Bispecific antibodies against IL-13 and TSLP and uses thereof

Abstract

Disclosed herein are bispecific antibodies against IL-13 and TSLP and uses thereof, in particular monoclonal antibodies against IL-13, bispecific antibodies against IL-13 and TSLP, nucleic acids comprising nucleotide sequences encoding the antibodies, vectors comprising the nucleic acids, and host cells comprising the nucleic acids or vectors. Also disclosed are pharmaceutical compositions and conjugates comprising the antibodies, as well as methods of treatment using the antibodies.

Description

[0001] This application claims priority to Chinese Patent Application No. 202410392611.9 filed on April 2, 2024 and Chinese Patent Application No. 202510130727.X filed on February 5, 2025, the entire contents of which are incorporated herein by reference for all purposes. TECHNICAL FIELD

[0002] The present application relates to the field of antibodies, in particular bispecific antibodies against IL-13 and TSLP, and the use of such antibodies, in particular their use in the treatment of inflammatory diseases. BACKGROUND

[0003] Thymic stromal lymphopoietin (TSLP) is a cytokine of the Interleukin-7 (IL-7) family, mainly secreted by epithelial cells, and is a key cytokine that regulates type 2 inflammatory responses. It acts on dendritic cells (DCs), T cells, B cells, mast cells, basophils, and eosinophils, and is involved in regulating cell proliferation and activation, etc. (Ziegler et al., 2013; Ebina-Shibuya et al., 2023). After being stimulated by allergens or pathogens, epithelial cells secrete cytokines such as TSLP, Interleukin-25 (IL-25), and Interleukin-33 (IL-33). TSLP directly acts on DCs cells, inducing DCs cells to express OX40L (also known as CD252 or TNFSF4), thereby promoting the differentiation of naive T cells into pro-inflammatory Th2 cells and the secretion of cytokines such as Interleukin-4 (IL-4), Interleukin-5 (IL-5), and Interleukin-13 (IL-13). At the same time, TSLP can also directly act on Type 2 innate lymphoid cells (ILC2s), secreting IL-5 and IL-13, and enhancing type 2 inflammatory responses. In addition, TSLP is also involved in the regulation of non-type 2 inflammatory responses. TSLP can induce DCs cells to secrete cytokines IL-6 (Interleukin-6, IL-6) and IL-23 (Interleukin-23, IL-23), which are crucial for the differentiation of naive T cells into Th17 cells (Pelaia et al., 2021).

[0004] IL-13 is one of the core cytokines of type 2 inflammatory response, mainly secreted by Th2, ILC2, mast cells, B cells, macrophages and other cells. IL-13 activates downstream signals by binding to a receptor complex composed of IL-13Ra1 and IL-4Ra, and its receptor is expressed on B cells, mast cells, eosinophils, monocytes, epithelial cells, fibroblasts, smooth muscle cells and other cells. In addition, IL-13 can also bind to IL-13Ra2, which has no obvious intracellular signal transduction domain and is considered to be a decoy receptor for IL-13. IL-13 can induce B cell proliferation and IgE secretion, promote the recruitment and activation of eosinophils. IL-13 can also act on endothelial cells, smooth muscle cells, fibroblasts, epithelial cells and other cells, triggering goblet cell proliferation, increased mucus secretion, tissue remodeling, etc. (McCormick et al., 2015; Bao et al., 2015).

[0005] Currently, the antibody targeting TSLP, Tezepelumab, has been approved for the treatment of asthma patients, and the antibodies targeting IL-13, Tralokinumab and Lebrikizumab, have been approved for the treatment of atopic dermatitis patients. However, there are still many patients who do not respond well or do not respond to these treatments. Meanwhile, targeting TSLP and IL-13 is expected to provide more effective treatment options for patients with type 2 inflammatory response related diseases. SUMMARY

[0006] In one aspect, the present disclosure provides a bispecific antibody or an antigen binding fragment thereof, comprising a first antigen binding region binding IL-13 and a second antigen binding region binding TSLP, the first antigen binding region comprising a first light chain variable region (VL1) and a first heavy chain variable region (VH1), the second antigen binding region comprising a second light chain variable region (VL2) and a second heavy chain variable region (VH2), wherein the VL1 comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 21-23, respectively, the VH1 comprises HCDR 1 having the amino acid sequence of SEQ ID NO: 24, HCDR 2 having the amino acid sequence of SEQ ID NO: 25 or 26, and HCDR 3 having the amino acid sequence of SEQ ID NO: 27, the VL2 comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 35-37, respectively, and the VH2 comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 30-32, respectively.

[0007] In some embodiments, the VL1 comprises LCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 21-23, respectively, the VH1 comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 24, 25, and 27, respectively, the VL2 comprises LCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 35-37, respectively, and the VH2 comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 30-32, respectively.

[0008] In some embodiments, the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 1, 3, 46, 53, the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 52, the VL2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 33 or 51, and the VH2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28 or 50.

[0009] In some embodiments, the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 1, 3, 46, 53, the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 5, 7, 9, 11, 13, 52, the VL2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 33, and the VH2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28.

[0010] In some embodiments, the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 53, and the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 52.

[0011] In some embodiments, the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, and the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9.

[0012] In some embodiments, the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, and the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 5.

[0013] In some embodiments, the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, and the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 7.

[0014] In some embodiments, the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, and the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11.

[0015] In some embodiments, the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 and the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 13.

[0016] In some embodiments, the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 46 and the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 5.

[0017] In some embodiments, the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 46 and the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 7.

[0018] In some embodiments, the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 46 and the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9.

[0019] In some embodiments, the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 46 and the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11.

[0020] In some embodiments, the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 3, and the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 5.

[0021] In some embodiments, the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 3, and the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 7.

[0022] In some embodiments, the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 3, and the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9.

[0023] In some embodiments, the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 3, and the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11.

[0024] In some embodiments, the VL1 comprises an amino acid sequence as set forth in SEQ ID NO: 53 and the VH1 comprises an amino acid sequence as set forth in SEQ ID NO: 52; and the VL2 comprises an amino acid sequence as set forth in SEQ ID NO: 33, and the VH2 comprises an amino acid sequence as set forth in SEQ ID NO: 28.

[0025] In some embodiments, the VL1 comprises an amino acid sequence as set forth in SEQ ID NO: 1 and the VH1 comprises an amino acid sequence as set forth in SEQ ID NO: 9; and the VL2 comprises an amino acid sequence as set forth in SEQ ID NO: 33 and the VH2 comprises an amino acid sequence as set forth in SEQ ID NO: 28.

[0026] In some embodiments, the VL1 comprises an amino acid sequence as set forth in SEQ ID NO: 1 and the VH1 comprises an amino acid sequence as set forth in SEQ ID NO: 5; and the VL2 comprises an amino acid sequence as set forth in SEQ ID NO: 33 and the VH2 comprises an amino acid sequence as set forth in SEQ ID NO: 28.

[0027] In some embodiments, the VL1 comprises an amino acid sequence as set forth in SEQ ID NO: 1 and the VH1 comprises an amino acid sequence as set forth in SEQ ID NO: 7; and the VL2 comprises an amino acid sequence as set forth in SEQ ID NO: 33 and the VH2 comprises an amino acid sequence as set forth in SEQ ID NO: 28.

[0028] In some embodiments, the VL1 comprises an amino acid sequence as set forth in SEQ ID NO: 1 and the VH1 comprises an amino acid sequence as set forth in SEQ ID NO: 11; and the VL2 comprises an amino acid sequence as set forth in SEQ ID NO: 33 and the VH2 comprises an amino acid sequence as set forth in SEQ ID NO: 28.

[0029] In some embodiments, the VL1 comprises an amino acid sequence as set forth in SEQ ID NO: 1 and the VH1 comprises an amino acid sequence as set forth in SEQ ID NO: 13; and the VL2 comprises an amino acid sequence as set forth in SEQ ID NO: 33 and the VH2 comprises an amino acid sequence as set forth in SEQ ID NO: 28.

[0030] In some embodiments, the antibody comprises

[0031] a first heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 42,

[0032] a first light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 44,

[0033] a second heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 38,

[0034] a second light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 40.

[0035] In some embodiments, the antibody comprises a first heavy chain having an amino acid sequence as set forth in SEQ ID NO: 42, a first light chain having an amino acid sequence as set forth in SEQ ID NO: 44, a second heavy chain having an amino acid sequence as set forth in SEQ ID NO: 38, and a second light chain having an amino acid sequence as set forth in SEQ ID NO: 40.

[0036] In another aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds IL-L3, comprising a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one selected from the group consisting of SEQ ID NO: 1, 3, 46, 53, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one selected from the group consisting of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 52.

[0037] In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 53, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 52.

[0038] In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9.

[0039] In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 5.

[0040] In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 7.

[0041] In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11.

[0042] In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 13.

[0043] In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 46 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 5.

[0044] In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 46, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 7.

[0045] In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 46, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9.

[0046] In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 46, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11.

[0047] In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 3, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 5.

[0048] In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 3, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 7.

[0049] In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 3 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9.

[0050] In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 3 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11.

[0051] In some embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 53 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 52.

[0052] In some embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 1 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 9.

[0053] In some embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 1 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 5.

[0054] In some embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 1 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 7.

[0055] In some embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 1 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 11.

[0056] In some embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 1 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 13.

[0057] In some embodiments, the antibody is of an isotype selected from the group consisting of IgG, IgA, IgM, IgE, and IgD.

[0058] In some embodiments, the antibody is of a subtype selected from the group consisting of IgGl, IgG2, IgG3, and IgG4.

[0059] In some embodiments, the antigen binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fv, scFv, and ds-scFv.

[0060] In some embodiments, the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody.

[0061] In some embodiments, the antibody is a bispecific antibody further comprising a second antigen binding region that binds to a second antigen.

[0062] In yet another aspect, the disclosure provides a nucleic acid comprising a nucleotide sequence encoding the bispecific antibody or antigen binding fragment thereof disclosed herein or the antibody or antigen binding fragment thereof disclosed herein.

[0063] In some embodiments, the nucleic acid comprises

[0064] a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 2, 4, 56,

[0065] a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 55,

[0066] a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 29, and

[0067] a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34.

[0068] In some embodiments, the nucleic acid comprises

[0069] a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 39,

[0070] a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 41,

[0071] a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 43, and

[0072] a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 45.

[0073] In another aspect, the present disclosure provides a vector comprising a nucleic acid disclosed herein.

[0074] In another aspect, the present disclosure provides a host cell comprising a nucleic acid disclosed herein or a vector disclosed herein.

[0075] In another aspect, the present disclosure provides a pharmaceutical composition comprising (i) a bispecific antibody or antigen-binding fragment thereof disclosed herein, or an antibody or antigen-binding fragment thereof disclosed herein; and (ii) a pharmaceutically acceptable carrier or excipient.

[0076] In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent.

[0077] In some embodiments, the second therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent, and a small molecule drug.

[0078] In another aspect, the present disclosure provides a conjugate comprising a bispecific antibody or antigen-binding fragment thereof disclosed herein, or an antibody or antigen-binding fragment thereof disclosed herein, and a chemical moiety conjugated thereto.

[0079] In some embodiments, the chemical moiety is selected from the group consisting of a therapeutic agent, a detectable moiety, and an immunostimulatory molecule.

[0080] In yet another aspect, the present disclosure provides a method for treating a disease associated with a type 2 inflammatory response in a subject, comprising administering to the subject an effective amount of a bispecific antibody or antigen-binding fragment thereof disclosed herein, an antibody or antigen-binding fragment thereof disclosed herein, a pharmaceutical composition disclosed herein, or a conjugate disclosed herein.

[0081] In some embodiments, the disease is an IL-13 and / or TSLP-mediated disease (e.g., an inflammatory disease and an autoimmune disease).

[0082] In some embodiments, the disease is selected from allergic inflammation (e.g., allergic rhinitis, allergic rhinosinusitis, allergic conjunctivitis), asthma, atopic dermatitis, urticaria, chronic obstructive pulmonary disease, eosinophilic esophagitis, rheumatoid arthritis, multiple sclerosis, idiopathic pulmonary fibrosis, eczema (e.g., hand eczema, asthmatic eczema), eosinophilic gastroenteritis, Crohn's disease, systemic sclerosis, ulcerative colitis, chronic rhino-sinusitis with nasal polyps.

[0083] In some embodiments, the method further comprises administering to the subject a second therapeutic agent.

[0084] In some embodiments, the second therapeutic agent is selected from an antibody, a chemotherapeutic agent, and a small molecule drug.

[0085] In another aspect, the present disclosure provides a method for inhibiting IgE antibody production in a subject, comprising administering to the subject an effective amount of the bispecific antibody or antigen binding fragment thereof disclosed herein, the antibody or antigen binding fragment thereof disclosed herein, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein. BRIEF DESCRIPTION OF DRAWINGS

[0086] An understanding of the features and advantages of the present application can be obtained by reference to the following detailed description in conjunction with the drawings, in which:

[0087] Figure 1 The results of the binding affinity detection of 228B / C-1 humanized antibody transient supernatant to hIL-13 are shown.

[0088] Figure 2 The results of the hIL-13-induced STAT6 activation inhibitory activity of 228B / C-1 humanized antibody are shown. Among them, A: H1K1 inhibits hIL-13-induced STAT6 activation activity; B: H2K1 inhibits hIL-13-induced STAT6 activation activity; C: H3K1 inhibits hIL-13-induced STAT6 activation activity.

[0089] Figure 3 The results of the hIL-13-induced TF-1 cell proliferation inhibitory activity of 228B / C-1 humanized antibody H3K1 are shown.

[0090] Figure 4 The results of the hIL-13-induced PBMC TARC release inhibitory activity of 228B / C-1 humanized antibody H3K1 are shown.

[0091] Figure 5The results of TSLP / IL-13 BsAb inhibiting hTSLP-induced activity are shown. Among them, A: TSLP / IL-13 BsAb inhibits hTSLP-induced STAT5 activation activity; B: TSLP / IL-13 BsAb inhibits hTSLP-induced Ba / f3-TSLPR-IL7Ra cell proliferation activity; C: TSLP / IL-13 BsAb inhibits hTSLP-induced PBMC release TARC activity.

[0092] Figure 6 The results of TSLP / IL-13 BsAb inhibiting hIL-13-induced activity are shown. Among them, A: TSLP / IL-13 BsAb inhibits hIL-13-induced STAT6 activation activity; B: TSLP / IL-13 BsAb inhibits hIL-13-induced TF-1 cell proliferation activity; C: TSLP / IL-13 BsAb inhibits hIL-13-induced PBMC release TARC activity.

[0093] Figure 7 The results of TSLP / IL-13 BsAb inhibiting hTSLP combined with hIL-13-induced PBMC release TARC activity are shown.

[0094] Figure 8 The results of TSLP / IL-13 BsAb in vivo efficacy in MC903-induced hTSLP / TSLPR humanized mouse dermatitis model are shown. Among them, A: The effect of TSLP / IL-13 BsAb on the change of ear thickness of hTSLP / TSLPR humanized mice; B: The effect of TSLP / IL-13 BsAb on the serum IgE level of hTSLP / TSLPR humanized mice; C: The effect of TSLP / IL-13 BsAb and ref2 nanobody on the change of ear thickness of hTSLP / TSLPR humanized mice.

[0095] Figure 9 The results of TSLP / IL-13 BsAb in vivo efficacy in MC903-induced hIL-13 humanized mouse dermatitis model are shown. Among them, A: The effect of TSLP / IL-13 BsAb on the change of ear thickness of hIL-13 humanized mice; B: The effect of TSLP / IL-13 BsAb on the serum IgE level of hIL-13 humanized mice.

[0096] Figure 10 The results of TSLP / IL-13 BsAb on the body weight of B-hIL13 mouse asthma model are shown. The data is shown as mean ± SEM, n = 6; Dunnett test after one-way ANOVA.

[0097] Figure 11 The results of the effect of TSLP / IL-13 BsAb on the sneezing and the number of scratching of the B-hIL13 mouse asthma model are shown. The data are shown as mean ± SEM, n = 6; #P < 0.05, ##P < 0.01, ###P < 0.001 compared with the Control group; *P < 0.05, **P < 0.01 compared with the Model group; one-way ANOVA followed by Dunnett's test, one-way ANOVA followed by Mann-Whitney U test.

[0098] Figure 12 The results of the effect of TSLP / IL-13 BsAb on the airway hyperresponsiveness of the B-hIL13 mouse asthma model are shown. The data are shown as mean ± SEM, n = 6; #P < 0.05 compared with the Control group; one-way ANOVA followed by Dunnett's test, one-way ANOVA followed by Mann-Whitney U test.

[0099] Figure 13 The results of the effect of TSLP / IL-13 BsAb on the number of inflammatory cells in the airway BALF of the B-hIL13 mouse asthma model are shown. The data are shown as mean ± SEM, n = 6; #P < 0.05, ##P < 0.01, ###P < 0.001 compared with the Control group; *P < 0.05, **P < 0.01, ***P < 0.001 compared with the Model group; one-way ANOVA followed by Dunnett's test, one-way ANOVA followed by Mann-Whitney U test. # P < 0.05, ## P < 0.01, ### P < 0.001; *P < 0.05, **P < 0.01, ***P < 0.001 compared with the Model group; one-way ANOVA followed by Dunnett's test, one-way ANOVA followed by Mann-Whitney U test.

[0100] Figure 14 The results of the effect of TSLP / IL-13 BsAb on the serum total IgE and OVA-sIgE levels of the B-hIL13 mouse asthma model are shown. The data are shown as mean ± SEM, n = 6; #P < 0.01 compared with the Control group; *P < 0.05 compared with the Model group; one-way ANOVA followed by Dunnett's test, one-way ANOVA followed by Mann-Whitney U test. ## P < 0.01; *P < 0.05 compared with the Model group; one-way ANOVA followed by Dunnett's test, one-way ANOVA followed by Mann-Whitney U test.

[0101] Figure 15 The results of the effect of TSLP / IL-13 BsAb on the lung tissue eosinophil infiltration pathology of the B-hIL13 mouse asthma model are shown. The data are shown as mean ± SEM, n = 6. ###P < 0.001 compared with the Control group; *P < 0.05 compared with the Model group. One-way ANOVA followed by Dunnett's test.

[0102] Figure 16 The results of the effect of TSLP / IL-13 BsAb on the pathological score of lung tissue H&E staining of the B-hIL13 mouse asthma model are shown. The data are shown as mean ± SEM, n = 6. Compared with the Control group, ###P < 0.001; compared with the Model group, *P < 0.05. One-way ANOVA followed by Dunnett's test.

[0103] Figure 17 The results of the effect of TSLP / IL-13 BsAb on the proliferation of goblet cell metaplasia in the B-hIL13 mouse asthma model are shown, where the green arrow is the positive expression of goblet cells, and the green circle is the mucus secretion. The data are shown as mean ± SEM, n = 6; compared with the Control group, ## P < 0.01; compared with the Model group, **P < 0.01; one-way ANOVA followed by Mann-Whitney U test.

[0104] Figure 18 The results of the effect of TSLP / IL-13 BsAb on the number and degranulation of mast cells in the lung tissue of the B-hIL13 mouse asthma model are shown, where the green arrow is the degranulation cell. The data are shown as mean ± SEM, n = 6; compared with the Control group, ##P < 0.01; compared with the Model group, **P < 0.01; one-way ANOVA followed by Mann-Whitney U test.

[0105] Figure 19 The results of the effect of TSLP / IL-13 BsAb on the degranulation rate of mast cells in the lung tissue of the B-hIL13 mouse asthma model are shown. The data are shown as mean ± SEM, n = 6; compared with the Control group, #P < 0.05; compared with the Model group, **P < 0.01; one-way ANOVA followed by Dunnett's test, one-way ANOVA followed by Mann-Whitney U test.

[0106] Figure 20 The results of the effect of TSLP / IL-13 BsAb on the increase in ear thickness and body weight of MC903-induced B-hIL-13 mouse dermatitis model are shown. One-way ANOVA analysis, compared with the Control group, ####P < 0.0001; compared with the Model group, **P < 0.01.

[0107] Figure 21Results showing the effect of TSLP / IL-13 BsAb on ear lesion of MC903-induced B-hIL-13 mouse dermatitis model. One-way ANOVA analysis, compared with Control group, #### P<0.0001; compared with Model group, ****P<0.0001.

[0108] Figure 22 Results showing the effect of TSLP / IL-13 BsAb on serum total IgE level of MC903-induced B-hIL-13 mouse dermatitis model. One-way ANOVA analysis.

[0109] Figure 23 Results showing the effect of TSLP / IL-13 BsAb on right ear thickness of MC903-induced B-hTSLP / hTSLPR mouse dermatitis model. One-way ANOVA analysis, compared with Control group #### P<0.0001; compared with Model group, **P<0.01.

[0110] Figure 24 Results showing the effect of TSLP / IL-13 BsAb on inhibiting hTSLP-induced STAT5 activation.

[0111] Figure 25 Results showing the effect of TSLP / IL-13 BsAb on inhibiting hTSLP-induced Ba / f3-TSLPR-IL7Ra cell proliferation.

[0112] Figure 26 Results showing the thermal stability detection profile of TSLP / IL-13 BsAb.

[0113] Figure 27 Results showing the effect of TSLP / IL-13 BsAb on TSLP-induced PBMC releasing TARC.

[0114] Figure 28 Results showing the effect of TSLP / IL-13 BsAb on TSLP combined with IL-13-induced PBMC releasing TARC.

[0115] Figure 29 Results showing the immunogenicity of TSLP / IL-13 BsAb.

[0116] Figure 30The results of the effect of TSLP / IL-13 BsAb on the body weight of mice in the OVA-induced B-hTSLP / hTSLPR mouse asthma model are shown. The data are shown as mean ± SEM, n = 12; Dunnett's test after one-way ANOVA, Mann-Whitney U test after one-way ANOVA.

[0117] Figure 31 The results of the effect of TSLP / IL-13 BsAb on the body weight of mice in the OVA-induced B-hTSLP / hTSLPR mouse asthma model are shown. The data are shown as mean ± SEM, n = 12; Dunnett's test after one-way ANOVA, Mann-Whitney U test after one-way ANOVA.

[0118] Figure 32 The results of the effect of TSLP / IL-13 BsAb on the body weight of mice in the OVA-induced B-hTSLP / hTSLPR mouse asthma model are shown. The data are shown as mean ± SEM, n = 12; Dunnett's test after one-way ANOVA, Mann-Whitney U test after one-way ANOVA.

[0119] Figure 33 The results of the effect of TSLP / IL-13 BsAb on the body weight of mice in the OVA-induced B-hTSLP / hTSLPR mouse asthma model are shown. The data are shown as mean ± SEM, n = 12; Dunnett's test after one-way ANOVA, Mann-Whitney U test after one-way ANOVA.

[0120] Figure 34The TSLP / IL-13 BsAb reduced the levels of total IgE and OVA-sIgE in the serum of OVA-induced B-hTSLP / hTSLPR mice asthma model. Data are shown as mean ± SEM, n = 12; Dunnett's test after one-way ANOVA, Mann-Whitney U test after one-way ANOVA. Compared with Control group: ###P < 0.001; Compared with Model group: *P < 0.05, **P < 0.01, ***P < 0.001. The serum OVA-sIgE levels of Day 20 and Day 28 were expressed as OD values. The serum of Control group mice was all negative (<Reference), and the serum of Model group, TSLP / IL-13 BsAb (3, 10, 30 mg / kg) group and KLH (30 mg / kg) group mice were all positive (>Reference).

[0121] Figure 35 The TSLP / IL-13 BsAb inhibited the expression of h-TSLP and MUC5AC mRNA in lung tissue of OVA-induced B-hTSLP / hTSLPR mice asthma model. Data are shown as mean ± SEM, n = 12; Dunnett's test after one-way ANOVA, Mann-Whitney U test after one-way ANOVA. Compared with Control group: ###P < 0.001; Compared with Model group: *P < 0.05, **P < 0.01.

[0122] Figure 36 The TSLP / IL-13 BsAb affected the results of H&E staining inflammation score in lung tissue of OVA-induced B-hTSLP / hTSLPR mice asthma model. Data are shown as mean ± SEM, n = 12; Dunnett's test after one-way ANOVA, Mann-Whitney U test after one-way ANOVA. Compared with Control group: ###P < 0.001; Compared with Model group: **P < 0.01.

[0123] Figure 37 The TSLP / IL-13 BsAb inhibited the infiltration of eosinophils in lung tissue of OVA-induced B-hTSLP / hTSLPR mice asthma model.

