining recombinant and modified fully human antibodies 43B1-H6L1 and 43B1-H2L2

[0464] The in vivo half-life of an antibody is closely related to its isoelectric point, affinity for FcRn, glycosylation modification, and immunogenicity. To prolong the half-life of the 43B1A8-hIgG antibody, the affinity for FcRn was enhanced through modification of the heavy and light chain antibody amino acid sequences.

[0465] Modification scheme 1: hIgG1 Fc (SEQ ID NO: 14) is replaced with IgG4 (SEQ ID NO: 70) containing two amino acid mutations. The mutation characteristics are that amino acid 434 of IgG4 (Eu numbering system) is changed from N to A, and amino acid 228 is changed from S to P (Eu numbering system).

[0466] Modification scheme 2: The 434th amino acid of hIgG1 Fc (SEQ ID NO: 14) is replaced with A (Eu numbering system) to obtain SEQ ID NO: 15; and the 16th R of FR1 of the heavy chain of 43B1A8-hIgG is mutated to G (Chothia numbering system); and the 79th R of FR3 of the light chain of 43B1A8-hIgG is mutated to Q (Chothia numbering system).

[0467] The antibodies obtained from the first and second modifications were named 43B1-H6L1 and 43B1-H2L2, respectively.

[0468] The heavy chain variable region sequence of 43B1-H2L2 is SEQ ID NO: 68; the heavy chain constant region sequence is SEQ ID NO: 15; the light chain variable region sequence is SEQ ID NO: 69; and the light chain constant region sequence is SEQ ID NO: 16.

[0469] The heavy chain variable region sequence of 43B1-H6L1 is SEQ ID NO: 40; the heavy chain constant region sequence is SEQ ID NO: 70; the light chain variable region sequence is SEQ ID NO: 41; and the light chain constant region sequence is SEQ ID NO: 16.

[0470] Table 12: Amino acid sequences and their SEQ ID NOs of antibodies 43B1-H6L1 and 43B1-H2L2

[0471] Antibody Heavy chain Heavy chain variable region Heavy chain constant region Light chain Light chain variable region Light chain constant region 43B1A8-hIgG 66 40 14 67 41 16 43B1-H2L2 71 68 15 72 69 16 43B1-H6L1 73 40 70 67 41 16

[0472] The heavy and light chains of the two modified molecules, 43B1-H2L2 and 43B1-H6L1, were used to construct pTT5 expression vectors. After plasmid extraction, the vectors were transfected into CHO-S cells (purchased from Thermo). After approximately 10 days of culture, the cell supernatant was purified using Protein A (MabSelect SuRe, GE). The protein content of the purified recombinant fully human antibody was quantified using spectrophotometry.

[0473] Example 17: Detection of dynamic affinity of recombinant fully human antibodies 43B1-H6L1 and 43B1-H2L2 with TSLP

[0474] The dynamic affinity of the fully human anti-TSLP antibody for TSLP was detected using Fortebio assays. The method was the same as described in Example 11 above. The results are shown in Table 13. The dynamic affinity of the 43B1-H6L1 and 43B1-H2L2 human antibody molecules for hTSLP was not weaker than that of the control antibody.

[0475] Table 13: Dynamic affinity analysis of modified fully human antibody molecules

[0476]

[0477] Example 18: Detection of the isoelectric point (PI) of recombinant fully human antibodies 43B1-H6L1 and 43B1-H2L2

[0478] The isoelectric points of antibodies 43B1-H6L1, 43B1-H2L2, and 43B1A8-hIgG were detected using isoelectric focusing. The method is briefly described below: The Maurice isoelectric focusing system from ProteinSimple, combined with its capillary cartridge, was used for analysis. To compare the differences in the isoelectric points of the three antibodies, they were diluted with water, and a pH gradient was formed using a 4% final concentration of the amphoteric electrolyte 3-10 (GE). The results are shown in Table 14. The isoelectric points of 43B1-H6L1 and 43B1-H2L2 were 1.3 and 0.4 lower than those of 43B1A8-hIgG, respectively, indicating the success of the modification scheme.

[0479] Table 14: Comparison of isoelectric points (PI) of recombinant fully human antibodies

[0480] Antibody 43B1-H2L2 43B1-H6L1 43B1A8-hIgG isoelectric point 8.2 7.3 8.6

[0481] Example 19: Detection of dynamic affinity between recombinant fully human antibodies 43B1-H6L1 and 43B1-H2L2 and FcRn

[0482] The dynamic affinity of a modified fully human anti-TSLP antibody for FcRn was detected using Fortebio. The method is briefly described below: The anti-TSLP antibody was serially diluted with PBST (pH 6.0) to obtain concentrations of 200 nM, 100 nM, 50 nM, 25 nM, 12.5 μM, 6.25 nM, 3.125 nM, 1.5625 nM, and 0 nM. The SA biosensor (Pall Life Sciences) was 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 equilibrated in PBST (pH 6.0) buffer for 60 s to obtain a baseline, then transferred to antibody dilution buffer for binding for 60 s, and then dissociated in PBST (pH 6.0) for 60 s. After one analytical cycle, the sensor was regenerated with PBST (pH 7.4). Using Date Analysis version 11.0 (Pall), a 1:1 model analysis was performed to determine the binding (Ka) and dissociation (Kd) rate constants and to calculate the dissociation equilibrium constant (KD).

[0483] As shown in Table 15, the dynamic affinity KD of FcRn of 43B1-H6L1 and 43B1-H2L2 is less than that of the control antibody, indicating that the affinity of FcRn is about twice that of the control antibody.

[0484] Table 15: FcRn affinity analysis of modified fully human antibody molecules

[0485]

[0486] 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

[0487] The activities of recombinant fully human antibodies 43B1-H6L1 and 43B1-H2L2 were detected using the Ba / F3 cell proliferation inhibition method. The method was the same as described in Example 8 above, and the EC50 of the antibody activity was analyzed based on the data. The results are shown in Table 16 and... Figure 7A , 7B As shown, 43B1-H2L2 and 43B1-H6L1 can inhibit the proliferation of Ba / F3-hTSLPR-hIL7Rα cells induced by human TSLP, and their EC50 values ​​are comparable to those of the control antibody, indicating that the fully human antibodies 43B1-H2L2 and 43B1-H6L1 have comparable ability to inhibit the cell activity of TSLP.

[0488] Table 16: Inhibitory activity of recombinant fully human antibody molecules against Ba / F3-hTSLPR-hIL7Rα cell proliferation

[0489]

[0490] Example 21: Detection of the activity of recombinant fully human antibody in inhibiting the secretion of macrophage-derived chemokines (MDCs) by human TSLP-induced PBMCs

[0491] The functional activity of fully human TSLP antibodies on primary cells was evaluated by inhibiting the secretion of MDC by human PBMCs induced by human TSLP. PBMCs contain dendritic cells (DCs). Studies have shown that TSLP can bind to the cell surface receptor TSLPR / IL7Rα, activating DCs, upregulating OX40L, and promoting the secretion of Th2 cytokines (such as TARC and MDC) by DCs. Anti-TSLP antibodies can block the binding of TSLP to TSLPR / IL7Rα on the surface of DCs, thereby blocking the activation of DCs and the secretion of Th2 cytokines by DCs.

[0492] The method is briefly described as follows: Human peripheral blood PBMCs were separated using Ficoll separation buffer (GE). hTSLP-His (+ / -) was incubated with recombinant fully human antibody or control antibody for 30 minutes at room temperature, and 2×10⁻⁶ antibodies were added. 5 PBMC cells / well were cultured for 120 hours. The supernatant was harvested and human MDC production was analyzed by ELISA, and the inhibition of MDC secretion by the antibody was determined. TARC levels in the supernatant were detected using an MDC ELISA kit (Raybiotech).

[0493] The results are shown in Table 17 and Figure 8 As shown, both 43B1-H2L2 and the control antibody significantly inhibited the production of TSLP-induced MDC factors, with EC50 values ​​of 7.84 pM and 5.63 pM, respectively.

[0494] Table 17: Recombinant fully human antibody inhibits human TSLP-induced MDC secretion by PBMCs

[0495] Antibody 43B1-H2L2 control antibody EC50(pM) 7.84 5.63

[0496] Example 22: Pharmacokinetic Analysis of Recombinant Fully Human Antibody in Cynomolgus Monkeys

[0497] The 43B1-H2L2 candidate molecule in this application carries an N434A mutation, the purpose of which is to affect its affinity for FcRn, thereby prolonging its drug metabolic half-life in vivo. Therefore, this embodiment uses subcutaneous injection to study the pharmacokinetics of the 43B1-H2L2 molecule in cynomolgus monkeys and compares it with the pharmacokinetics of the control antibody. The methods and results are as follows: Four cynomolgus monkeys (Macaca fascicularis, from Hainan Jingang Biotechnology Co., Ltd.) were selected and divided into two groups, one male and one female in each group. The subcutaneous injection dose was 5 mg / kg. Blood samples were collected at 0:00, and at 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. After the blood samples were left at room temperature for 1 hour to allow clotting, they were centrifuged to obtain serum samples, which were then frozen at -80℃ for testing. The antibody levels in serum were measured by ELISA, and the results are analyzed below: Pharmacokinetic parameters and curves for a single subcutaneous administration are shown in Table 18 and... Figure 9 These results show that the 43B1-H2L2 antibody has a longer half-life and a higher total blood drug AUC, approximately twice that of the control antibody.