[0124] Figure 38The results of the effect of TSLP / IL-13 BsAb on the positive rate of airway goblet cells in the OVA-induced B-hTSLP / hTSLPR mouse asthma model are shown. The data are shown as mean ± SEM, n = 12; Dunnett's test after one-way ANOVA, Mann-Whitney U test after one-way ANOVA. Compared with the Control group: ###P < 0.001; compared with the Model group: *P < 0.05, **P < 0.01.

[0125] Figure 39 The pathological pictures of TSLP / IL-13 BsAb reducing the metaplasia and proliferation of airway goblet cells in the OVA-induced B-hTSLP / hTSLPR mouse asthma model are shown.

[0126] Figure 40 The results of the effect of TSLP / IL-13 BsAb on the degranulation rate of lung tissue mast cells in the OVA-induced B-hTSLP / hTSLPR mouse asthma model are shown. The data are shown as mean ± SEM, n = 12; Dunnett's test after one-way ANOVA, Mann-Whitney U test after one-way ANOVA. Compared with the Control group: ###P < 0.001; compared with the Model group: *P < 0.05, **P < 0.01.

[0127] Figure 41 The pathological pictures of TSLP / IL-13 BsAb inhibiting the degranulation of lung tissue mast cells in the OVA-induced B-hTSLP / hTSLPR mouse asthma model are shown.

[0128] Figure 42 The results of the effect of TSLP / IL-13 BsAb on the positive rate of peribronchial collagen deposition in the lung tissue of the OVA-induced B-hTSLP / hTSLPR mouse asthma model are shown. The data are shown as mean ± SEM, n = 12; Dunnett's test after one-way ANOVA, Mann-Whitney U test after one-way ANOVA. Compared with the Control group: ###P < 0.001; compared with the Model group: *P < 0.05, **P < 0.01.

[0129] Figure 43 The pathological pictures of TSLP / IL-13 BsAb reducing the peribronchial collagen deposition in the lung tissue of the OVA-induced B-hTSLP / hTSLPR mouse asthma model are shown.

[0130] Figure 44Effect of TSLP / IL-13 BsAb on MC903-induced B-hTSLP / hTSLPR mice dermatitis model mice body weight. Data shown as mean ± SEM, n=11; Dunnett’s test after one way ANOVA, Mann-Whitney U test after one way ANOVA.

[0131] Figure 45 Effect of TSLP / IL-13 BsAb on MC903-induced B-hTSLP / hTSLPR mice ear thickness increase. Data shown as mean ± SEM, n=11; Dunnett’s test after one way ANOVA, Mann-Whitney U test after one way ANOVA. One way ANOVA, ####P<0.0001 compared to Control group, *P<0.05, ****P<0.0001 compared to Model group.

[0132] Figure 46 Effect of TSLP / IL-13 BsAb on MC903-induced B-hTSLP / hTSLPR mice skin lesion. Data shown as mean ± SEM, n=11; Dunnett’s test after one way ANOVA, Mann-Whitney U test after one way ANOVA. One way ANOVA, ####P<0.0001 compared to Control group, ****P<0.0001 compared to Model group.

[0133] Figure 47 Effect of TSLP / IL-13 BsAb on MC903-induced B-hTSLP / hTSLPR mice right ear draining side lymph node enlargement. Data shown as mean ± SEM, n=11; Dunnett’s test after one way ANOVA, Mann-Whitney U test after one way ANOVA. One way ANOVA, ####P<0.0001 compared to Control group, ***P<0.001, ****P<0.0001 compared to Model group.

[0134] Figure 48TSLP / IL-13 BsAb reduced MC903-induced serum total IgE levels in B-hTSLP / hTSLPR mice. Data shown as mean ± SEM, n=11; One way ANOVA followed by Dunnett’s test, Mann-Whitney U test after One way ANOVA. One way ANOVA, ##P<0.01 compared to Control group; *P<0.05 compared to Model group.

[0135] Figure 49 TSLP / IL-13 BsAb inhibited MC903-induced cytokine expression in ear tissue of B-hTSLP / hTSLPR mice. Data shown as mean ± SEM, n=11; One way ANOVA followed by Dunnett’s test, Mann-Whitney U test after One way ANOVA. One way ANOVA, ###P<0.001, ####P<0.0001 compared to Control group; ***P<0.001, ****P<0.0001 compared to Model group.

[0136] Figure 50 TSLP / IL-13 BsAb reduced MC903-induced ear tissue inflammation score in B-hTSLP / hTSLPR mice. Data shown as mean ± SEM, n=11; One way ANOVA followed by Dunnett’s test, Mann- Whitney U test after One way ANOVA. One way ANOVA, ####P<0.0001 compared to Control group; *P<0.05, **P<0.01, ***P<0.001 compared to Model group.

[0137] Figure 51 TSLP / IL-13 BsAb CM512 reduced MC903-induced ear tissue inflammation score in B-hTSLP / hTSLPR mice. Data shown as mean ± SEM, n=11; One way ANOVA followed by Dunnett’s test, Mann- Whitney U test after One way ANOVA. One way ANOVA, ####P<0.0001 compared to Control group; *P<0.05, **P<0.01, ***P<0.001 compared to Model group.

[0138] Figure 52 Biacore sensorgram showing TSLP / IL-13 BsAb binding to FcyRI.

[0139] Figure 53 Biacore sensorgram showing TSLP / IL-13 BsAb binding to FcyRIIA H131.

[0140] Figure 54 Biacore sensorgram showing TSLP / IL-13 BsAb binding to FcyRIIB.

[0141] Figure 55 Biacore sensorgram showing TSLP / IL-13 BsAb binding to FcyRIIIA V158.

[0142] Figure 56 Biacore sensorgram showing TSLP / IL-13 BsAb binding to FcyRIIIA F158.

[0143] Figure 57 Biacore sensorgram showing TSLP / IL-13 BsAb binding to FcRn at pH 7.4.

[0144] Figure 58 Biacore sensorgram showing TSLP / IL-13 BsAb binding to FcRn at pH 6.0.

[0145] Figure 59 Biacore sensorgram showing TSLP / IL-13 BsAb binding to Clq.

[0146] Figure 60 Biacore sensorgram showing TSLP / IL-13 BsAb binding activity to different species of IL-13.

[0147] Figure 61 Biacore sensorgram showing Anti-KLH hlgGl binding activity to different species of IL-13.

[0148] Figure 62 Biacore sensorgram showing TSLP / IL-13 BsAb binding activity to different species of TSLP.

[0149] Figure 63 Biacore sensorgram showing Anti-KLH hlgGl binding activity to different species of TSLP.

[0150] Figure 64 Biacore sensorgram showing TSLP / IL-13 BsAb blocking activity of IL-13 / IL-13Ra1 complex binding to IL-4Ra.

[0151] Figure 65The blocking activity of the TSLP / IL-13 BsAb against TSLP binding to TSLPR is shown.

[0152] Figure 66 The blocking activity of the TSLP / IL-13 BsAb against TSLP binding to TSLPR on the cell surface is shown. DETAILED DESCRIPTION

[0153] The above features and advantages of the present application, and additional features and advantages, will be more clearly understood from the following detailed description when read in conjunction with the accompanying drawings.

[0154] The embodiments described herein with reference to the drawings are explanatory, illustrative, and for general understanding of the present application. The embodiments should not be interpreted in a limiting manner on the scope of the present application. Identical or similar elements and elements having identical or similar functions are denoted by the same reference numerals throughout the specification.

[0155] In the present application, unless otherwise specified, the scientific and technical terms used herein have meanings commonly understood by a person skilled in the art. Also, the terms and procedures of protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology, and laboratory operations used herein are terms and procedures widely used in the corresponding fields. At the same time, in order to better understand the present application, the definitions and explanations of the relevant terms are provided below.

[0156] Definitions

[0157] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" includes a plurality of antibodies, and reference to "an antibody" in some embodiments includes a plurality of antibodies, and so forth.

[0158] Unless otherwise specified or defined, the terms "comprise", "comprising", and "comprises", and variations thereof such as "comprising" and "comprises", will be understood to imply the inclusion of a stated element or step or group of elements or steps but not the exclusion of any other element or step or group of elements or steps.

[0159] As used herein, the term "antibody" refers to an immunoglobulin molecule having the ability to specifically bind to a particular antigen. Such molecules typically comprise two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (or domain) (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (or domain) (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The variable regions of the antibody heavy and light chains contain the binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as Clq (the first component in the classical pathway of complement activation).

[0160] The heavy chain of an immunoglobulin can be divided into three functional regions: an Fd region, a hinge region, and an Fc region (fragment crystallizable). The Fd region comprises the VH and CH1 domains and, together with the light chain, forms the Fab (antigen binding fragment). The Fc fragment is responsible for the effector functions of the immunoglobulin, including, for example, complement binding and binding to Fc receptors on effector cells. The hinge region, found in the IgG, IgA, and IgD immunoglobulin classes, acts as a flexible spacer, allowing the Fab portions to move freely in space relative to the Fc region. The hinge domain is structurally diverse, differing in sequence and length between immunoglobulin classes and subclasses.

[0161] According to crystallographic studies, the immunoglobulin hinge region can be further subdivided into three regions based on structure and function: the upper hinge, the core hinge, and the lower hinge. The upper hinge includes amino acids from the carboxy-terminal end of CH1 to the first residue in the hinge that restricts movement, usually the first cysteine residue that forms an interchain disulfide bond between the two heavy chains. The length of the upper hinge region correlates with the flexibility of the fragment of the antibody. The core hinge region contains the inter-heavy chain disulfide bonds. The lower hinge region connects the amino-terminal end of the CH2 domain and includes residues in the CH2 domain. Structural and flexible allowed conformational changes in the immunoglobulin hinge region polypeptide sequence can influence the effector functions of the Fc portion of the antibody.

[0162] A "light chain variable region" (VL) or "heavy chain variable region" (VH) is comprised of "framework" regions separated by three "complementarity determining regions" or "CDRs." The framework regions serve to align the CDRs that are primarily responsible for specific antigenic epitope binding. The CDRs include the amino acid residues in the antibody that are primarily responsible for antigen binding. The VL domain and the VH domain each comprise, from amino-terminus to carboxy-terminus, the following framework regions (FRs) and CDRs: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The CDR1, CDR2, and CDR3 of a VL domain are also referred to herein as LCDR1, LCDR2, and LCDR3, respectively; the CDR1, CDR2, and CDR3 of a VH domain are also referred to herein as HCDR1, HCDR2, and HCDR3, respectively.

[0163] The amino acid arrangement of each VL domain and VH domain is consistent with any conventional definition of CDRs. Conventional definitions include the Kabat definition (Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991), the Chothia definition (Chothia and Lesk, J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:878-883, 1989); a composite of Chothia and Kabat CDRs, in which CDR-H1 is a composite of Chothia and Kabat CDRs; the AbM definition used by Oxford Molecular's antibody modeling software; and the CONTACT definition of Martin et al. (world wide web bioinfo.org.uk / abs). Kabat provides a widely used numbering convention (the Kabat numbering system), in which corresponding residues between different heavy chains or between different light chains are given the same number. The present disclosure can use CDRs defined according to any of these numbering systems, but preferred embodiments use CDRs defined according to the Kabat definition.

[0164] Based on the amino acid sequences of the constant regions of the heavy chains of the antibodies, immunoglobulin molecules can be assigned to five classes (isotypes): IgA, IgD, IgE, IgG, and IgM, and several subclasses, e.g., IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, etc. Based on the amino acid sequences of the light chains, the light chains of the antibodies can be assigned to either lambda (l) chain or kappa (K) chain.

[0165] As used herein, the term "antibody" shall be construed in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), antibody fragments, and multi-specific antibodies (e.g., bispecific antibodies) containing at least two antigen binding regions. Antibodies can contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations of glycosylation sites. Antibodies also include post-translationally modified antibodies, fusion proteins containing an antigenic determinant of an antibody, and immunoglobulin molecules containing any other modifications to the antigen recognition site, so long as the antibodies exhibit the desired biological activity.

[0166] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, each antibody in the population is identical except for possible naturally occurring mutations that can be present. Monoclonal antibodies are highly specific, being directed against a single antigen. The term "monoclonal antibody" herein is not limited to antibodies produced through hybridoma technology, and should not be construed as requiring production by any particular method.

[0167] The term "bispecific antibody" is understood in the context of the present application as an antibody having two different antigen binding regions defined by different antibody sequences. This can be understood as binding to different targets, but also includes binding to different epitopes of one target. The term "bispecific antibody" as used herein is to be construed in its broadest sense, including full-length bispecific antibodies and antigen-binding fragments thereof. Bispecific antibodies can contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations of glycosylation sites. Bispecific antibodies also include post-translationally modified antibodies, fusion proteins containing an antigenic determinant of an antibody, and immunoglobulin molecules containing any other modifications to the antigen recognition site, so long as the antibodies exhibit the desired biological activity.

[0168] As used herein, the term "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody.

[0169] Examples of antigen-binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fab' fragment, which is simply a Fab with part of the hinge region; (iv) a Fd fragment consisting of the VH and CH1 domains; (v) a Fd' fragment consisting of the VH and CH1 domains and one or more cysteine residues at the C-terminus of the CH1 domain; (vi) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (vii) a dAb fragment, which consists of a VH domain; (viii) an isolated complementarity determining region (CDR); (ix) a Nanobody, a heavy chain variable region containing a single variable domain and two constant domains. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv)). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Furthermore, the term also includes "linear antibodies", which comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) with dual variable domain (D3) antibodies, which form a pair of antigen binding regions with a complementary light chain polypeptide, as well as modifications of any of the foregoing structures.

[0170] These antigen-binding fragments can be obtained using conventional techniques known to those skilled in the art, and the utility of the fragments screened in the same manner as for whole antibodies.

[0171] As used herein, the term "binds" or "binds specifically to" refers to a nonrandom, binding reaction between two molecules, such as an antibody and its target antigen. The binding specificity of an antibody can be determined based on affinity and / or avidity. Affinity represents the equilibrium constant for dissociation of an antigen from an antibody (KD), a measure of the strength of binding between an antigenic determinant and an antigen binding site of an antibody: the smaller the value of KD, the stronger the binding strength between the antigenic determinant and the antibody. Alternatively, affinity can also be expressed as the affinity constant (KA), which is 1 / KD.

[0172] Avidity is a measure of the strength of binding between an antibody and the relevant antigen. Avidity involves both the affinity between the antigenic determinant and the antigen binding site of an antibody and the number of relevant binding sites present on the antibody. Typically, an antibody will bind an antigen with a dissociation constant (KD) of 10 -5 M to 10 -12 M or less, and preferably 10 -7 M to 10 -12 M or less, and more preferably 10-8 M to 10 -12 M, and / or have a binding affinity of at least 10 7 M -1 , preferably at least 10 8 M -1 , more preferably at least 10 9 M -1 , such as at least 10 12 M -1 . It is generally accepted that any K -4 M value greater than 10 D represents non-specific binding. Specific binding of an antibody to an antigen or antigenic determinant can be determined in any known suitable manner, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, as well as different variations thereof known in the art.

[0173] The term "epitope" refers to a site on an antigen to which an antibody binds. Epitopes can be formed both from contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of a protein or proteins. Epitopes formed from contiguous amino acids (also known as linear epitopes) are generally retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding (also known as conformational epitopes) are generally lost upon treatment with denaturing solvents. Epitopes usually comprise at least 3, more usually at least 5 or 8-10 amino acids in a unique spatial conformation. Epitopes define the minimum binding site of an antibody and are thus the specific targets of an antibody or antigen-binding fragment thereof.

[0174] As used herein, the term "sequence identity" refers to the extent to which two sequences (amino acid) have identical residues in the same positions after alignment. For example, "an amino acid sequence is X% identical to SEQ ID NO: Y" means that the amino acid sequence has X% identity to SEQ ID NO: Y and is set forth as X% of the residues in the amino acid sequence being identical to the residues of the sequence disclosed in SEQ ID NO: Y. Typically, such calculations are performed using a computer program. Exemplary programs for comparing and aligning pairs of sequences include ALIGN, FASTA, gapped BLAST, BLASTP, BLASTN, or GCG.

[0175] Furthermore, in determining the extent of sequence identity between two amino acid sequences, the skilled person can take into account so-called "conservative" amino acid substitutions, which can generally be described as amino acid substitutions of an amino acid residue for another amino acid residue having a similar chemical structure that has little or essentially no effect on the function, activity or other biological properties of the polypeptide. Such conservative amino acid substitutions are well known in the art.

[0176] Such conservative substitutions preferably are substitutions of one amino acid for another that is in the same group (a) to (e) : (a) small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gin; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, lie, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp.

[0177] Particularly preferred conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gin or to His; Asp to Glu; Cys to Ser; Gin to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gin; lie to Leu or to Val; Leu to lie or to Val; Lys to Arg, to Gin, or to Glu; Met to Leu, to Tyr, or to lie; Phe to Met, to Leu, or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, to lie, or to Leu.

[0178] As used herein, the term "vector" is intended to refer to a nucleic acid molecule that is capable of transporting another nucleic acid to which it has been linked.

[0179] As used herein, the term "host cell" refers to a cell into which an expression vector has been introduced.

[0180] The term "pharmaceutically acceptable" means that which the carrier or excipient is compatible with the other ingredients of the composition and not deleterious to the recipient thereof, and / or such carrier or excipient is approved or approvable by a regulatory agency of the Federal or a state government of the United States or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for inclusion in a drug product for administration to humans.

[0181] As used herein, the terms "treatment," "therapy," "treat," and the like, refer to the application of an agent or performance of a procedure for the purpose of effecting an outcome. The outcomes can be prophylactic, in terms of completely or partially preventing a disease or symptom thereof, and / or therapeutic, in terms of partially or completely curing a disease and / or symptoms of a disease. As used herein, "treatment" can include treatment of a disease or condition (e.g., an inflammatory disease) in a mammal, particularly in a human, and includes: (a) preventing the disease or symptom of the disease from occurring in an individual which can be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease. Treatment can refer to any successful indication in the treatment or amelioration or prevention of cancer, including any objective or subjective parameter, such as reduction of symptoms; alleviation of disease symptoms or making the disease condition more tolerable to the patient; slowing in rate of disease progression or degeneration; or improving quality of life. Treatment or amelioration of symptoms is based on one or more objective or subjective parameters; including physician-based measures. Thus, the term "treatment" includes the administration of an antibody or composition or conjugate disclosed herein to prevent or delay, alleviate or ameliorate a symptom or condition associated with a disease (e.g., an inflammatory disease). The term "therapeutic effect" refers to the reduction, elimination or prevention of a disease, a symptom of a disease, or a side effect of a disease in a subject.

[0182] As used herein, the term "effective amount" refers to an amount of a drug administered to a subject to treat a disease sufficient to effect treatment of the disease.

[0183] As used herein, the term "subject" refers to any mammalian subject in which diagnosis, treatment, or therapy is desired. A "mammal" for treatment purposes refers to any animal classified as a mammal, including humans, domestic and farm animals, and laboratory and sports or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, and the like.

[0184] Type 2 inflammatory response related diseases are inflammatory diseases mediated by Th2 cells, type 2 innate lymphoid cells and related cytokines. When the human body is exposed to different environmental or plant and animal allergens, type 2 inflammatory response will be formed in the body, which can involve multiple systems such as skin, respiratory tract and digestive tract, and cause type 2 inflammatory diseases. Type 2 inflammatory response related diseases have the following immunopathological characteristics in the affected tissues / organs:

[0185] i) increase of type 2 immune cells (Th2 and ILC2 cells, etc.), and increase of expression of related cytokines (IL-4, IL-5, IL-13, IL-25, IL-31, IL-33, TSLP, etc.);

[0186] ii) effector cell activation proliferation (e.g. B cell activation into plasma cells) or recruitment activation (e.g. mast cells, basophils and eosinophils (EOS));

[0187] iii) can be accompanied by an increase in total IgE and / or specific IgE;

[0188] iv) manifested as local organ microcirculation dilation, increased permeability, increased exudation, and related inflammatory responses such as cell infiltration.

[0189] The following reference antibodies are used herein:

[0190] ref1: IL-13 mAb Lebrikizumab, the preparation method of which is described in WO2005062967A2;

[0191] ref2: TSLP / IL-13 Nanobody Lunsekimig, the preparation method of which is described in US11208476B2;

[0192] ref3: TSLP mAb, the preparation method of which is described in CN112876564B.

[0193] Anti-IL-13 and TSLP bispecific antibodies

[0194] The present disclosure provides a bispecific antibody or antigen-binding fragment thereof, comprising a first antigen-binding region binding IL-13 and a second antigen-binding region binding TSLP, the first antigen-binding region comprising a first light chain variable region (VL1) and a first heavy chain variable region (VH1), the second antigen-binding region comprising a second light chain variable region (VL2) and a second heavy chain variable region (VH2), wherein the VL1 comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 21-23, respectively, the VH1 comprises HCDR 1 having the amino acid sequence of SEQ ID NO: 24, HCDR 2 having the amino acid sequence of SEQ ID NO: 25 or 26, and HCDR 3 having the amino acid sequence of SEQ ID NO: 27, the VL2 comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 35-37, respectively, and the VH2 comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 30-32, respectively.

[0195] In some embodiments, the VL1 comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 21-23, respectively, the VH1 comprises HCDR 1 having the amino acid sequence of SEQ ID NO: 24, HCDR 2 having the amino acid sequence of SEQ ID NO: 25 or 26, and HCDR 3 having the amino acid sequence of SEQ ID NO: 27, the VL2 comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 35-37, respectively, and the VH2 comprises HCDR 1-3 having the amino acid sequences of SEQ ID NO: 54 (TDYWMH), SEQ ID NO: 31, and SEQ ID NO: 32, respectively.

[0196] In some embodiments, the VL1 comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 21-23, respectively, the VH1 comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 24, 25, and 27, respectively, the VL2 comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 35-37, respectively, and the VH2 comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 30-32, respectively.

[0197] In some embodiments, the VL1 comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 21-23, respectively, the VH1 comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 24, 25, and 27, respectively, the VL2 comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 35-37, respectively, and the VH2 comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 54, 31, and 32, respectively.

[0198] In some embodiments, the VL1 comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 21-23, respectively, the VH1 comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 24, 26, and 27, respectively, the VL2 comprises LCDR 1-3 having the amino acid sequences of SEQ ID NOs: 35-37, respectively, and the VH2 comprises HCDR 1-3 having the amino acid sequences of SEQ ID NOs: 30-32, respectively.

[0199] In some embodiments, the VL1 comprises LCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 21-23, respectively, the VH1 comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 24, 26, and 27, respectively, the VL2 comprises LCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 35-37, respectively, and the VH2 comprises HCDR 1-3 having the amino acid sequences set forth in SEQ ID NOs: 54, 31, and 32, respectively.

[0200] In some embodiments, the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 1, 3, 46, 53, the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 52, the VL2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 33 or 51, and the VH2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28 or 50.

[0201] In some embodiments, the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 1, 3, 46, 53, the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 5, 7, 9, 11, 13, 52, the VL2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 33, and the VH2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28.

[0202] In some embodiments, the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 1, 3, 53, the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 52, the VL2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 33, and the VH2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28.

[0203] In some embodiments, the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 53, the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 52, the VL2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 33, and the VH2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28.

[0204] In some embodiments, the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 1, the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with any one selected from the group consisting of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 52, the VL2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 33, and the VH2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 28.

[0205] In some embodiments, the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 3, the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with any one selected from the group consisting of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 52, the VL2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 33, and the VH2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 28.

[0206] In some embodiments, the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 53, and the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 52.

[0207] In some embodiments, the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 and the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9.

[0208] In some embodiments, the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 and the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 5.

[0209] In some embodiments, the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 and the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 7.

[0210] In some embodiments, the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 and the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11.

[0211] In some embodiments, the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 and the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 13.

[0212] In some embodiments, the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 46, and the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 5.

[0213] In some embodiments, the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 46, and the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 7.

[0214] In some embodiments, the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 46, and the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9.

[0215] In some embodiments, the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 46, and the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11.

[0216] In some embodiments, the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 3, and the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 5.

[0217] In some embodiments, the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 3, and the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 7.

[0218] In some embodiments, the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 3, and the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9.

[0219] In some embodiments, the VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 3, and the VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11.