[0498] Table 18: Pharmacokinetic Study of Recombinant Fully Human Antibody Subcutaneously Administered to Cynomolgus Monkeys

[0499] PK parameters <![CDATA[t 1 / 2 (h)]]> <![CDATA[T max (h)]]> <![CDATA[C max (ng / ml)]]> <![CDATA[AUC (0-t) (hr*ng / ml)]]> 43B1-H2L2 549.3731 48 67373.4 47271741 control antibody 267.3537 72 75870.56 25600470

[0500] Although specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the present invention. The scope of protection of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. An antibody or antigen-binding fragment thereof that binds to thymic stromal lymphopoietin (TSLP), wherein, The antibody or its antigen-binding fragment comprises: (1) VH and VL, defined according to the IMGT numbering system: (1a) The VH comprises CDR-H1 with sequence SEQ ID NO: 42, CDR-H2 with sequence SEQ ID NO: 43, and CDR-H3 with sequence SEQ ID NO: 44; and, The VL includes CDR-L1 with the sequence SEQ ID NO: 45, CDR-L2 with the sequence SEQ ID NO: 46, and CDR-L3 with the sequence SEQ ID NO: 47; (1b) The VH comprises CDR-H1 with sequence SEQ ID NO: 3, CDR-H2 with sequence SEQ ID NO: 4, and CDR-H3 with sequence SEQ ID NO: 5; and, The VL includes CDR-L1 with the sequence SEQ ID NO: 6, CDR-L2 with the sequence SEQ ID NO: 7, and CDR-L3 with the sequence SEQ ID NO: 8; (1c) The VH comprises CDR-H1 with sequence SEQ ID NO: 19, CDR-H2 with sequence SEQ ID NO: 20, and CDR-H3 with sequence SEQ ID NO: 21; and, The VL includes CDR-L1 with the sequence SEQ ID NO: 22, CDR-L2 with the sequence SEQ ID NO: 23, and CDR-L3 with the sequence SEQ ID NO: 24; (1d) The VH comprises CDR-H1 with sequence SEQ ID NO: 32, CDR-H2 with sequence SEQ ID NO: 33, and CDR-H3 with sequence SEQ ID NO: 34; and, The VL includes CDR-L1 with the sequence SEQ ID NO: 35, CDR-L2 with the sequence SEQ ID NO: 23, and CDR-L3 with the sequence SEQ ID NO: 24; or (1e) The VH comprises CDR-H1 with sequence SEQ ID NO: 55, CDR-H2 with sequence SEQ ID NO: 56, and CDR-H3 with sequence SEQ ID NO: 57; and, The VL includes CDR-L1 with the sequence SEQ ID NO: 58, CDR-L2 with the sequence SEQ ID NO: 59, and CDR-L3 with the sequence SEQ ID NO: 60; (2) VH and VL, defined according to the AbM numbering system: (2a) The VH comprises CDR-H1 with sequence SEQ ID NO: 48, CDR-H2 with sequence SEQ ID NO: 49, and CDR-H3 with sequence SEQ ID NO: 50; and, The VL includes CDR-L1 with the sequence SEQ ID NO: 51, CDR-L2 with the sequence SEQ ID NO: 52, and CDR-L3 with the sequence SEQ ID NO: 47; (2b) The VH comprises CDR-H1 with sequence SEQ ID NO: 9, CDR-H2 with sequence SEQ ID NO: 10, and CDR-H3 with sequence SEQ ID NO: 11; and, The VL includes CDR-L1 with the sequence SEQ ID NO: 12, CDR-L2 with the sequence SEQ ID NO: 13, and CDR-L3 with the sequence SEQ ID NO: 8; (2c) The VH comprises CDR-H1 with sequence SEQ ID NO: 25, CDR-H2 with sequence SEQ ID NO: 26, and CDR-H3 with sequence SEQ ID NO: 27; and, The VL includes CDR-L1 with the sequence SEQ ID NO: 28, CDR-L2 with the sequence SEQ ID NO: 29, and CDR-L3 with the sequence SEQ ID NO: 24; (2d) The VH comprises CDR-H1 with sequence SEQ ID NO: 36, CDR-H2 with sequence SEQ ID NO: 37, and CDR-H3 with sequence SEQ ID NO: 38; and, The VL includes CDR-L1 with the sequence SEQ ID NO: 39, CDR-L2 with the sequence SEQ ID NO: 29, and CDR-L3 with the sequence SEQ ID NO: 24; or (2e) The VH comprises CDR-H1 with sequence SEQ ID NO: 61, CDR-H2 with sequence SEQ ID NO: 62, and CDR-H3 with sequence SEQ ID NO: 63; and, The VL includes CDR-L1 with sequence SEQ ID NO: 64, CDR-L2 with sequence SEQ ID NO: 65, and CDR-L3 with sequence SEQ ID NO:

60.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein, The antibody or its antigen-binding fragment binds to human TSLP and / or monkey TSLP.

3. The antibody or antigen-binding fragment thereof according to claim 2, wherein, The antibody or its antigen-binding fragment comprises: (a) VH of the sequence shown in SEQ ID NO: 68 and VL of the sequence shown in SEQ ID NO: 69; (b) VH of the sequence shown in SEQ ID NO: 1 and VL of the sequence shown in SEQ ID NO: 2; (c) VH of the sequence shown in SEQ ID NO: 17 and VL of the sequence shown in SEQ ID NO: 18; (d) VH of the sequence shown in SEQ ID NO: 30 and VL of the sequence shown in SEQ ID NO: 31; (e) VH of the sequence shown in SEQ ID NO: 40 and VL of the sequence shown in SEQ ID NO: 41; or (f) VH of the sequence shown in SEQ ID NO: 53 and VL of the sequence shown in SEQ ID NO:

54.

4. The antibody or its antigen-binding fragment according to claim 1, wherein, The antibody or its antigen-binding fragment is a chimeric antibody, a humanized antibody, or a fully human antibody.

5. The antibody or antigen-binding fragment thereof according to claim 1, wherein, The antibody or its antigen-binding fragment further comprises: (a) The heavy chain constant region (CH) of human immunoglobulins; and / or (b) Light chain constant region (CL) of human immunoglobulin.

6. The antibody or antigen-binding fragment thereof according to claim 5, wherein, The heavy chain constant region is the IgG heavy chain constant region.

7. The antibody or antigen-binding fragment thereof according to claim 5, wherein, The heavy chain constant region is the IgG1, IgG2, IgG3, or IgG4 heavy chain constant region.

8. The antibody or antigen-binding fragment thereof according to claim 5, wherein, The antibody or its antigen-binding fragment contains the constant region of the human IgG1 heavy chain.

9. The antibody or antigen-binding fragment thereof according to claim 5, wherein, The light chain constant region is the κ or λ light chain constant region.

10. The antibody or antigen-binding fragment thereof according to claim 5, wherein, The antibody or its antigen-binding fragment contains a constant region of the human κ light chain.

11. The antibody or antigen-binding fragment thereof according to claim 5, wherein, The heavy chain constant region includes: (1) The human IgG1 heavy chain constant region or a variant thereof, wherein, according to the EU numbering system, the variant is mutated at at least one site of sites 234, 235, 237, 265, 297, 331, 329, or 434; or (2) CH or a variant thereof shown in SEQ ID NO: 14, which, according to the EU numbering system, contains N297A and / or N434A; (3) CH as shown in SEQ ID NO: 15; or (4) The human IgG4 heavy chain constant region or a variant thereof, wherein, according to the EU numbering system, the variant is mutated at at least one site at sites 228 and 434; or (5) CH as shown in SEQ ID NO: 70; and / or The light chain constant region or a variant thereof: (6) Contains the κ light chain constant region; or (7) Contains the light chain constant region (CL) shown in SEQ ID NO:

16.

12. The antibody or antigen-binding fragment thereof according to claim 11, wherein, The human IgG1 heavy chain constant region variant contains at least one of the following mutations: L234A, L235A, D265A, N297A, L234F, L235E, P331S, P329G, N434A, N434Y, N434F, N434W, N434S, N434G, N434H, and N434Q.

13. The antibody or antigen-binding fragment thereof according to claim 11, wherein, The human IgG1 heavy chain constant region variant contains at least one of the following mutations: L234A, L235A, G237A, and N434A.