[0220] In some embodiments, VL1 comprises a functional variant of the amino acid sequence set forth in SEQ ID NO: 1, 3, 46, or 53, formed by insertion, deletion, and / or substitution of one or more amino acids therein, provided that the functional variant retains the ability to bind to IL-13. In some embodiments, VH1 comprises a functional variant of the amino acid sequence set forth in SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, or 52, formed by insertion, deletion, and / or substitution of one or more amino acids therein, provided that the functional variant retains the ability to bind to IL-13. In some embodiments, VL2 comprises a functional variant of the amino acid sequence set forth in SEQ ID NO: 33 or 51, formed by insertion, deletion, and / or substitution of one or more amino acids therein, provided that the functional variant retains the ability to bind to TSLP. In some embodiments, VH2 comprises a functional variant of the amino acid sequence set forth in SEQ ID NO: 28 or 50, formed by insertion, deletion, and / or substitution of one or more amino acids therein, provided that the functional variant retains the ability to bind to TSLP.

[0221] The functional variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to the amino acid sequence of the parent polypeptide.

[0222] In the context of functional variants, the number of inserted, deleted, and / or substituted amino acids is preferably not more than 40%, more preferably not more than 35%, more preferably 1% to 33%, more preferably 5% to 30%, more preferably 10% to 25%, more preferably 15% to 20% of the total number of amino acids in the parent amino acid sequence. For example, the number of inserted, deleted, and / or substituted amino acids can be 1 to 20, preferably 1 to 10, more preferably 1 to 7, still more preferably 1 to 5, most preferably 1 to 2. In preferred embodiments, the number of inserted, deleted, and / or substituted amino acids is 1, 2, 3, 4, 5, 6, or 7.

[0223] In some embodiments, the insertion, deletion, and / or substitution can be made in a framework (FR) region, e.g., in FR1, FR2, FR3, and / or FR4.

[0224] In some embodiments, the substitution of one or more amino acids can be a conservative substitution of one or more amino acids. Such conservative substitutions are preferably substitutions of one amino acid residue for another within the same group of (a) small aliphatic, nonpolar or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gin; (c) polar, positively charged residues: His, Arg, and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp.

[0225] Particularly preferred conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gin or to His; Asp to Glu; Cys to Ser; Gin to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gin; lie to Leu or to Val; Leu to lie or to Val; Lys to Arg, to Gin or to Glu; Met to Leu, to Tyr or to lie; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, to lie or to Leu.

[0226] In preferred embodiments, the VL1 comprises the amino acid sequence set forth in SEQ ID NO: 53 and the VH1 comprises the amino acid sequence set forth in SEQ ID NO: 52; and the VL2 comprises the amino acid sequence set forth in SEQ ID NO: 33 and the VH2 comprises the amino acid sequence set forth in SEQ ID NO: 28.

[0227] In preferred embodiments, the VL1 comprises the amino acid sequence set forth in SEQ ID NO: 1 and the VH1 comprises the amino acid sequence set forth in SEQ ID NO: 9; and the VL2 comprises the amino acid sequence set forth in SEQ ID NO: 33 and the VH2 comprises the amino acid sequence set forth in SEQ ID NO: 28.

[0228] In other preferred embodiments, the VL1 comprises the amino acid sequence set forth in SEQ ID NO: 1 and the VH1 comprises the amino acid sequence set forth in SEQ ID NO: 5; and the VL2 comprises the amino acid sequence set forth in SEQ ID NO: 33 and the VH2 comprises the amino acid sequence set forth in SEQ ID NO: 28.

[0229] In other preferred embodiments, the VL1 comprises the amino acid sequence set forth in SEQ ID NO: 1 and the VH1 comprises the amino acid sequence set forth in SEQ ID NO: 7; and the VL2 comprises the amino acid sequence set forth in SEQ ID NO: 33 and the VH2 comprises the amino acid sequence set forth in SEQ ID NO: 28.

[0230] In other preferred embodiments, the VL1 comprises the amino acid sequence set forth in SEQ ID NO: 1 and the VH1 comprises the amino acid sequence set forth in SEQ ID NO: 13; and the VL2 comprises the amino acid sequence set forth in SEQ ID NO: 33 and the VH2 comprises the amino acid sequence set forth in SEQ ID NO: 28.

[0231] In other preferred embodiments, the VL1 comprises the amino acid sequence set forth in SEQ ID NO: 1 and the VH1 comprises the amino acid sequence set forth in SEQ ID NO: 13; and the VL2 comprises the amino acid sequence set forth in SEQ ID NO: 33 and the VH2 comprises the amino acid sequence set forth in SEQ ID NO: 28.

[0232] In other preferred embodiments, the VL1 comprises the amino acid sequence set forth in SEQ ID NO: 46 and the VH1 comprises the amino acid sequence set forth in SEQ ID NO: 5; and the VL2 comprises the amino acid sequence set forth in SEQ ID NO: 33 and the VH2 comprises the amino acid sequence set forth in SEQ ID NO: 28.

[0233] In other preferred embodiments, the VL1 comprises the amino acid sequence set forth in SEQ ID NO: 46 and the VH1 comprises the amino acid sequence set forth in SEQ ID NO: 7; and the VL2 comprises the amino acid sequence set forth in SEQ ID NO: 33 and the VH2 comprises the amino acid sequence set forth in SEQ ID NO: 28.

[0234] In other preferred embodiments, the VL1 comprises the amino acid sequence set forth in SEQ ID NO: 46 and the VH1 comprises the amino acid sequence set forth in SEQ ID NO: 9; and the VL2 comprises the amino acid sequence set forth in SEQ ID NO: 33 and the VH2 comprises the amino acid sequence set forth in SEQ ID NO: 28.

[0235] In other preferred embodiments, the VL1 comprises the amino acid sequence set forth in SEQ ID NO: 46 and the VH1 comprises the amino acid sequence set forth in SEQ ID NO: 11; and the VL2 comprises the amino acid sequence set forth in SEQ ID NO: 33 and the VH2 comprises the amino acid sequence set forth in SEQ ID NO: 28.

[0236] In other preferred embodiments, the VL1 comprises the amino acid sequence set forth as SEQ ID NO: 3 and the VH1 comprises the amino acid sequence set forth as SEQ ID NO: 5; and the VL2 comprises the amino acid sequence set forth as SEQ ID NO: 33 and the VH2 comprises the amino acid sequence set forth as SEQ ID NO: 28.

[0237] In other preferred embodiments, the VL1 comprises the amino acid sequence set forth as SEQ ID NO: 3 and the VH1 comprises the amino acid sequence set forth as SEQ ID NO: 7; and the VL2 comprises the amino acid sequence set forth as SEQ ID NO: 33 and the VH2 comprises the amino acid sequence set forth as SEQ ID NO: 28.

[0238] In other preferred embodiments, the VL1 comprises the amino acid sequence set forth as SEQ ID NO: 3 and the VH1 comprises the amino acid sequence set forth as SEQ ID NO: 9; and the VL2 comprises the amino acid sequence set forth as SEQ ID NO: 33 and the VH2 comprises the amino acid sequence set forth as SEQ ID NO: 28.

[0239] In other preferred embodiments, the VL1 comprises the amino acid sequence set forth as SEQ ID NO: 3 and the VH1 comprises the amino acid sequence set forth as SEQ ID NO: 11; and the VL2 comprises the amino acid sequence set forth as SEQ ID NO: 33 and the VH2 comprises the amino acid sequence set forth as SEQ ID NO: 28.

[0240] The bispecific antibodies disclosed herein can comprise an Fc region of an antibody, which comprises a CH2 and CH3 domain.

[0241] The Fc region can be of any isotype, including but not limited to IgGl, IgG2, IgG3, and IgG4, and can comprise one or more mutations or modifications. In one embodiment, the Fc region is or is derived from an IgGl or IgG4 isotype, optionally with one or more mutations or modifications. In one embodiment, the Fc region is a human IgGl Fc. In one embodiment, the Fc region is a human IgG4 Fc.

[0242] In some embodiments, the Fc region comprises a modification or mutation that can inhibit Fc homo-dimerization, for example a knob-in-hole mutation in the CH3 of the Fc region.

[0243] Detailed descriptions of the knob-into-hole concept can be found, for example, in U.S. Patent Nos. 5,731,168 and 7,186,076; and Ridgway et al., Protein Engineering, Design and Selection, 1996, 9(7):617-621, Atwell et al., J Mol Biol, 1997, 270(1):26-35; Merchant et al., Nat Biotechnol, 1998, 16:677-681; and Carter, J. Immunological Methods, 2001, 24(1-2):7-15. Briefly, a knob can be created at the CH3 domain interface of a first IgG Fc chain by replacing a smaller one with a larger amino acid side chain (e.g., T366W); and a hole can be created in a juxtaposed position at the CH3 interface of a second IgG Fc chain by replacing a larger one with a smaller amino acid side chain (e.g., Y407V).

[0244] The amino acid residue forming the knob is typically a naturally occurring amino acid residue and is selected from arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). In some preferred embodiments, the amino acid residue is tryptophan and tyrosine. In one embodiment, the original residue forming the knob has a small side chain volume, such as alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine. Exemplary amino acid substitutions in the CH3 domain forming the knob include, but are not limited to, S354C, T366W, T366Y, or F405W substitutions (according to the EU numbering system).

[0245] The amino acid residue forming the hole is typically a naturally occurring amino acid residue and is selected from alanine (A), serine (S), threonine (T), and valine (V). In some preferred embodiments, the original residue forming the hole has a large side chain volume, such as tyrosine, arginine, phenylalanine, or tryptophan. Exemplary amino acid substitutions in the CH3 domain creating the hole include, but are not limited to, Y349C, T366S, L368A, F405A, Y407A, Y407T, and Y407V substitutions (according to the EU numbering system).

[0246] In preferred embodiments, the knob comprises a T366W substitution, and the hole comprises T366S, L368A, and Y407V substitutions. In some embodiments, the knob comprises S354C and T366W substitutions, and the hole comprises Y349C, T366S, L368A, and Y407V substitutions.

[0247] In some embodiments, the CH3 in one heavy chain comprises T366S, L368A, and Y407V mutations, and the CH3 in the other heavy chain comprises a T366W mutation. In some embodiments, the CH3 in one heavy chain comprises S354C and T366W substitutions, and the CH3 in the other heavy chain comprises Y349C, T366S, L368A, and Y407V substitutions.

[0248] In some embodiments, CH1 and CL comprise amino acid mutations that can promote dimerization of heavy and light chains, for example positively charged amino acids (e.g., K and R) in one of CH1 and CL are mutated to negatively charged amino acids (e.g., E, Q, D, and N), and negatively charged amino acids in the other are mutated to positively charged amino acids. In preferred embodiments, CH1 comprises K158E and K224E mutations, and CL comprises E143R and Q144K mutations. In some embodiments, CH1 comprises K147E and / or K213E (according to the EU numbering system). In preferred embodiments, CH1 in one heavy chain comprises K147E and K213E, and CH1 in the other heavy chain comprises K147E and K213E.

[0249] The bispecific antibodies of the present application can also comprise charge-pair mutations to improve association between particular domains and reduce bispecific antibody mispairing. Charge-pair mutations work by affecting the charge of amino acids in the domain surface, such that a domain with a charge-pair mutation preferentially associates with another domain with a complementary charge-pair mutation. Charge-pair mutations are described in greater detail in U.S. Application Nos. 8,592,562, 9,248,182, and 9,358,286, each of which is incorporated herein by reference.

[0250] In some embodiments, the charge-pair mutations can be in CH3. In particular embodiments, the charge-pair mutations are T366K in one heavy chain CH3 and L351D in the other heavy chain CH3.

[0251] In some embodiments, the charge-pair mutations can be in the VL and VH. In particular embodiments, the charge-pair mutations are charge-pair mutations at the VH / VL interface. In preferred embodiments, the charge-pair mutations at the VH / VL interface are Q39E in the VH and Q38K in the VL, or Q39K in the VH and Q38E in the VL (according to the Kabat numbering system). In preferred embodiments, the bispecific antibodies of the application comprise Q39E in the VH and Q38K in the VL. In other preferred embodiments, the bispecific antibodies of the application comprise Q39K in the VH and Q38E in the VL. In more preferred embodiments, the bispecific antibodies of the application comprise Q39E in the VH1, Q38K in the VL1, Q39K in the VH2, and Q38E in the VL2.

[0252] In some embodiments, the Fc region has reduced effector function, e.g., reduced ADCC, ADCP, CDC, and / or Clq, FcyRI, FcyRII, or FcyRIIIA binding. For example, the Fc region can be of the IgGl isotype, or of a non-IgGl type, e.g., IgG2, IgG3, or IgG4, which has been mutated such that the ability to mediate effector function is reduced or even eliminated. Such mutations have been described, e.g., in Dall’Acqua WF, et al., J Immunol. 177(2): 1129-1138 (2006) and Hezareh M, J Virol.; 75(24): 12161-12168 (2001). For example, the Fc region can comprise an amino acid sequence having one or more of the following amino acid substitutions compared to the wild-type sequence: E233P, L234A, L234F, L235A, L235E, G237A, N297A, N297D, P331S, and P329G. In preferred embodiments the Fc region comprises L234A and L235A (LA mutations).

[0253] In one embodiment, the Fc region comprises a mutation that removes the Asn-linked glycosylation receptor site or is otherwise manipulated to alter the glycosylation properties. For example, in an IgGl Fc region, the N297Q mutation can be used to remove the Asn-linked glycosylation site. Thus, in particular embodiments, the Fc region comprises an IgGl sequence with the N297Q mutation.

[0254] In further embodiments, the Fc region is glycoengineered to reduce fucose and thus enhance ADCC, for example by adding a compound to the culture medium during antibody production, as described in US2009317869 or as described in van Berkel et al. (2010) Biotechnol. Bioeng. 105:350, or by using FUT8 knockout cells, as described in Yamane-Ohnuki et al. (2004) Biotechnol. Bioeng 87:614. Alternatively, the ADCC can be optimized using the methods described in Shields et al. (1999) Nature Biotech 17:176. In another embodiment, the Fc region is engineered to enhance complement activation, for example as described in Natsume et al. (2009) Cancer Sci. 100:2411.

[0255] In some embodiments, the Fc region comprises a mutation that prolongs the half-life of the antibody. For example, the mutation can enhance the binding of the antibody to FcRn. In some embodiments, the mutation that prolongs the half-life of the antibody can be M428L and / or N434A (according to the EU numbering system). In some embodiments, the Fc in either of the two heavy chains comprises M428L and / or N434A. In some embodiments, the Fc in both of the two heavy chains comprises M428L and / or N434A. In preferred embodiments, the Fc in both of the two heavy chains comprises M428L and N434A.

[0256] In some embodiments, the antibody comprises:

[0257] a first heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 42,

[0258] a first light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 44,

[0259] a second heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 38,

[0260] a second light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 40.​

[0261] In some embodiments, the antibody comprises:

[0262] a first heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 57,

[0263] a first light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 44,

[0264] a second heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 58,

[0265] a second light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 40.

[0266] In some embodiments, the first heavy chain comprises a functional variant of the amino acid sequence set forth in SEQ ID NO: 42 formed by insertion, deletion, and / or substitution of one or more amino acids thereof, provided the functional variant retains the ability to bind to IL-13. In some embodiments, the first light chain comprises a functional variant of the amino acid sequence set forth in SEQ ID NO: 44 formed by insertion, deletion, and / or substitution of one or more amino acids thereof, provided the functional variant retains the ability to bind to IL-13. In some embodiments, the second heavy chain comprises a functional variant of the amino acid sequence set forth in SEQ ID NO: 38 formed by insertion, deletion, and / or substitution of one or more amino acids thereof, provided the functional variant retains the ability to bind to TSLP. In some embodiments, the second heavy chain comprises a functional variant of the amino acid sequence set forth in SEQ ID NO: 40 formed by insertion, deletion, and / or substitution of one or more amino acids thereof, provided the functional variant retains the ability to bind to TSLP.

[0267] In some embodiments, the first heavy chain comprises a functional variant of the amino acid sequence set forth in SEQ ID NO: 57, formed by insertion, deletion and / or substitution of one or more amino acids therein, provided that the functional variant retains the ability to bind to IL-13. In some embodiments, the first light chain comprises a functional variant of the amino acid sequence set forth in SEQ ID NO: 44, formed by insertion, deletion and / or substitution of one or more amino acids therein, provided that the functional variant retains the ability to bind to IL-13. In some embodiments, the second heavy chain comprises a functional variant of the amino acid sequence set forth in SEQ ID NO: 58, formed by insertion, deletion and / or substitution of one or more amino acids therein, provided that the functional variant retains the ability to bind to TSLP. In some embodiments, the second heavy chain comprises a functional variant of the amino acid sequence set forth in SEQ ID NO: 40, formed by insertion, deletion and / or substitution of one or more amino acids therein, provided that the functional variant retains the ability to bind to TSLP.

[0268] A functional variant comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to the amino acid sequence of the parent polypeptide.

[0269] In some embodiments, the number of inserted, deleted and / or substituted amino acids is preferably no more than 40%, more preferably no more than 35%, more preferably 1% to 33%, and more preferably 5% to 30%, more preferably 10% to 25%, more preferably 15% to 20% of the total number of amino acids in the parent amino acid sequence. For example, the number of inserted, deleted and / or substituted amino acids can be 1 to 50, preferably 1 to 20, more preferably 1 to 10, and still more preferably 1 to 5. In preferred embodiments, the number of inserted, deleted and / or substituted amino acids is 1, 2, 3, 4, 5, 6, or 7.

[0270] In some embodiments, the insertion, deletion and / or substitution can be in a framework (FR) region, e.g., FR1, FR2, FR3, and / or FR4; and / or a constant region, e.g., CL, CH1, CH2, and / or CH3.

[0271] In some embodiments, the substitution of one or more amino acids can be a conservative substitution of one or more amino acids. Examples of conservative substitutions are described above.

[0272] In preferred embodiments, the antibody comprises a first heavy chain having an amino acid sequence as set forth in SEQ ID NO: 42, a first light chain having an amino acid sequence as set forth in SEQ ID NO: 44, a second heavy chain having an amino acid sequence as set forth in SEQ ID NO: 38, and a second light chain having an amino acid sequence as set forth in SEQ ID NO: 40.

[0273] In preferred embodiments, the antibody comprises a first heavy chain having an amino acid sequence as set forth in SEQ ID NO: 57, a first light chain having an amino acid sequence as set forth in SEQ ID NO: 44, a second heavy chain having an amino acid sequence as set forth in SEQ ID NO: 58, and a second light chain having an amino acid sequence as set forth in SEQ ID NO: 40.

[0274] Anti-IL-13 antibodies

[0275] The present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds IL-L3, comprising a light chain variable region (VL) and a heavy chain variable region (VH), wherein the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one selected from the group consisting of SEQ ID NO: 1, 3, 46, 53, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one selected from the group consisting of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 52.

[0276] In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 1, 3, 53, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one selected from the group consisting of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 52.

[0277] In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 53, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 52.

[0278] In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 52.

[0279] In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 3, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 5, 7, 9, 11, 13, 15, 17, 19, 52.

[0280] In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 53, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 52.

[0281] In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9.

[0282] In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 5.

[0283] In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 7.

[0284] In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11.

[0285] In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 13.

[0286] In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 46 and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 5.

[0287] In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 46, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 7.

[0288] In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 46, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9.

[0289] In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 46, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11.

[0290] In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 3, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 5.

[0291] In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 3, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 7.

[0292] In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 3, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9.

[0293] In some embodiments, the VL comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 3, and the VH comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11.

[0294] In some embodiments, the VL comprises an amino acid sequence selected from any one of SEQ ID NOs: 1, 3, 46, 53, and the VH comprises an amino acid sequence selected from any one of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 52.

[0295] In some embodiments, the VL comprises an amino acid sequence selected from any one of SEQ ID NOs: 1, 3, 53, and the VH comprises an amino acid sequence selected from any one of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 52.

[0296] In some embodiments, the VL comprises an amino acid sequence selected from any one of SEQ ID NOs: 1, 3, 53, and the VH comprises an amino acid sequence selected from any one of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 52.

[0297] In some embodiments, the VL comprises an amino acid sequence selected from any one of SEQ ID NOs: 1, 3, 53, and the VH comprises an amino acid sequence selected from any one of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 52.

[0298] In some embodiments, the VL comprises an amino acid sequence selected from any one of SEQ ID NOs: 1, 3, 53, and the VH comprises an amino acid sequence selected from any one of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 52.

[0299] In some embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 53 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 52.

[0300] In some embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 1 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 9.

[0301] In some embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 1 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 5.

[0302] In some embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 1 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 7.

[0303] In some embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 1 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 11.

[0304] In some embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 1 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 13.

[0305] In some embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 46 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 5.

[0306] In some embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 46 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 7.

[0307] In some embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 46 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 9.

[0308] In some embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 46 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 11.

[0309] In some embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 3 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 5.

[0310] In some embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 3 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 7.

[0311] In some embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 3 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 9.

[0312] In some embodiments, the VL comprises an amino acid sequence as set forth in SEQ ID NO: 3 and the VH comprises an amino acid sequence as set forth in SEQ ID NO: 11.

[0313] Based on the amino acid sequences of the antibody heavy chain constant regions, immunoglobulin molecules can be assigned to five classes (isotypes): IgA, IgD, IgE, IgG, and IgM, and can be further divided into subclasses, e.g., IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, etc. Based on the amino acid sequences of the light chains, the light chains of antibodies can be assigned to either lambda (l) chain or kappa (K) chain. The antibodies disclosed herein can be of any of the above classes or subclasses.

[0314] In some embodiments, the antibody is of an isotype selected from the group consisting of IgG, IgA, IgM, IgE, and IgD. In some embodiments, the antibody is of a subclass selected from the group consisting of IgGl, IgG2, IgG3, and IgG4. In preferred embodiments, the antibody is an IgGl or IgG4 antibody.

[0315] The antibodies disclosed herein can be intact antibodies or antigen-binding fragments thereof. The antigen-binding fragments can be any fragment of an antibody that retains the ability to specifically bind to IL-13. Examples of antigen-binding fragments include, but are not limited to, Fab fragments; F(ab')2 fragments; Fab' fragments; Fd fragments; Fd' fragments; Fv fragments; scFv fragments; dAb fragments; isolated complementarity determining regions (CDRs); nanobodies; linear antibodies consisting of a pair of tandem Fd segments (VH-CH1-VH-CH1), and modified forms of any of the aforesaid fragments that retain antigen binding activity.

[0316] In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fv, scFv, and ds-scFv. In preferred embodiments, the antigen-binding fragment is Fab. In another preferred embodiment, the antigen-binding fragment is Fv. In another preferred embodiment, the antigen-binding fragment is scFv.

[0317] In some embodiments, the antibody is a monoclonal antibody, a bispecific antibody, or a multispecific antibody. In some embodiments, the antibody is a bispecific antibody further comprising a second antigen binding region that binds to a second antigen. In some embodiments, the second antigen is a proinflammatory cytokine, preferably a cytokine that promotes a type 2 inflammatory response, such as IL-4, IL-5, IL-13, IL-25, IL-31, IL-33, TSLP, and the like.

[0318] Nucleic acid

[0319] The present disclosure provides nucleic acids comprising a nucleotide sequence encoding a bispecific antibody or antigen-binding fragment thereof disclosed herein or an antibody or antigen-binding fragment thereof disclosed herein.

[0320] The term "nucleic acid" includes single- and double-stranded nucleotide polymers. The nucleic acid can be ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. The modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phenylphosphonothioate, phenylphosphonodithioate, and phosphoramidate.

[0321] In some embodiments, the present application provides a nucleic acid molecule encoding any one of the heavy chain variable region sequences disclosed herein. The present application also provides a nucleic acid molecule that is at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid encoding any one of the heavy chain variable region sequences disclosed herein. In some embodiments, the present application provides a nucleic acid molecule encoding any one of the light chain variable region sequences disclosed herein. The present application also provides a nucleic acid molecule that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid encoding any one of the light chain variable region sequences disclosed herein.

[0322] In some embodiments, the nucleic acid disclosed herein comprises

[0323] a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 2, 4, 56,

[0324] a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 55,

[0325] a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 29, and

[0326] a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 34.

[0327] In preferred embodiments, the nucleic acid disclosed herein comprises a nucleotide sequence as set forth in any one of SEQ ID NO: 2, 4, 56, a nucleotide sequence as set forth in any one selected from the group consisting of SEQ ID NO: 6, 8, 10, 12, 14, 16, 18, 20, 55, a nucleotide sequence as set forth in SEQ ID NO: 29, and a nucleotide sequence as set forth in SEQ ID NO: 34.

[0328] In some embodiments, the present application provides a nucleic acid molecule encoding a heavy chain sequence disclosed herein. The present application also provides a nucleic acid molecule that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid encoding a heavy chain sequence disclosed herein. In some embodiments, the present application provides a nucleic acid molecule encoding a light chain sequence disclosed herein. The present application also provides a nucleic acid molecule that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to a nucleic acid encoding a light chain sequence disclosed herein.

[0329] In some embodiments, the nucleic acid disclosed herein comprises

[0330] a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 39,

[0331] a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 41,

[0332] a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 43, and

[0333] a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 45.

[0334] In preferred embodiments, the nucleic acid disclosed herein comprises a nucleotide sequence as set forth in SEQ ID NO: 39, a nucleotide sequence as set forth in SEQ ID NO: 41, a nucleotide sequence as set forth in SEQ ID NO: 43, and a nucleotide sequence as set forth in SEQ ID NO: 45.

[0335] In some embodiments, the nucleic acid is a ribonucleic acid (RNA) or a deoxyribonucleic acid (DNA). In some embodiments, the present application provides a ribonucleic acid (RNA) comprising a nucleotide sequence encoding an antibody disclosed herein. In some embodiments, the present application provides a deoxyribonucleic acid (DNA) comprising a deoxyribonucleotide sequence encoding an antibody disclosed herein.

[0336] In some embodiments, the deoxyribonucleic acid (DNA) can be introduced into a human cell in vivo. In some embodiments, the deoxyribonucleic acid (DNA) of the present application is comprised in a vector or delivery agent. In some embodiments, the deoxyribonucleic acid (DNA) of the present application is integrated into the genome of the cell.

[0337] In some embodiments, the ribonucleic acid (RNA) can be introduced into a human cell in vivo. In some embodiments, the ribonucleic acid (RNA) of the present application is comprised in a vector or delivery agent.

[0338] Vectors

[0339] The present disclosure provides vectors comprising the nucleic acids disclosed herein.

[0340] In some embodiments, the vector is an expression vector capable of expressing a polypeptide comprising a heavy chain or light chain variable region of an antibody. For example, the present application provides an expression vector comprising any of the nucleic acid molecules described above.

[0341] Any vector can be suitable for use in the present disclosure. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, an SFG vector, a plasmid, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein Barr virus vector, a papovaviral vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus associated vector (AAV), a lentiviral vector, or any combination thereof. Suitable exemplary vectors include, for example, pGAR, pBABE-puro, pBABE-neo large T cDNA, pBABE-hygro-hTERT, pMKO.1 GFP, MSCV-IRES-GFP, pMSCV PIG (Puro IRES GFP empty plasmid), pMSCV-loxp-dsRed-loxp-eGFP-Puro-WPRE, MSCV IRES luciferase, pMIG, MDH1-PGK-GFP_2.0, TtRMPVIR, pMSCV-IRES-mCherry FP, pRetroX GFP T2A Cre, pRXTN, pLncEXP, and pLXIN-Luc.

[0342] The expression vector can be any suitable recombinant expression vector. Suitable vectors include those designed for propagation and amplification or for expression or both, such as plasmids and viruses. For example, the vector can be selected from the pUC series (Fermentas LifeSciences, Glen Burnie, Md.), the pBluescript series (Stratagene, La Jolla, Calif.), the pET series (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, Calif.). Bacteriophage vectors, such as lambda GT10, lambda GT11, lambda Zap II (Stratagene), lambda EMBL4, and lambda NM1149, can also be used. Examples of plant expression vectors useful in the present disclosure include pBI01, pBI101.2, pBI101.3, pBI121, and pBIN19 (Clontech). Examples of animal expression vectors useful in the present disclosure include pcDNA, pEUK-Cl, pMAM, and pMAMneo (Clontech).

[0343] Recombinant expression vectors can be prepared using standard recombinant DNA techniques described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Circular or linear expression vector constructs can be prepared to contain replication systems functional in prokaryotic or eukaryotic host cells. Replication systems can be derived, for example, from COLEL, 2μ plasmid, lambda, SV40, bovine papilloma virus, and the like.

[0344] For example, the vector can be an adenoviral vector comprising a nucleotide sequence encoding an antibody disclosed herein. The vector can be administered into a subject, then in vivo into a cell of the subject, thereby integrating the nucleotide sequence encoding the antibody disclosed herein into the genome of the cell, and subsequently the cell expresses the antibody disclosed herein.

[0345] Host cells

[0346] The present disclosure provides a host cell comprising a nucleic acid disclosed herein or a vector disclosed herein.

[0347] Any cell can be used as a host cell for the nucleic acids or vectors of the disclosure. In some embodiments, the cell can be a prokaryotic cell, a fungal cell, a yeast cell, or a higher eukaryotic cell such as a mammalian cell. Suitable prokaryotic cells include, but are not limited to, eubacteria, such as gram-negative or gram-positive organisms, for example, Enterobactehaceae, such as Escherichia, for example, E. coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, for example, Salmonella typhimurium; Serratia, for example, Serratia marcescans and Shigella; Bacilli, such as B. subtilis and B. licheniformis; Pseudomonas, such as P. aeruginosa; and Streptomyces. In some embodiments, the cell is a human cell. In some embodiments, the cell is an immune cell. In some embodiments, the host cell includes, for example, CHO cells, such as CHOS cells and CHO-K1 cells, or HEK293 cells, such as HEK293A, HEK293T, and HEK293FS.

[0348] The host cells of the present application are prepared by introducing the vectors disclosed herein or the nucleic acids disclosed herein in vitro or ex vivo. The host cells of the present application can be administered to a subject in vivo, and the host cells express the antibodies disclosed herein in vivo.

[0349] The present application provides host cells into which any of the above-described vectors have been introduced. The present application also provides methods of making the antibodies of the present application, comprising a) culturing the host cells disclosed herein under conditions suitable for production of the antibodies; and b) obtaining the antibodies from the culture.

[0350] Pharmaceutical compositions

[0351] The present disclosure provides pharmaceutical compositions comprising the bispecific antibodies or antigen-binding fragments thereof disclosed herein, or the antibodies or antigen-binding fragments thereof disclosed herein, and a pharmaceutically acceptable carrier or excipient.

[0352] The antibodies or antigen-binding fragments thereof or agents of the present application (also referred to herein as "active compounds") and derivatives, fragments, analogs, and homologs thereof can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise an antibody or antigen-binding fragment thereof or agent and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Preferred examples of such carriers or excipients include, but are not limited to, water, saline, Ringer's solutions, dextrose solutions, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils can also be used. Such vehicles and agents for use in

[0353] In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent, and a small molecule drug.

[0354] The pharmaceutical compositions of the present application can be formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic.

[0355] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL TM(BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of coating such as lecithin, by the maintenance of required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by the inclusion in the composition of agents which delay absorption, for example, aluminum monostearate and gelatin.

[0356] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze drying which yields a powder of the active ingredient plus any additional desired ingredient from the previously sterile-filtered solution thereof.

[0357] Oral composition generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash in which the compound is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0358] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressurized container or a dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide.

[0359] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into

[0360] The active compounds can also be prepared in the form of suppositories (e.g., using conventional suppository

[0361] In one embodiment, the active compounds are prepared with carriers that protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for

[0362] The present application provides therapeutic compositions comprising the antibodies or antigen-binding fragments thereof of the present application. The therapeutic compositions according to the present application will be administered with suitable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, etc. Many suitable formulations can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid-containing vesicles (cationic or anionic) such as LIPOFECTIN TM ), DNA conjugates, anhydrous absorption pastes, oil-in- water and water-in-oil emulsions, emulsions with

[0363] Conjugates

[0364] The present disclosure provides conjugates comprising the antibodies or antigen-binding fragments thereof disclosed herein or the bispecific antibodies or antigen-binding fragments thereof disclosed herein, and a chemical moiety conjugated thereto.

[0365] In the context of the present disclosure, a "conjugate" is an antibody or antibody fragment (such as an antigen-binding fragment) covalently linked to a chemical moiety. The chemical moiety can be, for example, a drug, a toxin, a therapeutic agent, a detectable label, a protein, a nucleic acid, a lipid, a nanoparticle, a carbohydrate, or a recombinant virus. Antibody conjugates are often referred to as "immunoconjugates." When the conjugate comprises an antibody linked to a drug (e.g., a cytotoxic agent), the conjugate is often referred to as an "antibody-drug conjugate" or "ADC."

[0366] The term "conjugate" or "linkage" can refer to joining two polypeptides into one continuous polypeptide molecule. In one embodiment, the antibody is linked to a chemical moiety. In another embodiment, the antibody linked to a chemical moiety is further linked to a lipid or other molecule to the protein or peptide to increase its half-life in vivo. The linkage can be by chemical or recombinant means. In one embodiment, the linkage is chemical, in which a reaction between the antibody moiety and the chemical moiety results in a covalent bond formed between the two molecules to form one molecule. A peptide linker (a short peptide sequence) can optionally be included between the antibody and the chemical moiety.

[0367] The chemical moiety can be linked to the antibody of the present application using any number of means known to those of skill in the art. Both covalent and non-covalent means of attachment can be used. The procedure for attaching the chemical moiety to the antibody varies depending on the chemical structure of the chemical moiety. Polypeptides typically contain a variety of functional groups; such as carboxylic acid (COOH), free amine (-NH2), or thiol (-SH) moieties, which can be used to react with a suitable functional group on the antibody to result in the binding of the chemical moiety. Alternatively, the antibody is derivatized to expose or attach additional reactive functional groups. The derivatization can involve the attachment of any of a number of known linker molecules. The linker can be any molecule that serves to link the antibody to the chemical moiety. The linker is capable of forming a covalent bond to both the antibody and the chemical moiety. Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight chain or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers. In the case where the antibody and the chemical moiety are polypeptides, the linker can be attached to a constituent amino acid through a side group (such as through a disulfide bond to a cysteine) or to the alpha carbon amino and carboxyl groups of terminal amino acids.

[0368] In certain cases, it is desirable to release the chemical moiety from the antibody when the immunoconjugate reaches its target site. Thus, in these cases, the immunoconjugate will comprise a linkage that is cleavable in the vicinity of the target site.

[0369] The conditions experienced by the enzymatic activity or immunoconjugate within the target cell or in the vicinity of the target site can prompt cleavage of the linker to release the chemical moiety from the antibody.

[0370] In view of the large number of methods reported for attaching various radiodiagnostic compounds, radiotherapeutic compounds, labels (such as enzymes or fluorescent molecules), drugs, toxins, and other agents to antibodies, one of skill in the art will be able to determine a suitable method for attaching a given agent to an antibody or other polypeptide.

[0371] Antibodies disclosed herein can be derivatized or linked to another molecule, such as another peptide or protein. Typically, the antibody or portion thereof is derivatized such that binding to the target antigen is not adversely affected by the derivatization or labeling. For example, an antibody can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody), a detector agent, a pharmaceutical agent, and / or a protein or peptide that can mediate association with another molecule, such as a streptavidin core region or a polyhistidine tag.

[0372] One type of derivatized antibody is produced by cross-linking two or more antibodies (of the same or different type). Suitable cross-linking agents include heterobifunctional or homobifunctional cross-linking agents having two distinctly reactive moieties separated by a suitable spacer region (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or a homobifunctional cross-linking agent (such as succinic acid bis-succinimidyl ester). Such linkers are commercially available.

[0373] In some embodiments of the conjugates disclosed herein, the chemical moiety is selected from a therapeutic agent, a detectable moiety, and an immunostimulatory molecule.

[0374] In some embodiments, the therapeutic agent includes, but is not limited to, an immunomodulatory agent, a radioactive compound, an enzyme (e.g., a perforin), a chemotherapeutic agent (e.g., cisplatin), or a toxin. In some embodiments, the therapeutic agent can be, for example, a maytansinoid, a geldanamycin, a tubulin inhibitor such as a tubulin binding agent (e.g., an auristatin) or a minor groove binder such as a calicheamicin.

[0375] In some embodiments, the detectable moiety can be selected from biotin, streptavidin, an enzyme or catalytically active fragment thereof, a radionuclide, a nanoparticle, a paramagnetic metal ion, or a fluorescent, phosphorescent, or chemiluminescent molecule. Detectable moieties for diagnostic purposes include, for example, fluorescent labels, radiolabels, enzymes, nucleic acid probes, and contrast agents.

[0376] Antibodies can be conjugated to detectable labels; for example, detectable labels that can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or diagnostic imaging techniques such as computed tomography (CT), computed axial tomography (CAT) scan, magnetic resonance imaging (MRI), nuclear magnetic resonance imaging NMRI), magnetic resonance tomography (MTR), ultrasound, fiberoptic examination, and laparoscopy. Specific, non-limiting examples of detectable labels include fluorophores, chemiluminescent agents, enzymatic linkages, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable labels include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-l-napthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, and the like. Bioluminescent labels such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP) can also be used.

[0377] Antibodies or antigen-binding fragments can also be conjugated to enzymes that are useful for detection, such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, and the like. When antibodies or antigen-binding fragments are conjugated to detectable enzymes, detection can be achieved by the addition of additional reagents that the enzyme uses to produce a recognizable reaction product. For example, when horseradish peroxidase reagents are present, the addition of hydrogen peroxide and diaminobenzidine results in a colored reaction product that can be detected visually. Antibodies or antigen-binding fragments can also be conjugated to biotin and detected by indirect measurement of avidin or streptavidin binding. It should be noted that avidin itself can be conjugated to an enzyme or fluorescent label.

[0378] Antibodies can be fused to self-labeling protein tags, such as HaloTag. For example, the protein tag can be cloned to the end of the constant region. HaloTag is a self-labeling protein tag derived from a bacterial enzyme (haloalkane dehalogenase) designed to covalently bind to synthetic ligands. In some cases, the synthetic ligand comprises a chloroalkane linker attached to a fluorophore, such as a near-infrared fluorophore (Los et al. (2008) ACS Chem Biol. 3(6):373-82).

[0379] Antibodies can be labeled with magnetic agents such as gadolinium. Antibodies can also be labeled with lanthanides (such as europium and dysprosium) and manganese.

[0380] Paramagnetic particles such as superparamagnetic iron oxide can also be used as labels. Antibodies can also be labeled with predetermined polypeptide epitopes recognized by a second reporter group (such as a leucine zipper pair sequence, a binding site for biotin, a metal binding domain, an epitope tag) that are recognized by a second reporter group. In some embodiments, the tag is attached by a spacer arm of various lengths to reduce potential steric hindrance.

[0381] Antibodies can also be labeled with radiolabeled amino acids. Radiolabels can be used for diagnostic and therapeutic purposes. For example, radiolabels can be used to detect expression of target antigens by x-ray, emission spectroscopy, or other diagnostic techniques. Examples of polypeptide labels include, but are not limited to, the following radioisotopes or radionucleotides: 3 H、 14 C、 15 N、 35 S、 90 Y、 99 Tc、 111 In、 125 I、 131 I.

[0382] In some embodiments, the immunostimulatory molecule is an immune effector molecule that stimulates an immune response. For example, the immunostimulatory molecule can be a cytokine such as IL-2 and IFN-gamma, a chemokine such as IL-8, platelet factor 4, melanoma growth stimulatory protein, a complement activator; a viral / bacterial protein domain, or a viral / bacterial peptide.

[0383] Therapeutic methods

[0384] The present disclosure provides methods for treating a disease associated with a type 2 inflammatory response in a subject, comprising administering to the subject an effective amount of a bispecific antibody or antigen-binding fragment thereof disclosed herein, an antibody or antigen-binding fragment thereof disclosed herein, a pharmaceutical composition disclosed herein, or a conjugate disclosed herein.

[0385] In some embodiments, the disease is an IL-13 and / or TSLP-mediated disease (e.g., an inflammatory disease and an autoimmune disease). In some embodiments, the disease is an IL-13-mediated inflammatory disease. In some embodiments, the disease is an IL-13-mediated autoimmune disease. In some embodiments, the disease is a TSLP-mediated inflammatory disease. In some embodiments, the disease is a TSLP-mediated autoimmune disease.

[0386] In some embodiments, the disease is selected from the group consisting of allergic inflammation (e.g., allergic rhinitis, allergic rhinosinusitis, allergic conjunctivitis), asthma, atopic dermatitis, urticaria, chronic obstructive pulmonary disease, eosinophilic esophagitis, rheumatoid arthritis, multiple sclerosis, idiopathic pulmonary fibrosis, eczema (e.g., hand eczema, asthmatic eczema), eosinophilic gastroenteritis, Crohn’s disease, systemic sclerosis, ulcerative colitis, chronic rhino-sinusitis with nasal polyps. In preferred embodiments, the disease is atopic dermatitis.

[0387] In some embodiments, the dosage administered to a subject can vary depending on the embodiment, the drug used, the method of administration, and the site and subject to be treated. However, the dosage should be sufficient to provide a therapeutic response. A clinician can determine the effective amount of the antibody to administer to a human or other subject to treat a medical condition. The precise amount required can depend on many factors such as the activity of the antibody and the route of administration.

[0388] A dose of an antibody, composition, or conjugate described herein can be administered to a mammal at one time or in a series of subdoses, over a suitable period of time, for example, on a daily, semi-weekly, weekly, biweekly, semimonthly, bimonthly, semiannual, or annual basis as needed. Dose units comprising an effective amount of an antibody, composition, or conjugate can be administered as a single daily dose, or the total daily dose can be administered in two, three, four, or more divided doses administered daily as needed.

[0389] A suitable mode of administration can be selected by a physician. The route of administration can be parenteral administration, for example, administration by injection, nasal administration, pulmonary administration, or transdermal administration. Systemic or local administration can be performed by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection. In some embodiments, the antibody, composition, or conjugate is selected for parenteral delivery, for inhalation, or for delivery through the digestive tract, for example, orally. The dose and method of administration can vary depending on the weight, age, condition, etc. of the subject, and can be appropriately selected.

[0390] In some embodiments, the method further comprises administering a second therapeutic agent to the subject. In certain embodiments, the antibody, composition, or conjugate disclosed herein is administered prior to, substantially simultaneously with, or after administration of the second therapeutic agent.

[0391] In some embodiments, the second therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent, and a small molecule drug.

[0392] The present disclosure also provides a method for inhibiting IgE antibody production in a subject, comprising administering to the subject an effective amount of the bispecific antibody or antigen-binding fragment thereof disclosed herein, the antibody or antigen-binding fragment thereof disclosed herein, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein.

[0393] One of the pathological features of type 2 inflammatory reaction related diseases is associated with an increase in IgE, thus inhibiting the production of IgE antibodies in the body of a subject can be used to prevent or treat type 2 inflammatory reaction related diseases, such as allergic inflammation (e.g., allergic rhinitis, allergic rhinosinusitis, allergic conjunctivitis), asthma, atopic dermatitis, urticaria, chronic obstructive pulmonary disease, eosinophilic esophagitis, rheumatoid arthritis, multiple sclerosis, idiopathic pulmonary fibrosis, eczema (e.g., hand eczema, asthmatic eczema), eosinophilic gastroenteritis, Crohn's disease, systemic sclerosis, ulcerative colitis, chronic rhino-sinusitis with nasal polyps.

[0394] Medical uses

[0395] The present disclosure provides the use of a bispecific antibody or antigen binding fragment thereof disclosed herein, an antibody or antigen binding fragment thereof disclosed herein, a pharmaceutical composition disclosed herein, or a conjugate disclosed herein in the manufacture of a medicament for treating a type 2 inflammatory reaction related disease in a subject.

[0396] The present disclosure also provides a bispecific antibody or antigen binding fragment thereof disclosed herein, an antibody or antigen binding fragment thereof disclosed herein, a pharmaceutical composition disclosed herein, or a conjugate disclosed herein for use in treating a type 2 inflammatory reaction related disease in a subject.

[0397] In some embodiments of the use disclosed herein, the disease is selected from allergic inflammation (e.g., allergic rhinitis, allergic rhinosinusitis, allergic conjunctivitis), asthma, atopic dermatitis, urticaria, chronic obstructive pulmonary disease, eosinophilic esophagitis, rheumatoid arthritis, multiple sclerosis, idiopathic pulmonary fibrosis, eczema (e.g., hand eczema, asthmatic eczema), eosinophilic gastroenteritis, Crohn's disease, systemic sclerosis, ulcerative colitis, chronic rhino-sinusitis with nasal polyps.

[0398] In some embodiments, the bispecific antibody or antigen binding fragment thereof disclosed herein, the antibody or antigen binding fragment thereof disclosed herein, the pharmaceutical composition disclosed herein, or the conjugate disclosed herein is used in combination with a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from an antibody, a chemotherapeutic agent, and a small molecule drug.

[0399] Kit

[0400] The present disclosure provides a pharmaceutical pack or kit comprising one or more containers containing one or more of the components of a pharmaceutical composition as described herein, such as an antibody or antigen binding fragment disclosed herein.

[0401] In particular embodiments, the kit includes a first container containing an antibody disclosed herein. In particular embodiments, the kit includes a first container, which is a vial containing the antibody as a lyophilized sterile powder under vacuum, and the kit further includes a second container containing a pharmaceutically acceptable fluid.

[0402] In particular embodiments, provided herein are injection devices containing an antibody. In particular embodiments, the injection device contains an antibody in a sterile solution. In particular embodiments, the injection device is a syringe.

[0403] In one embodiment, the kit includes instructional materials that disclose the manner in which the antibodies of the disclosure are used. The instructional materials can be written, electronic (e.g., computer diskette or CD-ROM), or visual (e.g., video cassette or DVD) and can take the form of a package insert. The kit can further comprise additional components to facilitate the use of the kit for which it is designed. Thus, for example, the kit can additionally contain tools (e.g., enzyme substrates for enzymatic labels, filter sets for detecting fluorescent labels, appropriate secondary labels such as a second antibody, etc.) for detecting the label. The kit can also include buffers and other reagents commonly used to practice the particular method for which the kit is designed. Such kits and suitable contents are well known to those of skill in the art.

[0404] Examples

[0405] The following examples are given for the purpose of illustrating various embodiments of the application and are not meant to limit the present application in any way. This example, as well as the methods described herein, are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the application. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the application as defined by the scope of the claims.

[0406] Example 1, IL-13 murine anti 228B / C-1 humanization

[0407] The IL-13 murine anti 228B / C-1 amino acid sequence is taken from patent WO2005062967A2 and is the murine parent molecule of Lebrikizumab. The light chain variable region (VL, also referred to as K0) and the heavy chain variable region (VH, also referred to as H0) of Lebrikizumab and their corresponding CDR sequences (according to the Kabat numbering system) are shown below:

[0408] LCDR1: RASKSVDSYGNSFMH (SEQ ID NO. 21)

[0409] LCDR2: LASNLES (SEQ ID NO. 22)

[0410] LCDR3: QQNNEDPRT (SEQ ID NO. 23)

[0411] HCDR1: AYSVN (SEQ ID NO. 24)

[0412] HCDR2: MIWGDGKIVYNSALKS (SEQ ID NO. 25)

[0413] HCDR3: DGYYPYAMDN (SEQ ID NO. 27)

[0414] VL amino acid sequence:

[0415] DIVMTQSPDSLSVSLGERATINCRASKSVDSYGNSFMHWYQQKPGQPPKLLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQNNEDPRTFGGGTKVEIK (SEQ ID NO. 46)

[0416] VL nucleotide sequence:

[0417] GACATCGTGATGACACAGAGCCCTGACAGCCTGTCCGTGTCTCTGGGAGAGAGAGCCACCATCAACTGCAGAGCCAGCAAGAGCGTGGACAGCTACGGCAACAGCTTCATGCACTGGTATCAGCAGAAGCCCGGCCAGCCTCCTAAGCTGCTGATCTACCTGGCCAGCAACCTGGAAAGCGGCGTGCCAGATAGATTCAGCGGCTCTGGCTCTGGCACCGACTTCACCCTGACAATCAGTTCCCTGCAGGCCGAGGATGTGGCCGTGTACTACTGCCAGCAGAACAACGAGGACCCCAGAACATTCGGCGGAGGCACCAAGgTGGAAATCAA (SEQ ID NO. 47)

[0418] VH amino acid sequence:

[0419] QVTLRESGPALVKPTQTLTLTCTVSGFSLSAYSVNWIRQPPGKALEWLAMIWGDGKIVYNSALKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCAGDGYYPYAMDNWGQGSLVTVSS (SEQ ID NO. 48)

[0420] VH nucleotide sequence:

[0421] CAAGTGACCCTGAGAGAGTCTGGACCCGCTCTGGTCAAGCCCACACAGACCCTGACACTGACCTGTACCGTGTCCGGCTTCAGCCTGTCTGCCTACAGCGTGAACTGGATCAGACAGCCTCCTGGCAAGGCCCTGGAATGGCTGGCTATGATCTGGGGCGACGGCAAGATCGTGTACAACAGCGCCCTGAAGTCCAGACTGACCATCAGCAAGGACACCAGCAAGAACCAGGTGGTGCTGACCATGACCAACATGGACCCTGTGGACACCGCCACCTACTACTGTGCTGGCGACGGCTACTACCCCTACGCCATGGATAATTGGGGCCAGGGCTCTCTGGTCACCGTGTCATCT (SEQ ID NO. 49)

[0422] The present inventors humanized the 228B / C-1 molecule by the strategy of antibody complementarity determining regions (CDRs) grafting to improve its developability. Based on computer homology modeling, the light chain selected human embryonic genes IMGT_hIGKV1_39, IMGT_hIGKV3-11 with high homology to the 228B / C-1 light chain framework region were subjected to CDR grafting, and FM4 selected the human IGKJ2*01 with the highest homology; the heavy chain selected human embryonic genes IMGT_IGHV4-59, IMGT_IGHV3-33 with high homology to the 228B / C-1 light chain framework region were subjected to CDR grafting, and FM4 selected the human IGHJ4*01 with the highest homology. At the same time, computer homology modeling was used to analyze the CDR region and its surrounding framework amino acid sequence to avoid the concentration distribution of molecular surface charge or hydrophobic region; by calculating the electrostatic force, van der Waals force, hydrophobicity and entropy, the key amino acid residues in the antibody gene sequence that bind to the target antigen and maintain the spatial framework were found, and on this basis, the back mutation sites were designed. A total of 8 humanized heavy chain variable regions H1-H8 and 2 humanized light chain variable regions K1-K2 were designed (Table 1).