14. The antibody or antigen-binding fragment thereof according to claim 11, wherein, The mutations in the human IgG1 heavy chain constant region variants are L234A, L235A, and G237A.

15. The antibody or antigen-binding fragment thereof according to claim 11, wherein, The mutations in the human IgG1 heavy chain constant region variants are L234A, L235A, G237A, and N434A.

16. The antibody or antigen-binding fragment thereof according to claim 11, wherein, The CH variant shown in SEQ ID NO: 14 contains N434A.

17. The antibody or antigen-binding fragment thereof according to claim 11, wherein, Human IgG4 heavy chain constant region variants contain S228P and / or N434A.

18. The antibody or antigen-binding fragment thereof according to claim 11, wherein, The human IgG4 heavy chain constant region variants include S228P and N434A.

19. The antibody or antigen-binding fragment thereof according to claim 11, wherein, The antibody or its antigen-binding fragment contains the heavy chain constant region (CH) shown in SEQ ID NO: 14 and the light chain constant region (CL) shown in SEQ ID NO:

16.

20. The antibody or antigen-binding fragment thereof according to claim 11, wherein, The antibody or its antigen-binding fragment contains the heavy chain constant region (CH) shown in SEQ ID NO: 15 and the light chain constant region (CL) shown in SEQ ID NO:

16.

21. The antibody or antigen-binding fragment thereof according to claim 11, wherein, The antibody or its antigen-binding fragment comprises the heavy chain constant region (CH) shown in SEQ ID NO: 70 and the light chain constant region (CL) shown in SEQ ID NO:

16.

22. The antibody or antigen-binding fragment thereof according to claim 11, wherein, The mutation causes the antibody or antigen-binding fragment to have no or reduced ADCP, ADCC, and / or CDC activity compared to the corresponding antibody or antigen-binding fragment without the mutation.

23. The antibody or antigen-binding fragment thereof according to claim 1, wherein, The antibodies include: (a) a heavy chain comprising VH as shown in SEQ ID NO: 68 and CH as shown in SEQ ID NO: 14, 15 or 70, and a light chain comprising VL as shown in SEQ ID NO: 69 and CL as shown in SEQ ID NO: 16; (b) Heavy chains including VH as shown in SEQ ID NO: 1 and CH as shown in SEQ ID NO: 14, 15 or 70, and light chains including VL as shown in SEQ ID NO: 2 and CL as shown in SEQ ID NO: 16; (c) Heavy chains including VH as shown in SEQ ID NO: 17 and CH as shown in SEQ ID NO: 14, 15 or 70, and light chains including VL as shown in SEQ ID NO: 18 and CL as shown in SEQ ID NO: 16; (d) Heavy chains including VH as shown in SEQ ID NO: 30 and CH as shown in SEQ ID NO: 14, 15 or 70, and light chains including VL as shown in SEQ ID NO: 31 and CL as shown in SEQ ID NO: 16; (e) includes heavy chains of VH shown in SEQ ID NO: 40 and CH shown in SEQ ID NO: 14, 15 or 70, and light chains of VL shown in SEQ ID NO: 41 and CL shown in SEQ ID NO: 16; or (f) Heavy chains including VH as shown in SEQ ID NO: 53 and CH as shown in SEQ ID NO: 14, 15 or 70, and light chains including VL as shown in SEQ ID NO: 54 and CL as shown in SEQ ID NO:

16.

24. The antibody or antigen-binding fragment thereof according to claim 1, wherein, The antibody or its antigen-binding fragment comprises: (a) Heavy chains and light chains, The heavy chain comprises: the sequence shown in SEQ ID NO: 71; and The light chain comprises the sequence shown in SEQ ID NO: 72; or (b) Heavy chains and light chains, The heavy chain comprises: the sequence shown in SEQ ID NO: 66 or 73; and The light chain comprises the sequence shown in SEQ ID NO:

67.

25. The antibody or antigen-binding fragment thereof according to claim 1, wherein, The antibody or its antigen-binding fragment is selected from scFv, Fab, Fab', F(ab')2, Fv fragment, disulfide-linked Fv (dsFv), and diabody.

26. The antibody or antigen-binding fragment thereof according to claim 1, wherein, The antibody or its antigen-binding fragment is labeled.

27. The antibody or antigen-binding fragment thereof according to claim 25, wherein, The antibody or its antigen-binding fragment carries a detectable marker.

28. The antibody or antigen-binding fragment thereof of claim 27, wherein the label is selected from enzymes, radionuclides, fluorescent dyes, luminescent substances or biotin.