[0423] Table 1. 228B / C-1 humanized antibody sequences

[0424]

[0425] The amino acid sequences and nucleotide sequences of the humanized light chain variable regions K1-K2 and the humanized heavy chain variable regions H1-H8 are as follows:

[0426] K1

[0427] amino acid sequence

[0428] DIQMTQSPSSLSASVGDRVTITC RASKSVDSYGNSFMH WYQQKPGKAPKLLIY LASNLES GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQNNEDPRT FGQGTKLEIK (SEQ ID NO. 1)

[0429] nucleotide sequence

[0430] GACATCCAGATGACACAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGAGACAGAGTGACCATCACCTGTAGAGCCAGCAAGAGCGTGGACAGCTACGGCAACAGCTTCATGCACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACCTGGCCAGCAACCTGGAAAGCGGCGTGCCATCTAGATTCAGCGGCTCTGGCTCTGGCACCGACTTCACCCTGACAATCTCTAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCAGAACAACGAGGACCCCAGAACATTCGGCCAGGGCACCAAGCTGGAAATCAAG (SEQ ID NO. 2)

[0431] K2

[0432] amino acid sequence

[0433] EIVLTQSPATLSLSPGERATLSC RASKSVDSYGNSFMH WYQQKPGQAPRLLIY LA SNLES GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQNNEDPRT FGQGTKLEIK (SEQ ID NO. 3)

[0434] nucleotide sequence

[0435] GAGATCGTGCTGACACAGAGCCCTGCCACACTGTCACTGTCTCCAGGCGAGAGAGCCACACTGAGCTGTAGAGCCAGCAAGAGCGTGGACAGCTACGGCAACAGCTTCATGCACTGGTATCAGCAGAAGCCCGGCCAGGCTCCTAGACTGCTGATCTACCTGGCCAGCAACCTGGAATCTGGCATCCCCGCTAGATTCAGCGGCTCTGGCTCTGGCACAGACTTCACCCTGACAATCAGCAGCCTGGAACCTGAGGACTTCGCCGTGTACTACTGCCAGCAGAACAACGAGGACCCCAGAACATTCGGCCAGGGCACCAAGCTGGAAATCAAG (SEQ ID NO. 4)

[0436] H1

[0437] amino acid sequence

[0438] QVQLQESGPGLVKPSETLSLTCTVSGFSLS AYSVN WIRQPPGKGLEWIG MIWGDGKIVYNSALKS RVTISKDTSKNQVVLKLSSVTAADTAVYYCAG DGYYPYAMDN WGQGTLVTVSS (SEQ ID NO. 5)

[0439] nucleotide sequence

[0440] CAGGTTCAGCTGCAAGAGTCTGGACCTGGCCTGGTCAAGCCTAGCGAGACACTGAGCCTGACCTGTACCGTGTCTGGCTTCAGCCTGAGCGCCTACAGCGTGAACTGGATCAGACAGCCTCCTGGCAAAGGCCTGGAATGGATCGGAATGATCTGGGGCGACGGCAAGATCGTGTACAACAGCGCCCTGAAGTCCAGAGTGACCATCAGCAAGGACACCAGCAAGAACCAGGTGGTGCTGAAGCTGAGCAGCGTGACAGCCGCTGATACCGCCGTGTACTATTGTGCTGGCGACGGCTACTACCCCTACGCCATGGATAATTGGGGCCAGGGCACACTGGTCACCGTGTCATCT (SEQ ID NO. 6)

[0441] H2

[0442] amino acid sequence

[0443] QVQLVESGGGVVQPGRSLRLSCAVSGFSLS AYSVN WVRQAPGKGLEWVA MIWGDGKIVYNSAVKS RLTISKDNSKNTVYLQMNSLRAEDTAVYYCAG DGYYPYAMDN WGQGTLVTVSS (SEQ ID NO. 7)

[0444] nucleotide sequence

[0445] CAGGTGCAGCTGGTTGAATCTGGTGGCGGAGTGGTGCAGCCTGGCAGATCTCTGAGACTGAGCTGTGCCGTGTCTGGCTTCAGCCTGTCTGCCTACTCTGTGAACTGGGTCCGACAGGCTCCTGGCAAAGGACTGGAATGGGTCGCCATGATCTGGGGCGACGGCAAGATCGTGTACAACAGCGCCGTGAAGTCCAGACTGACCATCAGCAAGGACAACAGCAAGAACACCGTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCTGGCGACGGCTACTACCCCTACGCCATGGATAATTGGGGCCAGGGCACACTGGTCACCGTGTCATCT (SEQ ID NO. 8)

[0446] H3

[0447] amino acid sequence

[0448] QVQLQESGPGLVKPSETLSLTCTVSGFSLS AYSVN WIRQPPGKGLEWIG MIWGDGKIVYNSALKS RVTISKDTSKNQVSLKLSSVTAADTAVYYCAG DGYYPYAMDN WGQGTLVTVSS (SEQ ID NO. 9)

[0449] nucleotide sequence

[0450] CAGGTTCAGCTGCAAGAGTCTGGACCTGGCCTGGTCAAGCCTAGCGAGACACTGAGCCTGACCTGTACCGTGTCTGGCTTCAGCCTGAGCGCCTACAGCGTGAACTGGATCAGACAGCCTCCTGGCAAAGGCCTGGAATGGATCGGAATGATCTGGGGCGACGGCAAGATCGTGTACAACAGCGCCCTGAAGTCCAGAGTGACCATCAGCAAGGACACCAGCAAGAACCAGGTGAGCCTGAAGCTGAGCAGCGTGACAGCCGCTGATACCGCCGTGTACTATTGTGCTGGCGACGGCTACTACCCCTACGCCATGGATAATTGGGGCCAGGGCACACTGGTCACCGTGTCATCT (SEQ ID NO. 10)

[0451] H4

[0452] amino acid sequence

[0453] QVQLQESGPGLVKPSETLSLTCTVSGFSLS AYSVN WIRQPPGKGLEWIG MIWGDGKIVYNSALKS RVTISKDTSKNQFSLKLSSVTAADTAVYYCAG DGYYPYAMDN WGQGTLVTVSS (SEQ ID NO. 11)

[0454] nucleotide sequence

[0455] CAGGTTCAGCTGCAAGAGTCTGGACCTGGCCTGGTCAAGCCTAGCGAGACACTGAGCCTGACCTGTACCGTGTCTGGCTTCAGCCTGAGCGCCTACAGCGTGAACTGGATCAGACAGCCTCCTGGCAAAGGCCTGGAATGGATCGGAATGATCTGGGGCGACGGCAAGATCGTGTACAACAGCGCCCTGAAGTCCAGAGTGACCATCAGCAAGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACAGCCGCTGATACCGCCGTGTACTATTGTGCTGGCGACGGCTACTACCCCTACGCCATGGATAATTGGGGCCAGGGCACACTGGTCACCGTGTCATCT (SEQ ID NO. 12)

[0456] H5

[0457] amino acid sequence

[0458] QVQLQESGPGLVKPSETLSLTCTVSGFSLS AYSVN WIRQPPGKGLEWIG MIWGDGKIVYNSALKS RVTISVDTSKNQFSLKLSSVTAADTAVYYCAG DGYYPYAMDN WGQGTLVTVSS (SEQ ID NO. 13)

[0459] nucleotide sequence

[0460] CAGGTTCAGCTGCAAGAGTCTGGACCTGGCCTGGTCAAGCCTAGCGAGACACTGAGCCTGACCTGTACCGTGTCTGGCTTCAGCCTGAGCGCCTACAGCGTGAACTGGATCAGACAGCCTCCTGGCAAAGGCCTGGAATGGATCGGAATGATCTGGGGCGACGGCAAGATCGTGTACAACAGCGCCCTGAAGTCCAGAGTGACCATCAGCGTGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACAGCCGCTGATACCGCCGTGTACTATTGTGCTGGCGACGGCTACTACCCCTACGCCATGGATAATTGGGGCCAGGGCACACTGGTCACCGTGTCATCT (SEQ ID NO. 14)

[0461] H6

[0462] amino acid sequence

[0463] QVQLVESGGGVVQPGRSLRLSCAVSGFSLS AYSVN WVRQAPGKGLEWVA MIWGDGKIVYNSAVKS RLTISKDNSKNTLYLQMNSLRAEDTAVYYCAG DGYYPYAMDN WGQGTLVTVSS (SEQ ID NO. 15)

[0464] nucleotide sequence

[0465] CAGGTGCAGCTGGTTGAATCTGGTGGCGGAGTGGTGCAGCCTGGCAGATCTCTGAGACTGAGCTGTGCCGTGTCTGGCTTCAGCCTGTCTGCCTACTCTGTGAACTGGGTCCGACAGGCTCCTGGCAAAGGACTGGAATGGGTCGCCATGATCTGGGGCGACGGCAAGATCGTGTACAACAGCGCCGTGAAGTCCAGACTGACCATCAGCAAGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCTGGCGACGGCTACTACCCCTACGCCATGGATAATTGGGGCCAGGGCACACTGGTCACCGTGTCATCT (SEQ ID NO. 16)

[0466] H7

[0467] amino acid sequence

[0468] QVQLVESGGGVVQPGRSLRLSCAVSGFSLS AYSVN WVRQAPGKGLEWVA MIWGDGKIVYNSAVKS RLTISRDNSKNTLYLQMNSLRAEDTAVYYCAG DGYYPYAMDN WGQGTLVTVSS (SEQ ID NO. 17)

[0469] nucleotide sequence

[0470] CAGGTGCAGCTGGTTGAATCTGGTGGCGGAGTGGTGCAGCCTGGCAGATCTCTGAGACTGAGCTGTGCCGTGTCTGGCTTCAGCCTGTCTGCCTACTCTGTGAACTGGGTCCGACAGGCTCCTGGCAAAGGACTGGAATGGGTCGCCATGATCTGGGGCGACGGCAAGATCGTGTACAACAGCGCCGTGAAGTCCAGACTGACCATCAGCAGAGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCTGGCGACGGCTACTACCCCTACGCCATGGATAATTGGGGCCAGGGCACACTGGTCACCGTGTCATCT(SEQ ID NO.18)

[0471] H8

[0472] 氨基酸序列

[0473] QVQLVESGGGVVQPGRSLRLSCAVSGFSLS AYSVN WVRQAPGKGLEWVA MIWGDGKIVYNSAVKS RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAG DGYYPYAMDN WGQGTLVTVSS(SEQ ID NO.19)

[0474] 核苷酸序列

[0475] CAGGTGCAGCTGGTTGAATCTGGTGGCGGAGTGGTGCAGCCTGGCAGATCTCTGAGACTGAGCTGTGCCGTGTCTGGCTTCAGCCTGTCTGCCTACTCTGTGAACTGGGTCCGACAGGCTCCTGGCAAAGGACTGGAATGGGTCGCCATGATCTGGGGCGACGGCAAGATCGTGTACAACAGCGCCGTGAAGTCCAGATTCACCATCAGCAGAGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTATTGTGCTGGCGACGGCTACTACCCCTACGCCATGGATAATTGGGGCCAGGGCA

[0476] CACTGGTCACCGTGTCATCT (SEQ ID NO. 20)

[0477] In the above humanized variable region sequences, the six CDRs of K1, K2, H1, H3, H4, and H5 are the same as the CDR sequences of the parent antibody Lebrikizumab; the HCDR2 (MIWGDGKIVYNSAVKS, SEQ ID NO. 26) of H2, H6, H7, and H8 is different from the HCDR2 of the parent antibody Lebrikizumab, and the other five CDRs are the same as Lebrikizumab.

[0478] Example 2, Expression of 228B / C-1 Humanized Antibodies

[0479] 1) Pairing verification of 228B / C-1 humanized light chain and heavy chain

[0480] After pairing the Lebrikizumab light chain variable region (K0) and two humanized light chain variable regions (K1 and K2) with the Lebrikizumab heavy chain variable region (H0) and eight humanized heavy chain variable regions (H1-H8) respectively, the HEK293 cells were transiently transfected, the supernatant was collected, and the biofilm interference technology (BLI / gator prime) was used to capture the antibodies with ProA probes, then the probes were transferred to the diluted human IL-13 antigen (hIL-13), and according to the signal change detected on the probe, the real-time binding signal of the antigen-antibody was obtained, finally the atlas was subjected to 1:1 kinetic fitting to obtain the binding affinity of the antibody in the transient supernatant to the antigen (KD). Figure 1 The light and heavy chain pairs with higher affinity, H1K0, H2K0, H3K0, H4K0, H1K1, H2K1, H3K1, H4K1, H5K1, H1K2, H2K2, H3K2, and H4K2 were selected for further transient expression and purification.

[0481] 2) Expression and purification of 228B / C-1 humanized antibodies

[0482] The light chain and the heavy chain were transfected into ExpiCHO cells at a ratio of 1:1 using a transfection reagent, and cultured at 37°C, 8% CO2, and 80% humidity for 7 days. After the culture ended, the fermentation broth was centrifuged at 4°C and 4500 rpm for 30 min, and the supernatant was filtered with a 0.22 μm needle filter. Protein A was used for purification in a gravity column. The transient production of humanized antibodies is shown in Table 2. The results show that the optimized transient production of 228B / C-1 humanized antibodies is better than that of the parent antibody Lebrikizumab.

[0483] Table 2. Transient production of 228B / C-1 humanized antibodies

[0484] Antibody Yield (mg / L) Antibody Yield (mg / L) Lebrikizumab 32.1 H3K1 156.4 H1K0 145.9 H4K1 215.9 H2K0 123.5 H5K1 190.8 H3K0 140.4 H1K2 137.2 H4K0 110.7 H2K2 80.7 H1K1 168.3 H3K2 103.1 H2K1 217.5 H4K2 104.8

[0485] Example 3, 228B / C-1 Humanized Antibody Activity

[0486] 1) Binding affinity to human IL-13 (hIL-13)

[0487] Using surface plasmon resonance technology (SPR / Biacore T200), a CM5 chip was selected, goat anti-hFc antibody was coupled to chip Fc1-4 channel, and humanized antibody was captured to the chip surface by specific high affinity binding of goat anti-hFc to humanized antibody. Then gradient diluted hIL-13 antigen was used as analyte, flowed through the chip surface, to determine the affinity and kinetic information of humanized antibody to hIL-13. The data was fitted by Biacore T200 Evaluation software 3.1 software, in Langmuir 1:1 kinetics or steady state mode, to obtain the affinity of humanized antibody to antigen interaction. The affinity of H1K1 and H4K1 to hIL-13 was comparable to ref1 (Lebrikizumab), while the affinity of H3K1 to hIL-13 was slightly better than ref1 (Table 3).

[0488] Table 3. 228B / C-1 Humanized Antibody Affinity

[0489] Humanized antibody Affinity (KD, M) Humanized antibody Affinity (KD, M) ref1 1.22E-14 H3K1 7.11E-15 H1K1 2.70E-14 H4K1 1.14E-14 H2K1 1.47E-13

[0490] 2) Inhibition of hIL-13 induced STAT6 activation

[0491] HEK293-STAT6 cells were cultured with their corresponding complete culture medium at 37°C, 5% CO2, and the cell concentration was controlled to contain 2x10 5 -1x10 6 cells per 1 mL. After collecting the cells at 4°C, 300g centrifugation for 5 minutes, the supernatant was discarded, the cells were resuspended with an appropriate amount of 10% FBS / DMEM solution and counted, and the cells were prepared to contain 6x10 5Cell suspension. Add 50 μL cell suspension to each well of a 96-well fluorescence detection plate, and incubate at 37 °C in a 5% CO2incubator for 16-24 h. Mix the gradient dilution of humanized antibody with 2 ng / ml of hIL-13 in equal volume, and pre-incubate at 37 °C for 30 min. Add 50 μl of the mixture to each well, and incubate at 37 °C in 5% CO2for 6 h. Take the fluorescence detection reagent from the refrigerator 2 h in advance, melt and equilibrate to room temperature (avoid light). Take the cell plate 30 min in advance, and equilibrate to room temperature. Use a multichannel pipette to suck 100 μL of fluorescence substrate solution directly into the cells (avoid light), and detect in a microplate reader after reaction at room temperature for 5-10 min. The results show that the activities of H1K1, H2K1 and H3K1 in inhibiting hIL-13-induced STAT6 activation are equivalent to those of the parent antibody Lebrikizumab (ref 1) Figure 2 ).

[0492] 3) Inhibition of hIL-13-induced TF-1 cell proliferation

[0493] TF-1 cells are cultured with 2 ng / ml of GM-CSF cell culture solution at 37 °C in 5% CO2, and the cell concentration is controlled to be 1 x 10 ℃ -1 x 10 5 -1 x 10 6 Cells are collected by centrifugation at 300 g for 10 min at 4 °C, and the cell culture solution is prepared into a cell suspension containing 1 x 10 5 Each well of a flat-bottom 96-well cell culture plate is inoculated with 100 μl, and incubated at 37 °C in 5% CO2for 20 h. Different concentrations of humanized antibodies are mixed with 40 ng / ml of hIL-13 in equal volume, 100 μl of the mixture is added to each well, and incubated at 37 °C in 5% CO2for 96 h. Cell viability is determined by using a Cell Titer-Glo Luminescent Cell Viability Assay kit. The results show that the activity of H3K1 in inhibiting hIL-13-induced TF-1 cell proliferation is better than that of the parent antibody Lebrikizumab (ref 1) Figure 3 ).

[0494] 4) Inhibition of hIL-13-induced PBMC release of TARC

[0495] PBMC are prepared into a cell suspension containing 2.5 x 10 6A cell suspension of 100 μl per well was inoculated in a flat-bottom 96-well cell culture plate. The gradient-diluted humanized antibody was mixed with 20 ng / ml of hIL-13 in equal volume, and incubated at 37°C for 30 minutes. 100 μl of the mixture was added to the cells, and incubated at 37°C, 5% CO2 for 72 hours. The cell culture supernatant was taken, and the TARC release content in the cell culture supernatant was detected by Human TARC / CCL17 ELISA Kit. The results showed that the H3K1 inhibited hIL-13-induced PBMC release TARC activity was comparable to the parent antibody Lebrikizumab (ref1) Figure 4

[0496] Example 4, Construction and Expression of Vector Encoding Anti-TSLP / IL-13 Bispecific Antibody

[0497] Using 228B / C-1H3K1 as the IL-13 binding domain of the bispecific antibody, and the anti-TSLP antibody in patent CN112876564B as the TSLP binding domain of the bispecific antibody, an anti-TSLP / IL-13 bispecific antibody TSLP / IL-13 BsAb was constructed. By introducing charge-pairing mutations in VH and VL, and introducing knob-into-hole mutations in Fc to reduce bispecific antibody mispairing, in addition, LA mutations (M428L and N434A, according to EU numbering system) were introduced in the antibody Fc to extend the half-life of the bispecific antibody.