29. The antibody or antigen-binding fragment thereof according to claim 28, wherein, The enzyme in question is horseradish peroxidase.

30. The antibody or antigen-binding fragment thereof according to claim 28, wherein, The luminescent material is a chemiluminescent material.

31. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof has at least one of the following characteristics: (a) K with less than 50 nM D Combined with TSLP; the K D Detected by Fortebio or ELISA; (b) TSLP is bound with EC50 of less than 50 nM; said EC50 is measured by flow cytometry or ELISA. (c) Inhibit the binding of TSLP to IL7Rα / TSLPR with an IC50 of less than 50 nM; the IC50 was determined by ELISA. (d) Inhibit or block TSLP-induced activation and / or proliferation of mast cells, DCs, and NKT cells; (e) Inhibit or block TSLP-induced OX40L expression; (f) Inhibit or block TSLP-induced osteoprotegerin (OPG) secretion; (g) Inhibit or block TSLP-induced secretion of Th2 cytokines; (h) Good affinity for binding with FcRn; (i) The isoelectric point (PI) is between 6.5 and 8.

5.

32. The antibody or antigen-binding fragment thereof according to claim 31, wherein, TSLP is a person TSLP.

33. The antibody or antigen-binding fragment thereof according to claim 31, wherein, The cytokines are selected from TARC, CCL22, IL-4, IL-13 or IL-5.

34. The antibody or antigen-binding fragment thereof according to claim 31, wherein, K D Less than 20 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, 1 pM, 0.1 pM or lower.

35. The antibody or antigen-binding fragment thereof according to claim 31, wherein, EC50 is less than 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 smaller.

36. The antibody or antigen-binding fragment thereof according to claim 31, wherein, IC50 is less than 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 smaller.

37. The antibody or antigen-binding fragment thereof according to claim 31, wherein, The isoelectric point (PI) is 6.5, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.7, 7.9, 8.0, 8.2 or 8.

5.

38. An isolated nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof as described in any one of claims 1-37.

39. A vector comprising the isolated nucleic acid molecule of claim 38.

40. The carrier of claim 39, wherein, The vector is a cloning vector or an expression vector.

41. A host cell comprising the isolated nucleic acid molecule of claim 38 or the vector of claim 39 or 40.

42. A method for preparing an antibody or antigen-binding fragment thereof according to any one of claims 1-37, comprising culturing a host cell according to claim 41 under conditions allowing expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.

43. A multispecific antibody comprising an antibody or antigen-binding fragment thereof that binds to TSLP as described in any one of claims 1-37, and additional antibodies or fragments thereof or antibody analogs thereof.

44. The multispecific antibody of claim 43, wherein, The multispecific antibody is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

45. A conjugate comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 1-37 and a conjugated portion, wherein the conjugated portion is a detectable label or a therapeutic agent.

46. ​​The conjugate of claim 45, wherein, The marker is selected from radioactive isotopes, fluorescent substances, luminescent substances, colored substances, or enzymes.

47. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as claimed in any one of claims 1-37, an isolated nucleic acid molecule as claimed in claim 38, a carrier as claimed in claim 39 or 40, a host cell as claimed in claim 41, a multispecific antibody as claimed in claim 43 or 44, and / or a conjugate as claimed in claim 45 or 46, and a pharmaceutically acceptable carrier and / or excipient.

48. The pharmaceutical composition of claim 47, wherein the pharmaceutical composition is used to induce at least one of the following biological activities in a subject: (a) Inhibit or block the binding of TSLP to IL7Rα-TSLPR, (b) Downregulate or eliminate TSLP activity, (c) Downregulate or block OX40L expression (d) Inhibit or block TSLP-induced activation and / or proliferation of mast cells, DCs, and NKT cells. (e) Inhibit or block TSLP-induced osteoprotegerin (OPG) secretion, (f) Inhibit or block the secretion of TSLP-induced Th2 cytokines.

49. The pharmaceutical composition of claim 48, wherein, The cytokines are selected from TARC, CCL22, IL-4, IL-13 or IL-5.

50. A kit comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1-37, and / or a nucleic acid as described in claim 38, and / or a vector as described in claim 39 or 40, and / or a host cell as described in claim 41, and / or a multispecific antibody as described in claim 43 or 44, and / or a conjugate as described in claim 45 or 46, and / or a pharmaceutical composition as described in any one of claims 47-49.

51. The kit of claim 50, further comprising instructions for use.

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