[0498] The VH, VL and CDR sequences of the anti-TSLP antibody in CN112876564B (according to the Kabat numbering system) are as follows:

[0499] VH amino acid sequence

[0500] QVQLVQSGAEVKKPGSSVKVSCKASGYTFT DYWMH WVRQAPGQGLEWMG IIDPSDSDTSLNQKFQG RVTITADTSTSTAYMELSSLRSEDTAVYYCAR SLDGYYDY WGQGTLVTVSS (SEQ ID NO. 50)

[0501] HCDR1: DYWMH (SEQ ID NO. 30)

[0502] HCDR2: IIDPSDSDTSLNQKFQG (SEQ ID NO. 31)

[0503] HCDR3: SLDGYYDY (SEQ ID NO. 32)​

[0504] VL amino acid sequence

[0505] DIQMTQSPSSLSASVGDRVTITC RTSENIYSYLA WYQQKPGKAPKLLIY FAKTLT D GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QHHYGTPWT FGQGTKVEIK (SEQ ID NO. 51)

[0506] LCDR1: RTSENIYSYLA (SEQ ID NO. 35)

[0507] LCDR1: FAKTLTD (SEQ ID NO. 36)

[0508] LCDR3: QHHYGTPWT (SEQ ID NO. 37)

[0509] The anti-TSLP VH and VL of the present application are obtained by introducing Q39E in the anti-TSLP VH of CN112876564B and Q38K in the VL (according to the Kabat numbering system) charge pair mutation, the amino acid sequence and nucleotide sequence thereof are as follows:

[0510] VH amino acid sequence

[0511] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYWMHWVR E APGQGLEWMGIIDPSDSDTSLNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCARSLDGYYDYWGQGTLVTVSS (SEQ ID NO. 28)

[0512] VH nucleotide sequence

[0513] CAGGTTCAGCTGGTTCAGTCTGGCGCCGAAGTGAAGAAACCTGGCAGCAGCGTGAAGGTGTCCTGCAAGGCTAGCGGCTACACATTCACCGACTACTGGATGCACTGGGTCCGAGAGGCTCCAGGACAGGGACTTGAGTGGATGGGCATCATCGACCCCAGCGACAGCGACACAAGCCTGAACCAGAAATTCCAGGGCAGAGTGACCATCACCGCCGACACCTCTACAAGCACCGCCTACATGGAACTGAGCAGCCTGAGAAGCGAGGACACCGCCGTGTACTACTGTGCCAGATCTCTGGACGGCTACTACGATTACTGGGGCCAGGGAACCCTGGTCACCGTTTCTTCT (SEQ ID NO. 29)

[0514] VL amino acid sequence

[0515] DIQMTQSPSSLSASVGDRVTITCRTSENIYSYLAWYQ K KPGKAPKLLIYFAKTLTD GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYGTPWTFGQGTKVEIK (SEQ ID NO. 33)

[0516] VL nucleotide sequence

[0517] GACATCCAGATGACACAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGAGACAGAGTGACCATCACCTGTAGAACCAGCGAGAACATCTACAGCTACCTGGCCTGGTATCAGAAGAAGCCTGGCAAGGCTCCCAAGCTGCTGATCTACTTCGCCAAGACACTGACCGACGGCGTGCCCTCTAGATTCAGCGGATCTGGCTCTGGCACCGACTTCACCCTGACAATCTCTAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCACCACTACGGCACACCTTGGACATTCGGCCAGGGCACCAAGGTGGAAATCAAG (SEQ ID NO. 34)

[0518] A new anti-IL-13 VH and VL were obtained by introducing Q39K in the above H3K1 VH and Q38E in the VL (according to the Kabat numbering system) charge pair mutations for use in the anti-TSLP / IL-13 bispecific antibodies of the present application, the amino acid sequences and nucleotide sequences of which are shown below:

[0519] VH amino acid sequence

[0520] QVQLQESGPGLVKPSETLSLTCTVSGFSLSAYSVNWIR K PPGKGLEWIGMIWGDGKIVYNSALKSRVTISKDTSKNQVSLKLSSVTAADTAVYYCAGDGYYPYAMDNWGQGTLVTVSS (SEQ ID NO. 52)

[0521] VH nucleotide sequence

[0522] CAGGTTCAGCTGCAAGAGTCTGGACCTGGCCTGGTCAAGCCTAGCGAGACACTGAGCCTGACCTGTACCGTGTCTGGCTTCAGCCTGAGCGCCTACAGCGTGAACTGGATCAGAAAGCCTCCTGGCAAAGGCCTGGAATGGATCGGAATGATCTGGGGCGACGGCAAGATCGTGTACAACAGCGCCCTGAAGTCCAGAGTGACCATCAGCAAGGACACCAGCAAGAACCAGGTGAGCCTGAAGCTGAGCAGCGTGACAGCCGCTGATACCGCCGTGTACTATTGTGCTGGCGACGGCTACTACCCCTACGCCATGGATAATTGGGGCCAGGGCACACTGGTCACCGTGTCATCT (SEQ ID NO. 55)

[0523] VL amino acid sequence

[0524] DIQMTQSPSSLSASVGDRVTITCRASKSVDSYGNSFMHWYQ E KPGKAPKLLIYLA SNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNNEDPRTFGQGTKLEIK (SEQ ID NO. 53)

[0525] VL nucleotide sequence

[0526] GACATCCAGATGACACAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGAGACAGAGTGACCATCACCTGTAGAGCCAGCAAGAGCGTGGACAGCTACGGCAACAGCTTCATGCACTGGTATCAGGAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACCTGGCCAGCAACCTGGAAAGCGGCGTGCCATCTAGATTCAGCGGCTCTGGCTCTGGCACCGACTTCACCCTGACAATCTCTAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCAGAACAACGAGGACCCCAGAACATTCGGCCAGGGCACCAAGCTGGAAATCAAG (SEQ ID NO. 56)

[0527] The amino acid sequences and nucleotide sequences of the heavy chain and light chain of the anti-TSLP / IL-13 bispecific antibody TSLP / IL-13 BsAb of the present application are shown below:

[0528] Heavy chain 1 (anti-IL-13)

[0529] Amino acid sequence

[0530] QVQLQESGPGLVKPSETLSLTCTVSGFSLSAYSVNWIRKPPGKGLEWIGMIWGDGKIVYNSALKSRVTISKDTSKNQVSLKLSSVTAADTAVYYCAGDGYYPYAMDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDERVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVLHEALHAHYTQKSLSLSPGK (SEQ ID NO. 42) Nucleotide sequence

[0531]

[0532] Light chain 1 (anti-IL-13)

[0533] Amino acid sequence

[0534] DIQMTQSPSSLSASVGDRVTITCRASKSVDSYGNSFMHWYQEKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNNEDPRTFGQGTKLEIKRTVAAPSVFIFPPSDKQLKSGKASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO. 44) Nucleotide sequence

[0535] GACATCCAGATGACACAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGAGACAGAGTGACCATCACCTGTAGAGCCAGCAAGAGCGTGGACAGCTACGGCAACAGCTTCATGCACTGGTATCAGGAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACCTGGCCAGCAACCTGGAAAGCGGCGTGCCATCTAGATTCAGCGGCTCTGGCTCTGGCACCGACTTCACCCTGACAATCTCTAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCAGAACAACGAGGACCCCAGAACATTCGGCCAGGGCACCAAGCTGGAAATCAAGCGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATAAACAGTTGAAATCTGGAAAAGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT (SEQ ID NO. 45)

[0536] Heavy chain 2 (anti-TSLP)

[0537] Amino acid sequence

[0538] QVQLVQSGAEVKKPGSSVKVSCKASGYTF TDYWMH WVREAPGQGLEWMG IIDPSDSDTSLNQKFQG RVTITADTSTSTAYMELSSLRSEDTAVYYCAR SLDGYYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVLHEALHAHYTQKSLSLSPGK (SEQ ID NO. 38)

[0539] Nucleotide sequence

[0540]

[0541] Light chain 2 (anti-TSLP)

[0542] Amino acid sequence

[0543] DIQMTQSPSSLSASVGDRVTITC RTSENIYSYLA WYQKKPGKAPKLLIY FAKTLTD GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QHHYGTPWT FGQGTKVEIKGQPKAAPSVTLFPPSSEELQANEATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO. 40) Nucleotide sequence

[0544] GACATCCAGATGACACAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGGAGACAGAGTGACCATCACCTGTAGAACCAGCGAGAACATCTACAGCTACCTGGCCTGGTATCAGAAGAAGCCTGGCAAGGCTCCCAAGCTGCTGATCTACTTCGCCAAGACACTGACCGACGGCGTGCCCTCTAGATTCAGCGGATCTGGCTCTGGCACCGACTTCACCCTGACAATCTCTAGCCTGCAGCCTGAGGACTTCGCCACCTACTACTGCCAGCACCACTACGGCACACCTTGGACATTCGGCCAGGGCACCAAGGTGGAAATCAAGGGACAGCCCAAGGCGGCGCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACGAGGCCACACTGGTGTGTCTCATAAGTGACTTCTATCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA (SEQ ID NO. 41)

[0545] TSLP / IL-13 BsAb plasmid (1 :2:1 :2 of heavy chain 1 :light chain 1 :heavy chain 2:light chain 2) was transfected into ExpiCHO cells using transfection reagent and cultured for 7 days at 37°C, 8% CO2, 80% humidity. After the end of the culture, the fermentation broth was centrifuged at 4°C, 4500 rpm for 30 min, and the supernatant was filtered with a 0.22 μm needle filter. Protein A was used to purify by gravity column, through loading, balancing, elution and elution. Then CEX was used to remove antibody aggregates and fragments to improve the purity of protein monomers. The quality of TSLP / IL-13 BsAb was detected by SEC and mass spectrometry, and the results are shown in Table 4.

[0546] Table 4. TSLP / IL-13 BsAb expression yield and quality

[0547]

[0548] Example 5, TSLP / IL-13 BsAb binding affinity to TSLP and IL-13

[0549] Using surface plasmon resonance technology (SPR / Biacore T200), ProA chip was selected, TSLP / IL-13 BsAb was captured to the chip surface, then gradient dilution of human TSLP (hTSLP), cynomolgus TSLP (cyTSLP), hIL-13 and cynomolgus IL-13 (cyIL-13) antigens were used as analytes, flowed through the chip surface, to determine the affinity and kinetic information of TSLP / IL-13 BsAb to each antigen. The data was fitted by Biacore T200 Evaluation software 3.1 software, Langmuir 1:1 kinetics or steady state mode, to obtain the affinity of antibody and antigen interaction. TSLP mAb (ref3) and IL-13 mAb Lebrikizumab (ref1) were used as controls. The results showed that TSLP / IL-13 BsAb bound to human and cynomolgus TSLP with high affinity, and the affinity was comparable to TSLP mAb (ref3); TSLP / IL-13 BsAb bound to human and cynomolgus IL-13 with high affinity, and the affinity was comparable to IL-13 mAb Lebrikizumab (ref1) (Table 5).

[0550] Table 5. TSLP / IL-13 BsAb affinity to antigens

[0551]

[0552] Example 6, TSLP / IL-13 BsAb inhibits hTSLP-induced activity

[0553] 1) Inhibition of hTSLP-induced STAT5 activation

[0554] Ba / f3-hTSLPR-IL7Rα-STAT5 luc cells were cultured with cell culture medium at 37°C, 5% CO2. Centrifuged at 4°C, 300g for 5 minutes, discarded the supernatant, collected the cells, washed once with 10ml PBS, resuspended the cells with appropriate amount of 10% FBS / RPMI1640 solution (maintain the cell density at 1.5×10 6cells / mL) without antibiotics and murine IL-3 (mIL-3) cytokine, starved overnight at 37 °C, 5% CO2. Next day, collect cells, centrifuge at 300g for 5 min, discard supernatant, resuspend cells with appropriate amount of assay buffer (1% FBS / RPMI1640), take 100 μL of cell suspension for counting. Adjust cell density to 1 x 10 6 cells / mL, take 50 μL of cell suspension per well into 96-well sterile fluorescence assay white plate, 5 x 10 4 cells per well. Incubate at 37 °C, 5% CO2 incubator. Mix TSLP / IL-13 BsAb (18 μg / ml, 1:3 equi- dilution) with 12 ng / ml of hTSLP in equal volume, pre-incubate at 37 °C for 30 min. Add 50 μl of mixture per well into cells, incubate at 37 °C, 5% CO2 for 6 hours. Take fluorescence assay reagent from freezer 2 hours in advance, thaw and equilibrate to room temperature (dark). Take cell plate half an hour in advance, equilibrate to room temperature. Use multichannel pipette to pipette 100 μL of fluorescence substrate solution directly into cells (dark), incubate at room temperature for 5-10 min, then put into microplate reader for detection. Use TSLP mAb (ref 3) and Tezepelumab analogue as controls. The results show that TSLP / IL-13 BsAb inhibits hTSLP-induced STAT5 activation activity slightly weaker than TSLP mAb (ref 3) and Tezepelumab analogue (A), which might be due to the lack of one binding region for TSLP in TSLP / IL-13 BsAb. TSLP / IL-13 BsAb inhibits hTSLP-induced STAT5 activation activity better than Tezepelumab analogue (B). Figure 5 A), which might be due to the lack of one binding region for TSLP in TSLP / IL-13 BsAb. TSLP / IL-13 BsAb inhibits hTSLP-induced STAT5 activation activity better than Tezepelumab analogue (B) Figure 24

[0555] 2) Inhibition of hTSLP-induced Ba / f3-TSLPR-IL7Ra cell proliferation

[0556] Ba / f3-TSLPR-IL7Ra cells were cultured with cell culture medium at 37 °C, 5% CO2. Centrifuge at 4 °C, 300g for 10 min, discard supernatant, collect cells, wash once with 10 ml PBS, prepare cell suspension containing 1 x 10 5 cells / mL with RPMI-1640 + 10% FBS culture medium, inoculate 100 μl per well into flat-bottom 96-well cell culture plate, starve overnight at 37 °C, 5% CO2. Mix TSLP / IL-13 BsAb (120 μg / ml, 3-fold gradient dilution) with 20 ng / ml of hTSLP in equal volume, pre-incubate at 37 °C for 30 min. Add 100 μl of mixture per well into cells, incubate at 37 °C, 5% CO2 for 72 hours, use ​Luminescent Cell Viability Assay kit. TSLP mAb (ref 3) and Tezepelumab analogue were used as controls. The results show that TSLP / IL-13 BsAb inhibits hTSLP-induced Ba / f3-TSLPR-IL7Ra cell proliferation activity comparable to TSLP mAb, and superior to Tezepelumab analogue Figure 5 B and Figure 25 ).

[0557] 3) Inhibition of hTSLP-induced TARC release by PBMC

[0558] PBMC were prepared in cell culture medium to contain 2.5 x 10 6 cells per 1 ml, and 100 μl was seeded into each well of a flat-bottomed 96-well cell culture plate. Gradient-diluted TSLP / IL-13 BsAb was mixed with 20 ng / ml of hTSLP in equal volume, and incubated at 37°C for 30 minutes. 100 μl of the mixture was added to each well, and incubated at 37°C, 5% CO2for 72 hours. The cell culture supernatant was taken, and the content of TARC release in the cell culture supernatant was detected by human TARC / CCL17 ELISA kit. TSLP mAb (ref 3) was used as control. The results show that TSLP / IL-13 BsAb inhibits hTSLP-induced TARC release activity comparable to TSLP mAb Figure 5 C).

[0559] Example 7, Inhibition of hIL-13-induced activity by TSLP / IL-13 BsAb

[0560] 1) Inhibition of hIL-13-induced STAT6 activation

[0561] HEK293-STAT6 cells were cultured in their respective complete culture medium at 37°C, 5% CO2, and the cell concentration was controlled to contain 2 x 10 5 - 1 x 10 6 cells per 1 ml. After the cells were collected by centrifugation at 300g for 5 minutes at 4°C, the supernatant was discarded, and the cells were resuspended with an appropriate amount of 10% FBS / DMEM solution and counted. The cells were prepared to contain 6 x 10 5A cell suspension of 1 x 105cells / ml was prepared. 50 μl of the cell suspension was added to each well of a 96-well fluorescence detection plate and incubated at 37°C in a 5% CO2incubator for 16-24 hours. The gradient dilution of TSLP / IL-13 BsAb was mixed with 2 ng / ml of hIL-13 in equal volume and pre-incubated at 37°C for 30 minutes. 50 μl of the mixture was added to each well and incubated at 37°C in 5% CO2for 6 hours. The fluorescence detection reagent was taken out of the freezer 2 hours in advance, thawed and equilibrated to room temperature (protected from light). The cell plate was taken out half an hour in advance and equilibrated to room temperature. 100 μl of the fluorescence substrate solution was pipetted directly into the cells using a multichannel pipette (protected from light) and incubated at room temperature for 5-10 minutes before being placed in the microplate reader for detection. IL-13 monoclonal antibody Lebrikizumab (ref 1) and known TSLP / IL-13 bispecific nanobody Lunsekimig (ref 2) were used as controls. The results showed that the TSLP / IL-13 BsAb inhibited hIL-13-induced STAT6 activation activity slightly weaker than IL-13 monoclonal antibody (this could be due to the lack of one binding region for IL-13 in TSLP / IL-13 BsAb than IL-13 monoclonal antibody), better than ref 2 nanobody Figure 6 A).

[0562] 2) Inhibition of hIL-13-induced TF-1 cell proliferation

[0563] The TF-1 cells were incubated with 2 ng / ml of GM-CSF cell culture solution at 37°C in 5% CO2, and the cell concentration was controlled to be 1 x 105 5 ~ 1 x 105 6 cells per 1 ml. Centrifugation was performed at 300g per minute for 10 minutes at 4°C, and the cells were collected. The cell culture solution was prepared to contain 1 x 105 5Cell suspension of 100 μl cells was inoculated into a flat-bottom 96-well cell culture plate in each well and cultured at 37°C and 5% CO2 for 20 hours. TSLP / IL-13 BsAb of different concentrations was mixed with 40 ng / ml hIL-13 in equal volumes, 100 μl of the mixture was added to each well, and cultured at 37°C and 5% CO2 for 96 hours. Cell viability was determined using the Cell Titer-GloLuminescent Cell Viability Assay kit. IL-13 monoclonal antibody Lebrikizumab (ref1) and TSLP / IL-13 bispecific nanoantibody Lunsekimig (ref2) were used as controls. The results showed that the TSLP / IL-13 BsAb's ability to inhibit IL-13-induced TF-1 cell proliferation was slightly weaker than that of the ref1 monoclonal antibody (this may be because TSLP / IL-13BsAb lacks a binding region for IL-13 compared to the IL-13 monoclonal antibody), but was superior to the ref2 nanoantibody ( Figure 6 B).

[0564] 3) Inhibit hIL-13-induced TARC release from PBMC

[0565] PBMC cell culture medium was prepared to contain 2.5×10 6 A cell suspension of 100 μl cells was inoculated into a flat-bottom 96-well cell culture plate at 100 μl per well. The gradiently diluted TSLP / IL-13 BsAb was mixed with an equal volume of 20 ng / ml hIL-13 and incubated at 37°C for 30 minutes. 100 μl of the mixture was added to the cells per well and cultured at 37°C and 5% CO2 for 72 hours. The cell culture supernatant was taken and the TARC release content in the cell culture supernatant was detected using a human TARC / CCL17 ELISA kit. The IL-13 monoclonal antibody Lebrikizumab (ref1) and the TSLP / IL-13 bispecific nanoantibody Lunsekimig (ref2) were used as controls. The results showed that the TSLP / IL-13 BsAb's inhibition of hIL-13-induced PBMC release of TARC activity was slightly weaker than that of the IL-13 monoclonal antibody (this may be because the TSLP / IL-13 BsAb lacks a binding region for IL-13 compared to the IL-13 monoclonal antibody), and was better than the ref2 nanoantibody ( Figure 6 C).

[0566] Example 8: TSLP / IL-13 BsAb inhibits hTSLP combined with hIL-13-induced TARC release from PBMCs

[0567] The inhibition of TSLP / IL-13 BsAb on the synergistic activity of TSLP and IL-13 was evaluated by the induction of TARC release from PBMC by hTSLP combined with hIL-13. PBMC were prepared into a cell suspension containing 2.5 x 10 6 cells per 1 ml of cell culture solution, and 100 μl was inoculated into each well of a flat-bottom 96-well cell culture plate. 40 ng / ml hIL-13 was mixed with 40 ng / ml hTSLP in equal volumes, and gradient-diluted TSLP / IL-13 BsAb was mixed with 20 ng / ml of the hIL-13 and hTSLP mixture in equal volumes, and incubated at 37°C for 30 minutes. 100 μl of the mixture was added to each well of the cells, and incubated at 37°C in 5% CO2 for 72 hours. The cell culture supernatant was taken, and the TARC release content in the cell culture supernatant was detected using a human TARC / CCL17 ELISA kit. IL-13 monoclonal antibody Lebrikizumab (ref 1), TSLP / IL-13 bispecific nanobody Lunsekimig (ref 2), and TSLP monoclonal antibody (ref 3) were used as controls. The results showed that TSLP / IL-13 BsAb and ref 2 nanobody can completely inhibit the release of TARC from PBMC induced by hTSLP combined with hIL-13, and the inhibitory activity of TSLP / IL-13 BsAb is slightly better than that of ref 2 nanobody, while ref 1 monoclonal antibody and ref 3 monoclonal antibody cannot completely inhibit the release of TARC from PBMC induced by hTSLP combined with hIL-13 Figure 7 ). This indicates that TSLP / IL-13 BsAb can exert a better therapeutic effect than monoclonal antibodies by simultaneously blocking the TSLP and IL-13 signaling pathways.

[0568] Example 9, In vivo efficacy of TSLP / IL-13 BsAb in a MC903-induced mouse dermatitis model

[0569] 1) MC903-induced hTSLP / TSLPR humanized mouse dermatitis model

[0570] 8-10 week old hTSLP / TSLPR humanized mice were randomly divided into 5 groups (control group, model group, 3 mg / kg TSLP / IL-13 BsAb dosing group, 30 mg / kg TSLP / IL-13 BsAb dosing group and 30 mg / kg irrelevant antibody Anti-KLH (prepared according to US8734796B) dosing group), 11 mice in each group. The model group and dosing group mice were smeared with 20 μl MC903 (50 μM) on the right ear on D1, D2, D3, D4, D5, D8, D9, D10, D11 and D12 days, and the control group was smeared with 20 μl absolute ethanol. Each dosing group was given the corresponding dose of TSLP / IL-13 BsAb or Anti-KLH by subcutaneous injection on the day before modeling (D0), D3, D6, D8, D10 and D12. The change in right ear thickness of the mice was detected during modeling and dosing. On D14, the serum of the mice was taken, and the IgE level in the serum of the mice was detected by mouse IgE ELISA kit.

[0571] The results show that in the hTSLP / TSLPR humanized mouse dermatitis model, 3 mg / kg and 30 mg / kg TSLP / IL-13 BsAb can significantly inhibit the increase of mouse ear thickness, and significantly reduce the serum IgE level of mice (A and 8B). Figure 8

[0572] The effect of TSLP / IL-13 BsAb and TSLP / IL-13 bispecific nanobody Lunsekimig (ref2) was further compared in the same mouse dermatitis model. 8-10 week old hTSLP / TSLPR humanized mice were randomly divided into 4 groups (control group, model group, 4 mg / kg TSLP / IL-13 BsAb dosing group and 4 mg / kg ref2 antibody dosing group). The model group and dosing group mice were smeared with 20 μl MC903 (50 μM) on the right ear on D1, D2, D3, D4, D5, D8, D9, D10, D11, D12, D14 and D15 days, and the control group was smeared with 20 μl absolute ethanol. Each dosing group was given the corresponding dose of TSLP / IL-13 BsAb or ref2 antibody by subcutaneous injection on the day before modeling (D0), D3, D6, D8, D10, D12 and D14, and the change in right ear thickness of the mice was detected during modeling and dosing.

[0573] The results show that 4 mg / kg TSLP / IL-13 BsAb inhibits the increase of mouse ear thickness better than ref2 antibody (C). Figure 8

[0574] 2) MC903 induced hIL-13 humanized mouse dermatitis model

[0575] ​​8-10 week old hIL-13 humanized mice were randomly divided into 5 groups (control group, model group, 2 mg / kg TSLP / IL-13 BsAb dosing group, 10 mg / kg TSLP / IL-13 BsAb dosing group and 10 mg / kg irrelevant antibody Anti-KLH dosing group), 12 mice in each group. The model group and dosing group mice were smeared with 20 μl MC903 (50 μM) on the right ear on D1, D2, D3, D4, D5, D8, D9, D10, D11 and D12 days, and the control group was smeared with 20 μl absolute ethanol. Each dosing group was given the corresponding dose of TSLP / IL-13 BsAb or Anti-KLH by subcutaneous injection on the day before modeling (D0), D3, D6, D8, D10 and D12. The change of right ear thickness of mice was detected during modeling and dosing. On D14, the serum of mice was taken, and the IgE level in the serum of mice was detected by mouse IgE ELISA kit.

[0576] The results showed that in the hIL-13 humanized mouse dermatitis model, the 10 mg / kg TSLP / IL-13 BsAb dosing group could significantly inhibit the increase of mouse ear thickness, and the 2 mg / kg and 10 mg / kg TSLP / IL-13 BsAb dosing groups could observe the decrease of mouse serum IgE level Figure 9 A and 9B).

[0577] Example 10, Pharmacokinetics (PK) of TSLP / IL-13 BsAb in hFcRn humanized mice

[0578] 6-8 week old female hFcRn humanized mice were given 10 mg / kg TSLP / IL-13 BsAb, IL-13 monoclonal antibody Lebrikizumab (ref1), TSLP monoclonal antibody (ref3) and TSLP / IL-13 BsAb without LA mutation (TSLP / IL-13 BsAb-WT) by single subcutaneous injection, 4 mice in each group, and the mouse blood was collected at 0h, 4h, D2, D3, D4, D6, D8, D15, D22, D29, D36, D43, D54 after dosing for PK detection. On D54, the half-lives of TSLP / IL-13 BsAb, TSLP / IL-13 BsAb-WT, ref1 monoclonal antibody and ref3 monoclonal antibody were 16.8 days, 11.2 days, 11.0 days and 13.3 days respectively. It is shown that the half-life of TSLP / IL-13 BsAb is better than that of other molecules, which is expected to reduce the dosing frequency of clinical patients.

[0579] The light chain and heavy chain amino acid sequences of TSLP / IL-13 BsAb-WT are as follows:

[0580] Anti-IL-13 heavy chain

[0581] QVQLQESGPGLVKPSETLSLTCTVSGFSLSAYSVNWIRKPPGKGLEWIGMIWGDGKIVYNSALKSRVTISKDTSKNQVSLKLSSVTAADTAVYYCAGDGYYPYAMDNWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDERVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO. 57)

[0582] Anti-IL-13 light chain

[0583] DIQMTQSPSSLSASVGDRVTITCRASKSVDSYGNSFMHWYQEKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQNNEDPRTFGQGTKLEIKRTVAAPSVFIFPPSDKQLKSGKASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO. 44)

[0584] Anti-TSLP heavy chain

[0585] QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYWMHWVREAPGQGLEWMGIIDPSDSDTSLNQKFQGRVTITADTSTSTAYMELSSLRSEDTAVYYCARSLDGYYDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO. 58)

[0586] Anti-TSLP heavy chain

[0587] DIQMTQSPSSLSASVGDRVTITCRTSENIYSYLAWYQKKPGKAPKLLIYFAKTLTDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHHYGTPWTFGQGTKVEIKGQPKAAPSVTLFPPSSEELQANEATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO. 40)

[0588] Example 11, Pharmacodynamic effects of TSLP / IL-13 BsAb on asthma in B-hIL13 mice

[0589] Eighteen B-hIL13 mice were randomly divided into 3 groups according to body weight, 6 mice in each group, which were Control group, Model group and 30 mg / kg TSLP / IL-13 BsAb administration group respectively. The first day of modeling, multi-point injection of OVA Al(OH)3 mixture was used for sensitization (counted as Day 0), and on Day 14, intraperitoneal injection of OVA Al(OH)3 mixture was used for reinforcement sensitization, and from Day 21 to Day 27, OVA solution was used for aerosol inhalation. The first administration was one day before modeling (Day-1), and from Day-1 to Day 19, administration was performed every 4 days, a total of 5 times; from Day 21 to Day 27, administration was performed every 1 day, a total of 4 times; 2 hours before OVA challenge, subcutaneous injection was performed. The model group was synchronously injected subcutaneously with 0.9% sodium chloride injection.

[0590] Effect on mouse body weight

[0591] During the experiment, general symptom observation was performed every day, and the body weight of the mice was measured before each administration, and the body weight results are shown in Table 1. Figure 10 The results showed that there was no significant difference in the body weight of mice among the Control group, the Model group and the 30 mg / kg TSLP / IL-13 BsAb administration group (P>0.05).

[0592] Effect on nose scratching and sneezing

[0593] On Day 21 and Day 27, the number of times of scratching nose and sneezing within 30 minutes after OVA challenge was counted, and the results are shown in Table 6-Table 7. Figure 11 The results showed that compared with the Control group, the number of times of scratching nose within 30 minutes after OVA challenge on Day 21 and Day 27 of the mice in the Model group was significantly increased (P<0.01-0.001), and the number of times of sneezing was not significantly changed (P>0.05); compared with the Model group, the number of times of scratching nose of the mice in the 30 mg / kg TSLP / IL-13 BsAb administration group on Day 21 and Day 27 was significantly reduced (P<0.05-0.01).

[0594] Table 6. The number of times of scratching nose and sneezing within 30 minutes after OVA challenge on Day 21

[0595]

[0596] The data is shown as mean ± SEM, n=6. ##P<0.01 compared with the Control group; **P<0.05 compared with the Model group. Dunnett test after one-way ANOVA, Mann-Whitney U test after one-way ANOVA.

[0597] Table 7. Number of nose scratching and sneezing within 30 min after OVA challenge on Day 27

[0598]

[0599]

[0600] Data are shown as mean ± SEM, n = 6. Compared with Control group, ### P < 0.001; compared with Model group, *P < 0.05. Dunnett's test after one-way ANOVA, Mann-Whitney U test after one-way ANOVA.

[0601] Effect on airway hyperresponsiveness (AHR)

[0602] Day 28, the mice were aerosolized with 0, 1, 2, 4, 8, 12, 24 or 48 mg / mL methacholine (Mch) and the Mch-induced airway hyperresponsiveness in mice was detected by EMKA animal lung function monitoring system, and the results are shown in Figure 12 The results showed that compared with the Control group, the mice in the Model group had a trend of increasing airway resistance (Penh) when aerosolized with each concentration of Mch (P > 0.05); the percentage of Penh increase was significantly increased when aerosolized with 12 and 24 mg / mL Mch (P < 0.05); compared with the Model group, the mice in the 30 mg / kg TSLP / IL-13 BsAb administration group had a decreasing trend in the percentage of Penh increase when aerosolized with 1, 2, 4, 8, 12, 24 and 48 mg / mL Mch, and the inhibition rates were 68.2%, 57.7%, 55.4%, 47.4%, 14.1%, 29.7% and 19.8%, respectively.

[0603] Effect on inflammatory cell number in BALF

[0604] Day 28, bronchoalveolar lavage was performed on the mice, and the total number of white blood cells, eosinophils, macrophages and lymphocytes in the bronchoalveolar lavage fluid (BALF) was counted under a microscope, and the results are shown in Figure 13 The results showed that compared with the Control group, the number of white blood cells, eosinophils, macrophages and lymphocytes in the BALF of the mice in the Model group was significantly increased (P < 0.05-0.001); compared with the Model group, the number of white blood cells, eosinophils and macrophages in the BALF of the mice in the 30 mg / kg TSLP / IL-13 BsAb administration group was significantly decreased (P < 0.05-0.001), and the inhibition rates were 44.2%, 80.3% and 52.7%, respectively.

[0605] Effect on serum total IgE and OVA-sIgE levels

[0606] Day 20、Day 28, the blood of mice was collected from retro-orbital venous plexus, and the total IgE and OVA-sIgE in serum were detected by ELISA, and the results were shown in Figure 14 The results showed that compared with the Control group, the total IgE and OVA-sIgE in serum of the Model group were significantly increased on Day 20 and Day 28 (P<0.01); compared with the Model group, the total IgE in serum of the 30mg / kg TSLP / IL-13 BsAb administration group was significantly reduced on Day 28 (P<0.05), and the OVA-sIgE had a decreasing trend (P>0.05).

[0607] Effect on lung tissue eosinophil infiltration

[0608] Day 28, after the lung tissue was completely fixed with 10% neutral buffered formalin solution, paraffin-embedded section was performed, and H&E staining was used to observe the eosinophil infiltration in lung tissue, and pathological score was performed, and the eosinophil infiltration pathological chart was shown in Figure 15 , and the pathological score results were shown in Figure 16 . The H&E staining results showed that the Model group mice had visible eosinophil infiltration in the airway and blood vessels of lung tissue (P<0.001), and the 30mg / kg TSLP / IL-13 BsAb administration group could significantly inhibit the eosinophil infiltration (P<0.05).

[0609] Effect on airway goblet cell metaplasia proliferation

[0610] Day 28, PAS staining was performed on the lung tissue of mice, and the proliferation of goblet cells in the airway of lung tissue was observed, and the positive rate was counted, and the results were shown in Figure 17 . The PAS staining results showed that the Model group mice had obvious goblet cell metaplasia and proliferation in the airway (P<0.01), and the 30mg / kg TSLP / IL-13 BsAb administration group could significantly reduce the mucus secretion of airway epithelial cells and the goblet cell metaplasia and proliferation (P<0.01).

[0611] Effect on lung tissue mast cell number and degranulation

[0612] Day 28, the lung tissue of mice was subjected to toluidine blue staining, and the degranulation of mast cells in lung tissue was observed, and the degranulation rate was counted, and the results were shown in Figure 18 and 19The toluidine blue staining results showed that the total number of mast cells in the lung tissue of the model group mice was significantly increased (P<0.05), and the degranulation of mast cells tended to increase (P>0.05); compared with the model group, the 30 mg / kg TSLP / IL-13 BsAb administration group could significantly reduce the total number of mast cells (P<0.01) and had an effect of reducing the degranulation of mast cells (P>0.05).

[0613] Conclusion

[0614] In this embodiment, an OVA-induced B-hIL13 mouse asthma model was established, and the 30 mg / kg TSLP / IL-13 BsAb administration group was subcutaneously injected to obviously reduce the number of inflammatory cells in the BALF, reduce the serum total IgE level, reduce the infiltration of eosinophils in the lung tissue, inhibit the increase of mucus secretion of airway epithelial cells, and reduce the proliferation of goblet epithelial cells; and had a trend of improving airway hyperresponsiveness and reducing the degranulation of mast cells.

[0615] Example 12, Pharmacodynamic evaluation of TSLP / IL-13 BsAb on MC903-induced B-hIL-13 mouse dermatitis model

[0616] The B-hIL-13 mice were randomly divided into 6 groups, 12 mice in each group, namely a blank control group (Control group), a model group (Model group), a TSLP / IL-13 BsAb administration group (0.4, 2.0, 10.0 mg / kg), and a negative control Anti-KLH hIgG1 (10.0 mg / kg) group. On the first day of modeling, 20 μL of MC903 was applied to the right ear of the mice, 10 μL on the ear ventral side and 10 μL on the ear dorsal side (counted as Day 1), and the application was continuously performed for 5 days, and then stopped for 2 days. From Day 8 to Day 12, the mice were continuously applied with 5 days of application to the right ear, and then stopped for 1 day, and the model was ended on Day 14. The blank control group was synchronously applied with anhydrous ethanol. The first administration was performed on the day before modeling (Day 0), and from Day 0 to Day 7, the administration was performed once every 2 days, and the administration time was Day 0, Day 3, and Day 6, a total of 3 times; from Day 8 to Day 14, the administration was performed once every 1 day, and the administration time was Day 8, Day 10, and Day 12, a total of 3 times, and the administration was performed by subcutaneous injection. The blank control group and the model group were synchronously injected with the vehicle TSLP / IL-13 BsAb blank solution.

[0617] Effect on mouse body weight

[0618] The body weight of the mice was measured before each administration during the test, Figure 20 The results showed that there was no significant difference in the body weight of the mice in each group (P>0.05).

[0619] Effect on mouse ear thickness increase

[0620] The right ear thickness of each group of mice was measured using a micrometer from Day 0 to Day 14, and the results are shown in Table 1. Figure 20 The results showed that, compared with the blank control group, the ear thickness of the model group mice was significantly increased (P < 0.0001); compared with the model group, the TSLP / IL-13 BsAb (10.0 mg / kg) group could significantly inhibit the increase of the right ear thickness of the mice (P < 0.01), and the TSLP / IL-13 BsAb 0.4 mg / kg group, 2.0 mg / kg group and 10.0 mg / kg group showed a dose-dependent inhibition of the increase of the right ear thickness of the mice, with inhibition rates of 13.85%, 18.76% and 27.11%, respectively.

[0621] Effect on mouse ear skin lesion

[0622] The right ear lesion condition of each group of mice was clinically scored before each administration from Day 0 to Day 14, and the results are shown in Table 2. Figure 21 The results showed that, compared with the blank control group, the ventral side of the right ear of the model group mice was significantly dry, the color was deepened, and the ear was dark red, scaly and edematous, the dorsal side of the ear was significantly dilated, and the clinical disease score was significantly increased (P < 0.0001); compared with the model group, the ventral side of the right ear of the TSLP / IL-13 BsAb (10.0 mg / kg) group mice was significantly weakened, the dorsal side of the ear was not significantly dilated, and the clinical disease score was significantly decreased (P < 0.0001), and the inhibition rates of the TSLP / IL-13 BsAb 0.4 mg / kg group, 2.0 mg / kg group and 10.0 mg / kg group on the right ear lesion condition of the mice were 2.61%, 11.4% and 26.32%, respectively.

[0623] Effect on mouse serum total IgE level

[0624] The total IgE level in the serum of each group of mice at the end of the model (Day 14) was detected by ELISA, and the results are shown in Table 3. Figure 22 The results showed that, compared with the blank control group, the total IgE level in the serum of the model group mice showed a trend of increase; compared with the model group, the total IgE level in the serum of the TSLP / IL-13 BsAb (2.0, 10.0 mg / kg) group mice showed a trend of decrease, and the inhibition rates of the TSLP / IL-13 BsAb (2.0, 10.0 mg / kg) group on the total IgE level in the serum of the mice were 24.06% and 25.95%, respectively.

[0625] Example 13, Pre-test of the pharmacodynamic evaluation of TSLP / IL-13 BsAb on the MC903-induced dermatitis model of B-hTSLP / hTSLPR mice

[0626] The B-hTSLP / hTSLPR mice were randomly divided into 8 groups, namely, a blank control group (Control group), a model group (Model group), TSLP / IL-13 BsAb (4.0, 20.0 mg / kg) administration groups, Lunsekimig analogue (4.0, 20.0 mg / kg) administration groups, and anti-TSLP (Ref3, 4.0, 20.0 mg / kg) administration groups. There were 3 mice in the blank control group, and 4 mice in each of the model group and the administration groups. On the first day of modeling, 20 ml of 50 mM C903 was applied to the right ear of the mice, 10 ml to the ear ventral side and 10 ml to the ear dorsal side (counted as Day 1), and the application was continuously performed for 5 days, followed by a 2-day stop. From Day 8 to Day 12, the right ear of the mice was continuously applied for 5 days, followed by a 1-day stop. From Day 14 to Day 15, the right ear of the mice was continuously applied for 2 days, and the modeling was completed on Day 16. The blank control group was synchronously applied with anhydrous ethanol. The first administration was performed on the day before modeling (Day 0), and the administration was performed every 2 days from Day 0 to Day 7, with a total of 3 times on Day 0, Day 3, and Day 6. The administration was performed every 1 day from Day 8 to Day 16, with a total of 4 times on Day 8, Day 10, Day 12, and Day 14, and the administration was performed by subcutaneous injection. The blank control group and the model group were synchronously injected with a TSLP / IL-13 BsAb blank solution. From Day 0 to Day 16, the right ear thickness of the mice in each group was measured using a screw micrometer, and the results are shown in Table 1. Figure 23

[0627] The results showed that, compared with the blank control group, the ear thickness of the mice in the model group was significantly increased (P < 0.0001). Compared with the model group, the TSLP / IL-13 BsAb (4.0, 20.0 mg / kg) groups could significantly inhibit the increase of the right ear thickness of the mice (P < 0.01), with inhibition rates of 25.43% and 27.03%, respectively. The Lunsekimig analogue (4.0, 20.0 mg / kg) groups could significantly inhibit the increase of the right ear thickness of the mice (P < 0.01), with inhibition rates of 22.34% and 26.92%, respectively. The anti-TSLP (Ref3) (4.0 mg / kg) group could inhibit the increase of the right ear thickness of the mice, with an inhibition rate of 19.36%, but no statistical difference was observed (P > 0.05). The anti-TSLP (Ref3, 20.0 mg / kg) group could significantly inhibit the increase of the ear thickness of the mice (P < 0.01), with an inhibition rate of 21.08%.

[0628] Example 14, TSLP / IL-13 BsAb thermal stability

[0629] ​In a 1.5 mL centrifuge tube, TSLP / IL-13 BsAb was diluted to 1 mg / mL with 1x PBS buffer, and then centrifuged at 12000 rpm for 5 minutes using a centrifuge. A capillary tube was used to reach the bottom of the centrifuge tube, and the sample was siphoned to fill the capillary tube. The capillary tube was placed in the capillary tray, covered with a capillary cover, and the capillary drawer was closed. Scanning was performed using the nanoDSF method, followed by size analysis and thermal unfolding analysis. Anti-TSLP (Ref3), Lebrikizumab analog, Tezepelumab analog, and Lunsekimig analog were used as controls. The detection results are shown in Table 8, and the detection spectrum is shown in Figure 26 Table 8. nanoDSF thermal stability detection results

[0630] Table 8. nanoDSF thermal stability detection results

[0631]

[0632] Example 15, TSLP / IL-13 BsAb inhibits TSLP-induced release of TARC from PBMC

[0633] TSLP / IL-13 BsAb was evaluated for its inhibition of TSLP by measuring the release of TARC from PBMC induced by TSLP. TSLP can induce the release of TARC from PBMC, and after TSLP / IL-13 BsAb binds to TSLP, it inhibits the release of TARC. The change in the content of TARC in the culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA), and the half-effective inhibitory concentration (IC 50 ) of TSLP / IL-13 BSAB was calculated according to the change, thereby evaluating the biological activity of TSLP / IL-13 BsAb in inhibiting TSLP-induced release of TARC from PBMC. The recovered PBMC were prepared to contain 2.5x10 6A cell suspension of 2 x 105cells per well was inoculated in a flat-bottom 96-well cell culture plate. After TSLP / IL-13 BsAb, Anti-TSLP (Ref3) and Tezepelumab analogues were diluted to 4 pg / mL with cell culture solution, they were diluted by 3-fold gradient, a total of 8 dilutions, 2 wells for each dilution; TSLP was diluted to 20 ng / mL with cell culture solution. The gradient-diluted TSLP / IL-13 BSAB, Anti-TSLP (Ref3) and Tezepelumab analogues were mixed with TSLP in equal volume, respectively, and 100 pL of the mixture was added to each well of the cells, which were incubated at 37°C, 5% CO2 for 72 hours. The cell culture supernatant was taken, and the TARC content in the cell culture supernatant was detected using a human TARC ELISA kit. The linear regression equation was obtained from the standard concentration of TARC and its corresponding absorbance, and the TARC content in the culture supernatant was calculated from the linear regression equation and multiplied by the dilution factor to obtain the final content. The results are shown in Table 2. Figure 27

[0634] The results show that TSLP / IL-13 BsAb, Anti-TSLP (Ref3) and Tezepelumab analogues can all inhibit TSLP-induced release of TARC from PBMC, and the IC 50 values are 0.32 nM, 0.26 nM and 1.12 nM, respectively. This indicates that TSLP / IL-13 BSAB can effectively inhibit TSLP-induced release of TARC from PBMC, and the inhibitory activity is better than that of Tezepelumab analogues.

[0635] Example 16, TSLP / IL-13 BsAb Inhibits TSLP Combined with IL-13 Induced Release of TARC from PBMC

[0636] TSLP / IL-13 BSAB was evaluated for its inhibition of TSLP and IL-13 synergistic activity by TSLP combined with IL-13 induced release of TARC from PBMC. Both TSLP and IL-13 can induce release of TARC from PBMC, and after TSLP / IL-13 BsAb binds to TSLP and IL-13 with high affinity, it can inhibit the release of TARC. The change in TARC content in the culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA), and the maximum inhibition rate of TSLP / IL-13 BsAb was calculated according to the change, so as to evaluate the biological activity of TSLP / IL-13 BsAb in inhibiting TSLP and IL-13 combined induction of PBMC cells to release TARC. The recovered PBMC were prepared into a cell suspension of 2 x 105cells per 1 ml with cell culture solution, and 100 pL of the cell suspension was inoculated in each well of a flat-bottom 96-well cell culture plate. After TSLP / IL-13 BsAb was diluted to 4 pg / mL with cell culture solution, it was diluted by 3-fold gradient, a total of 8 dilutions, 2 wells for each dilution; TSLP was diluted to 20 ng / mL with cell culture solution. The gradient-diluted TSLP / IL-13 BSAB and TSLP were mixed in equal volume, respectively, and 100 pL of the mixture was added to each well of the cells, which were incubated at 37°C, 5% CO2 for 72 hours. The cell culture supernatant was taken, and the TARC content in the cell culture supernatant was detected using a human TARC ELISA kit. The linear regression equation was obtained from the standard concentration of TARC and its corresponding absorbance, and the TARC content in the culture supernatant was calculated from the linear regression equation and multiplied by the dilution factor to obtain the final content. The results are shown in Table 2. 6 ​A cell suspension of 100 μl was inoculated into a flat-bottom 96-well cell culture plate. TSLP / IL-13 BsAb, Anti-TSLP (Ref3), Lebrikizumab analogue, Lunsekimig analogue and Tezepelumab analogue were diluted to 26.7 nM with cell culture solution, 3-fold gradient dilution, a total of 8 dilutions, 2 holes for each dilution; IL-13 and TSLP were diluted to 40 ng / mL with cell culture solution, respectively. Equal volume of 40 ng / mL TSLP and IL-13 was mixed. Gradient dilution of each antibody was mixed with TSLP and IL-13 mixture in equal volume, 100 μL of the mixture was added to each well, and incubated at 37°C, 5% CO2 for 72 hours. The cell culture supernatant was taken, and the TARC content in the cell culture supernatant was detected by human TARC ELISA kit. The linear regression equation was obtained by the absorbance corresponding to the standard concentration of TARC. The TARC content in the culture supernatant was calculated from the linear regression equation and multiplied by the dilution factor to obtain the content value and calculate the inhibition rate. The results are shown in Figure 28

[0637] The results show that TSLP / IL-13 BSAB, Anti-TSLP (Ref3), Lebrikizumab analogue, Lunsekimig analogue and Tezepelumab analogue can all inhibit the release of TARC from PBMC induced by TSLP and IL-13 combination, with maximum inhibition rates of 93.1%, 58.4%, 77.3%, 94.0% and 49.4%, respectively. This indicates that TSLP / IL-13 BsAb can effectively inhibit the release of TARC from PBMC induced by TSLP and IL-13 combination, and the inhibition effect is better than Anti-TSLP (Ref3), Lebrikizumab analogue and Tezepelumab analogue.

[0638] Example 17, Immunogenicity evaluation of TSLP / IL-13 BsAb

[0639] By detecting the activation of CD4 + T cells by TSLP / IL-13 BsAb, the immunogenicity risk of TSLP / IL-13 BsAb was preliminarily evaluated. The clinical immunogenicity of drugs is related to CD134 (OX40) and CD137 (4-1BB) positive CD4 + ​T cell ratio showed correlation. The ratio of CD134 and CD137 positive cells in CD4+ T cells was detected by flow cytometry after co-culturing TSLP / IL-13 BsAb with peripheral blood mononuclear cells (PBMC) isolated from peripheral blood of healthy volunteers for 48 hours, to evaluate the immunogenicity risk of TSLP / IL-13 BsAb. The density of the isolated and collected cells was adjusted to 2.5 x 10 6 / mL with X-vivo15 medium, and 100 μL was added to a 96-well U-shaped plate. Each antibody and positive control was diluted to 200 μg / mL with X-vivo15 medium. 100 μL of the diluted sample was added to the cells, and the cells were cultured at 37°C, 5% CO2 for 48 hours. KLH was used as a positive control, and the well without sample was used as a negative control. After 48 hours, the cells were washed and stained with antibodies (50 μL / well of FITC anti-human CD4, APC anti-human CD137, and PE anti-human CD134) for 45 minutes, and 50 μL of PI solution was added for 5 minutes. After washing with flow cytometry buffer, the cells were resuspended in 100 μL of PBS and analyzed by flow cytometry. The results are shown in Figure 29

[0640] The results showed that the stimulation index of TSLP / IL-13 BsAb, Anti-TSLP (Ref3, Lebrikizumab analog, Tezepelumab analog, and Lunsekimig analog) was 0.91, 0.97, 0.90, 1.11, and 1.12, respectively, and the immunogenicity risk of each group was equivalent, which was much smaller than the stimulation index of the positive control group KLH (3.51). This indicated that the immunogenicity risk of TSLP / IL-13 BsAb, Anti-TSLP (Ref3), Lebrikizumab analog, Tezepelumab analog, and Lunsekimig analog was equivalent, and all were low immunogenicity.

[0641] ​Example 18, Pharmacodynamic Effect of TSLP / IL-13 BsAb on OVA-induced Asthma in B-hTSLP / hTSLPR Mice The B-hTSLP / hTSLPR mice were randomly divided into 6 groups according to body weight, 12 mice in each group, namely, a control group, a model group, a KLH negative control group (30 mg / kg), a TSLP / IL-13 BsAb low-dose group (3 mg / kg), a TSLP / IL-13 BsAb middle-dose group (10 mg / kg), and a TSLP / IL-13 BsAb high-dose group (30 mg / kg). On the first day of modeling, the mice were sensitized by multiple point injection of 2 mg / mL OVA Al(OH)3 mixture, a total of 0.5 mL per mouse (counted as Day 0), and on Day 14, the mice were intraperitoneally injected with 0.2 mL of OVA Al(OH)3 mixture for reinforcement sensitization. From Day 21 to Day 27, the mice were inhaled with OVA solution (10 mg / mL prepared by 0.9% sodium chloride injection). The first administration was one day before modeling (Day -1), and from Day -1 to Day 19, the mice were administered every 4 days for a total of 5 times; from Day 21 to Day 27, the mice were administered every 1 day for a total of 4 times; and the mice were subcutaneously injected with the drug 2 hours before OVA challenge. The control group and the model group were synchronously subcutaneously injected with the vehicle TSLP / IL-13 BsAb blank solution.

[0642] Effect on body weight of mice

[0643] During the administration period of modeling, the general symptoms were observed every day, and the body weight of the mice in each group was measured before each administration, and the results are shown in Table 1. Figure 30 The results showed that there was no significant difference in the body weight of the mice in the model group during the modeling and administration periods compared with the control group (P>0.05). Compared with the model group, there was no significant difference in the body weight of the mice in the TSLP / IL-13 BsAb (3, 10, 30 mg / kg) group and the KLH (30 mg / kg) group during the modeling and administration periods (P>0.05). No obvious abnormalities were observed in the general symptoms of the mice in each test group.

[0644] Effect on the number of flexural nose and sneezing

[0645] On Day 27, the number of times of scratching the nose and sneezing within 30 minutes after OVA challenge was counted, and the results are shown in Table 2. Figure 31The results showed that compared with the blank control group, the number of nose scratching and sneezing of the model group mice was significantly increased (P<0.001). Compared with the model group, the number of nose scratching and sneezing of the TSLP / IL-13 BsAb (10, 30 mg / kg) group mice was significantly reduced (P<0.05-0.001), and there was no significant difference between the TSLP / IL-13 BsAb (3 mg / kg) group and the KLH (30 mg / kg) group (P>0.05); the number of nose scratching and sneezing of the TSLP / IL-13 BsAb (3, 10, 30 mg / kg) group mice was reduced in a dose-dependent manner, and the inhibition rates of nose scratching were 7.08%, 26.15% and 49.54%, respectively, and the inhibition rates of sneezing were 9.89%, 26.37% and 36.26%, respectively.

[0646] Effect on Mch-induced airway hyperresponsiveness (AHR)

[0647] On day 28, the mice were nebulized with 0, 1, 2, 4, 8, 12, 24 or 48 mg / mL acetylcholine (Mch) and the Mch-induced airway hyperresponsiveness (AHR) of the mice was detected by the EMKA animal lung function monitoring system. After the detection of airway hyperresponsiveness, the detection data were exported by IOX recording and analysis software, and 3-5 maximum values were selected continuously at the baseline value and after nebulization with Mch, respectively, to calculate the measured value and increase rate of airway resistance (Penh) and PC100, and the results were as follows Figure 32The results showed that compared with the blank control group, the Penh value and the Penh increase rate of the model group were significantly increased at Mch 12, 24, 48 mg / mL (P<0.01-0.001). Compared with the model group, the TSLP / IL-13 BsAb (10, 30 mg / kg) group significantly reduced the increase of Penh value (P<0.05-0.001), the TSLP / IL-13 BsAb (3 mg / kg) group significantly reduced the Penh value at Mch 24 mg / mL (P<0.05), and the KLH (30 mg / kg) group had no significant difference in the increase of Penh value at each concentration of Mch (P>0.05); the inhibition rates of the TSLP / IL-13 BsAb (3, 10, 30 mg / kg) group on the Penh value at Mch 48 mg / mL were 23.72%, 46.33% and 57.04%, respectively. The PC100 of the blank control group, the model group, the TSLP / IL-13 BsAb (3, 10, 30 mg / kg) group and the KLH (30 mg / kg) group were 10.43, 5.82, 6.75, 9.41, 13.99 and 5.21 mg / mL, respectively, indicating that after the OVA-induced mouse asthma model, the PC100 of the model group decreased, the airway sensitivity increased, and the PC100 of the TSLP / IL-13 BsAb (3, 10, 30 mg / kg) group increased in a dose-dependent manner, and the airway sensitivity decreased.

[0648] Effect on the number of inflammatory cells in bronchoalveolar lavage fluid (BALF)

[0649] On day 28, after blood collection, the mice were sacrificed by cervical dislocation, the left lung and accessory lobe were ligated, and 0.5 mL bronchoalveolar lavage fluid (BALF) was used for lavage, which was repeated twice. The three lavage fluids were mixed and evenly mixed, and the white blood cells, eosinophils, macrophages and lymphocytes were counted under a microscope. The results are shown in Table 2. Figure 33The results showed that compared with the blank control group, the number of leukocytes, eosinophils, macrophages and lymphocytes in BALF of the model group were significantly increased (P<0.001). Compared with the model group, TSLP / IL-13 BsAb (10, 30 mg / kg) group could significantly inhibit the increase of the number of leukocytes and eosinophils (P<0.01-0.001), TSLP / IL-13 BsAb (3 mg / kg) group could significantly inhibit the increase of the number of eosinophils (P<0.05), TSLP / IL-13 BsAb (30 mg / kg) group could significantly inhibit the increase of the number of macrophages (P<0.001), TSLP / IL-13 BsAb (3, 10, 30 mg / kg) group had no effect on the number of lymphocytes (P>0.05), and KLH (30 mg / kg) group had no effect on the number of leukocytes, eosinophils, macrophages and lymphocytes (P>0.05); the inhibition rates of TSLP / IL-13 BsAb (3, 10, 30 mg / kg) group on the number of leukocytes were 17.16%, 28.82% and 41.03% respectively, and the inhibition rates on the number of eosinophils were 32.39%, 58.74% and 69.87% respectively.

[0650] Effect on serum total IgE and OVA-sIgE levels

[0651] Day 20, Day 28, the blood of mice was collected from retro-orbital venous plexus, and the total IgE and OVA-sIgE in serum were detected by ELISA, and the results were shown in Table 2. Figure 34 The results showed that compared with the blank control group, the total IgE and OVA-sIgE in serum of Day 20 and Day 28 were significantly increased (P<0.001). Compared with the model group, Day 20, TSLP / IL-13 BsAb (10, 30 mg / kg) group could significantly reduce the level of serum total IgE (P<0.05-0.01); Day 28, TSLP / IL-13 BsAb (10, 30 mg / kg) group could significantly reduce the levels of serum total IgE and OVA-sIgE (P<0.01-0.001); Day 20 and Day 28, the level of serum total IgE of TSLP / IL-13 BsAb (3, 10, 30 mg / kg) group was reduced in a dose-dependent manner, and the inhibition rates were 25.04%, 31.90% and 46.88% respectively on Day 20, and the inhibition rates were 7.81%, 49.91% and 56.12% respectively on Day 28.

[0652] Effect on lung tissue h-TSLP and MUC5AC mRNA expression

[0653] The lung tissue was taken, 1 mL TRIzon Reagent was added, homogenized, RNA was extracted, and the RNA was reversely transcribed into cDNA, and the expression levels of IL-4, IL-5, IL-13, h-TSLP and MUC5AC mRNA in the lung tissue were detected by using q-PCR method, and the results are shown in Figure 35 The results show that, compared with the blank control group, the expression levels of h-TSLP and MUC5AC mRNA in the lung tissue of the model group mice were significantly increased (P<0.001), and the expression levels of IL-4, IL-5 and IL-13 mRNA had a trend of increase but no significant difference (P>0.05). Compared with the model group, the TSLP / IL-13 BsAb (10, 30 mg / kg) group could significantly inhibit the increase of the expression levels of h-TSLP and MUC5AC mRNA in the lung tissue (P<0.05-0.01), and the inhibition rates of h-TSLP were 35.85% and 24.73% respectively, and the inhibition rates of MUC5AC were 48.81% and 57.21% respectively.

[0654] Effect on lung tissue eosinophil infiltration

[0655] On day 28, after the alveolar lavage, the left lung hilar part was fixed with 10% neutral buffered formalin solution, and after complete fixation, paraffin embedding sectioning was performed, H&E staining was performed to observe the eosinophil infiltration of the lung tissue, and pathological scoring was performed, and the results of the pathological scoring are shown in Figure 36 , and the pathological picture of eosinophil infiltration is shown in Figure 37 The results show that, compared with the blank control group, a large number of eosinophil infiltrations in the airway and perivascular of the lung tissue of the model group mice were observed (P<0.001). Compared with the model group, the TSLP / IL-13 BsAb (10, 30 mg / kg) group significantly inhibited the eosinophil infiltration in the airway and perivascular of the lung tissue of the mice in a dose-dependent manner (P<0.01).

[0656] Effect on airway goblet cell hyperplasia proliferation

[0657] On day 28, the lung tissue of the mice was subjected to PAS staining, the airway pericyte cell metaplasia and proliferation of the lung tissue were observed, the positive rate was counted, and the results of the positive rate are shown in Figure 38 , and the results of the pathological picture of airway pericyte cell metaplasia and proliferation are shown in Figure 39The PAS staining results showed that compared with the blank control group, the model group mice had a large number of goblet cell metaplasia and mucus secretion in the inner side of the airway lumen of the lung tissue (P<0.001), and the positive rate of goblet cells was 46.37%. Compared with the model group, the TSLP / IL-13 BsAb (10, 30 mg / kg) groups significantly inhibited the goblet cell metaplasia and mucus secretion in the airway of mice in a dose-dependent manner (P<0.05-0.01), and the inhibition rates were 21.36% and 46.06%, respectively.

[0658] Effect on lung tissue mast cell degranulation

[0659] On day 28, the lung tissues of the mice were subjected to toluidine blue staining, and the degranulation of the lung tissue mast cells was observed. The degranulation rate was calculated, and the degranulation rate results are shown in Figure 40 , and the mast cell degranulation pathological chart results are shown in Figure 41 . The results showed that compared with the blank control group, the model group mice had more lung tissue mast cell degranulation (P<0.001), and the degranulation rate was 66.10%. Compared with the model group, the TSLP / IL-13 BsAb (3, 10, 30 mg / kg) groups significantly inhibited the lung tissue mast cell degranulation of mice in a dose-dependent manner (P<0.05-0.01), and the inhibition rates were 30.69%, 43.27% and 65.33%, respectively; the KLH (30 mg / kg) group had no significant difference (P>0.05).

[0660] Effect on lung tissue peribronchial collagen deposition

[0661] On day 28, the lung tissues of the mice were subjected to Masson's staining, and the airway remodeling and airway pericollagen deposition were observed under a microscope. The area of the positive expression region was calculated, and the collagen deposition positive rate results are shown in Figure 42 , and the airway pericollagen deposition pathological chart results are shown in Figure 43 . The results showed that compared with the blank control group, the model group mice had more collagen deposition in the peribronchial lung tissue (P<0.001). Compared with the model group, the TSLP / IL-13 BsAb (3, 10, 30 mg / kg) groups significantly improved the peribronchial collagen deposition of the lung tissue of mice in a dose-dependent manner (P<0.05-0.01), and the inhibition rates were 30.63%, 50.62% and 57.32%, respectively; the KLH (30 mg / kg) group had no significant difference (P>0.05).

[0662] Conclusion

[0663] In this embodiment, the OVA-induced B-hTSLP / hTSLPR mouse asthma model was successfully established, and subcutaneous injection of TSLP / IL-13 BsAb could significantly inhibit Mch-induced airway hyperresponsiveness; alleviate the symptoms of allergic rhinitis; reduce the increase in the total number of white blood cells, the number of eosinophils, and the number of macrophages in BALF; reduce the levels of total IgE and OVA-sIgE in serum; inhibit the expression of h-TSLP and MUC5AC mRNA in lung tissue; reduce eosinophil infiltration in lung tissue; inhibit the increase in mucus secretion of airway epithelial cells, alleviate the proliferation of goblet epithelial cells; reduce mast cell degranulation in lung tissue; and reduce collagen deposition around the airway. These results demonstrate that TSLP / IL-13 BsAb has a significant anti-asthma effect.

[0664] Example 19, Pharmacodynamic evaluation of TSLP / IL-13 BsAb on MC903-induced B-hTSLP / hTSLPR mouse dermatitis model

[0665] The B-hTSLP / hTSLPR mice were randomly divided into 6 groups, 11 mice in each group, namely, a blank control group (Control group), a model group (Model group), TSLP / IL-13 BsAb (0.3, 3.0, 30.0 mg / kg) groups, and a negative control Anti-KLH hIgG1 (30.0 mg / kg) group. The first day of modeling was counted as Day 0, and 20 μL of MC903 (50 μM) was applied to the right ear of the mice, 10 μL on each of the ear ventral and ear dorsal sides, for 5 consecutive days, and then stopped for 2 days; from Day 7 to Day 11, the mice were continuously applied to the right ear for 5 days, and then stopped for 2 days, and the model ended on Day 13; the blank control group was synchronously applied with anhydrous ethanol. The first administration was one day before modeling (Day-1), and from Day-1 to Day 6, the administration was performed once every 2 days, and the administration time was Day-1, Day 2, and Day 5, a total of 3 times; from Day 7 to Day 13, the administration was performed once every day, and the administration time was Day 7, Day 9, and Day 11, a total of 3 times, and the administration was performed by subcutaneous injection. The blank control group and the model group were synchronously injected with the vehicle TSLP / IL-13 BsAb blank solution.

[0666] Body weight and general symptom observation

[0667] During the modeling and administration period, general symptom observation was performed every day, and the body weight of the mice in each group was measured before each administration, and the results are shown in Table 1. Figure 44The results showed that, compared with the blank control group, the body weight of the model group mice had no significant difference during the modeling and dosing period (P>0.05). Compared with the model group, the body weight of the TSLP / IL-13 BsAb (0.3, 3.0, 30.0 mg / kg) group and the Anti-KLH hIgG1 (30.0 mg / kg) group mice had no significant difference during the modeling and dosing period (P>0.05).

[0668] Inhibitory effect on the increase of ear thickness of mice

[0669] On Day-1~Day 13, the right ear thickness of each group of mice was measured before each administration and on Day 12 and Day 13, and the results are shown in Figure 45 The results showed that, compared with the blank control group, the right ear thickness of the model group mice was significantly increased (P<0.0001). Compared with the model group, the TSLP / IL-13 BsAb (3.0, 30.0 mg / kg) group could significantly inhibit the increase of the right ear thickness of the mice (P<0.05~0.0001); the TSLP / IL-13 BsAb (0.3, 3.0, 30.0 mg / kg) group showed a dose-dependent inhibition of the increase of the right ear thickness of the mice, and the inhibition rates were 5.45%, 17.29% and 40.48%, respectively.

[0670] Inhibitory effect on the skin lesion of mice

[0671] On Day-1~Day 13, the right ear skin lesion condition of each group of mice was scored before each administration and on Day 12 and Day 13, and the results are shown in Figure 46 The results showed that, compared with the blank control group, the right ear ventral side of the model group mice was significantly dry, darkened in color, dark red, scaly and edematous, the blood vessels on the dorsal side of the ear were significantly dilated, and the clinical disease score was significantly increased (P<0.0001). Compared with the model group, the right ear ventral side skin lesion of the TSLP / IL-13 BsAb (30.0 mg / kg) group mice was significantly weakened, the blood vessels on the dorsal side of the ear showed no significant dilation trend, and the clinical disease score was significantly reduced (P<0.0001), and the TSLP / IL-13 BsAb (0.3, 3.0, 30.0 mg / kg) group reduced the right ear skin lesion of the mice, and the inhibition rates were 11.58%, 10.53% and 40.26%, respectively.

[0672] Inhibition of the enlargement of right ear draining lymph nodes of mice

[0673] On Day 13, the right ear draining lymph nodes of the mice were taken and weighed, and the results are shown in Figure 47The results showed that the weight of right ear draining side lymph nodes of the model group mice was significantly increased compared with the blank control group (P<0.0001). Compared with the model group, the right ear draining side lymph nodes of the TSLP / IL-13 BsAb (3.0, 30.0 mg / kg) group mice were not significantly enlarged, and the weight was not significantly increased (P<0.001-0.0001). There was no significant difference in the weight of right ear draining side lymph nodes between the TSLP / IL-13 BsAb (0.3 mg / kg) group and the Anti-KLH hIgG1 (30.0 mg / kg) group (P>0.05). TSLP / IL-13 BsAb (0.3, 3.0, 30.0 mg / kg) could dose-dependently inhibit the enlargement of right ear draining side lymph nodes of the mice induced by MC903, and the inhibition rates were 19.03%, 40.48% and 50.65%, respectively.

[0674] Reduction of serum total IgE levels of mice

[0675] On day 13, the total IgE levels in the serum of the mice in each group were detected by ELISA, and the results are shown in FIG. 6. Figure 48 The results showed that the total IgE level in the serum of the mice in the model group was significantly increased compared with the blank control group (P<0.01). Compared with the model group, the total IgE level in the serum of the mice in the TSLP / IL-13 BsAb (3.0, 30.0 mg / kg) group was significantly decreased (P<0.05). The TSLP / IL-13 BsAb (3.0, 30.0 mg / kg) group significantly decreased the total IgE level in the serum of the mice, and the inhibition rates were 47.79% and 46.61%, respectively.

[0676] Inhibition of cytokine expression in ear tissue of mice

[0677] The IL-4, IL-5 and IL-6 mRNA expressions in the ear tissues of the mice were detected by qPCR, and the results are shown in FIG. 7. Figure 49 The results showed that the IL-4 and IL-6 mRNA levels in the ear tissues of the mice in the model group were significantly increased compared with the blank control group (P<0.001), and the IL-5 mRNA level was not significantly increased (P>0.05). Compared with the model group, the IL-4 mRNA level in the ear tissues of the mice in the TSLP / IL-13 BsAb (3.0, 30.0 mg / kg) group was significantly decreased (P<0.001), and the inhibition rates on IL-4 were 78.38% and 85.95%, respectively. The IL-6 mRNA level in the ear tissues of the mice in the TSLP / IL-13 BsAb (0.3, 3.0, 30.0 mg / kg) group was significantly decreased (P<0.001), and the inhibition rates on IL-6 were 48.02%, 73.17% and 84.49%, respectively.

[0678] Reduction of inflammation score in ear tissue of mice

[0679] Day 13, the right ear tissues of mice were stained with H&E and inflammation scores were performed, and the results are shown in Figure 50 and 51 The pathological results of the right ear tissues of mice showed that compared with the blank control group, the inflammation score of the ear tissues of the mice in the model group was significantly increased (P <0.0001). Compared with the model group, the inflammation score of the ear tissues of the mice in the TSLP / IL-13 BsAb (3.0, 30.0 mg / kg) groups was significantly reduced (P <0.01-0.001). The TSLP / IL-13 BsAb (0.3, 3.0, 30.0 mg / kg) groups showed a dose-dependent reduction in the inflammation score of the ear tissues of mice, and the inhibition rates were 16.7%, 25.0% and 33.3%, respectively.

[0680] Conclusion

[0681] In this experimental example, the MC903-induced B-hTSLP / hTSLPR mouse dermatitis model was successfully established, and subcutaneous injection of TSLP / IL-13 BsAb could significantly inhibit the increase of ear thickness in mice; improve the ear lesions of mice, reduce the clinical disease score; inhibit the enlargement of the right ear draining lymph nodes; reduce the total IgE level in serum; inhibit the expression of IL-4 and IL-6 mRNA in ear tissues; and inhibit the inflammatory reaction in ear tissues. These results demonstrate that TSLP / IL-13 BsAb has obvious anti-dermatitis effect.

[0682] Example 20, Study on the affinity of TSLP / IL-13 BsAb with FcγR, FcRn and C1q

[0683] Binding affinity to FcγRI, FcγRII and FcγRIII

[0684] SPR / Biacore T200 was used to couple His-Tag antibody on the surface of CM5 chip, and FcyRI, FcyRIIAH131, FcyRIIB, FcyRIIIAV158 and FcyRIIIAF158 were captured by the mutual binding of His tag and His-Tag antibody. The capture time was set to 40 s, the flow rate was 10 μL / min, and the chip was washed with buffer until the baseline was stable. The gradient-diluted TSLP / IL-13 BsAb was flowed through the chip at a flow rate of 30 μL / min. When detecting the affinity of TSLP / IL-13 BsAb and FcyRI, the binding time of TSLP / IL-13 BsAb was set to 120 s, and the dissociation time was set to 200 s. When detecting the affinity of TSLP / IL-13 BsAb and FcyRII and FcyRIII, the binding time of TSLP / IL-13 BsAb was set to 90 s, and the dissociation time was set to 120 s. Finally, 10 mM Glycine-HCl at pH 2.0 was used for regeneration for 30 s at a flow rate of 30 μL / min. The measured data was fitted to obtain the equilibrium dissociation constant (KD). The results are shown in Figures 52-56 .

[0685] Figure 52 It is shown that the binding of TSLP / IL-13 BsAb and positive control antibody Anti-KLH hIgG1 to FcyRI conforms to Langmuir 1:1 kinetic theory model, which is monovalent binding. The affinity of TSLP / IL-13 BsAb to FcyRI is 1.53 nM, and the affinity of positive control antibody Anti-KLH hIgG1 to FcyRI is 0.66 nM.

[0686] Figure 53 and Figure 54 It is shown that the binding of TSLP / IL-13 BsAb and positive control antibody Anti-KLH hIgG1 to FcyRII conforms to Steady state steady-state model, which is monovalent binding. The affinity of TSLP / IL-13 BsAb to FcyRIIAH131 is 2.36 μM, and the affinity of positive control antibody Anti-KLH hIgG1 to FcyRIIAH131 is 1.11 μM Figure 53 . Both TSLP / IL-13 BsAb and positive control antibody Anti-KLH hIgG1 weakly bind to FcyRIIB Figure 54 .

[0687] Figure 55 and Figure 56The binding of TSLP / IL-13 BsAb and positive control antibody Anti-KLH hlgGl to FcyRIII was shown to fit the Steady state model, which was monovalent binding. The affinity of TSLP / IL-13 BsAb to FcyRIII AV158 was 843.7 nM, and the affinity of positive control antibody Anti-KLH hlgGl to FcyRIII AV158 was 258.3 nM Figure 55 ). The affinity of TSLP / IL-13 BsAb to FcyRIII AF158 was 1.28 μΜ, and the affinity of positive control antibody Anti-KLH hlgGl to FcyRIII AF158 was 995.2 nM Figure 56 ).

[0688] Binding affinity to FcRn

[0689] Using SPR / Biacore T200, FcRn was captured to the surface of CM5 chip which was coupled with Tis-Tag antibody, the capture time was set as 40 s, the flow rate was 10 μL / min, the chip was washed with buffer until the baseline was stable, the gradient diluted TSLP / IL-13 BsAb and positive control antibody Anti-KLH hlgGl were flowed through the chip at a flow rate of 30 μL / min, the binding time was set as 80 s, the dissociation time was set as 110 s, 10 mM Glycine-HCl at pH 2.0 was used for regeneration for 30 s at a flow rate of 30 μL / min. The measured data were fitted to obtain the equilibrium dissociation constant. The results are shown in Figures 57-58 .

[0690] Figure 57 and Figure 58 The binding of TSLP / IL-13 BsAb and positive control antibody Anti-KLH hlgGl to FcRn was shown to be pH-dependent. At pH 7.4, TSLP / IL-13 BsAb and positive control antibody Anti-KLH hlgGl weakly bound to FcRn Figure 57 ). At pH 6.0, the binding of TSLP / IL-13 BsAb and positive control antibody Anti-KLH hlgGl to FcRn fit the Steady state model, which was monovalent binding; the affinity of TSLP / IL-13 BsAb to FcRn was 451.8 nM, and the affinity of positive control antibody Anti-KLH hlgGl to FcRn was 599.3 nM Figure 58 .

[0691] Binding affinity to Clq

[0692] SPR / Biacore T200 was used to capture TSLP / IL-13 BsAb and positive control antibody Anti-KLH hlgG1 to Fc2 and Fc4 channels of Protein L chip, with a capture time of 40 s, a flow rate of 10 μL / min, and the chip was flushed with buffer until the baseline was stable. Gradient-diluted C1q was sequentially flowed through the chip at a flow rate of 30 μL / min, with a binding time of 80 s, a dissociation time of 110 s, and a regeneration time of 30 s with 10 mM Glycine-HCl at pH 2.0, and a flow rate of 30 μL / min. The measured data were fitted to obtain the equilibrium dissociation constant. Analysis was performed using Biacore T200 Evaluation Software 3.1, and the binding of the antibody to FcγRI was fitted using Kinetics 1:1 binding mode, and other weak interactions (fast binding and fast dissociation) were fitted using SteadyState mode to obtain affinity data. The results are shown in Table 1. Figure 59

[0693] Figure 59 It was shown that the binding of TSLP / IL-13 BsAb to C1q conforms to the Steady state model. The affinity of TSLP / IL-13 BsAb to C1q is 79.18 nM, and the affinity of positive control antibody Anti-KLH hlgG1 to C1q is 53.62 nM.

[0694] Conclusion

[0695] TSLP / IL-13 BsAb can bind to FcγRI, FcγRIIAH131, FcγRIIIAV158, and FcγRIIIAF158 and C1q, and weakly bind to FcγRIIB; and binds to FcRn in a pH-dependent manner, weakly binds to FcRn at pH 7.4, and the affinity to FcRn is enhanced at pH 6.0, and the affinity is stronger than that of positive control antibody Anti-KLH IgG1.

[0696] Example 21, Study on the binding of TSLP / IL-13 BsAb to different species of IL-13

[0697] ELISA was used to coat human, cynomolgus monkey, mouse, and rat IL-13 recombinant proteins on 96-well enzyme-labeled plates, and after blocking, different concentrations of TSLP / IL-13 BsAb stock solution were added for co-incubation, and HRP-labeled anti-human Fc secondary antibody was used for labeling, TMB substrate was used for color development, and the absorbance at 450 nm wavelength was measured to calculate the EC50, and the binding activity of TSLP / IL-13 BsAb to human, cynomolgus monkey, mouse, and rat IL-13 was evaluated. ​

[0698] Human, cynomolgus monkey, rat and mouse IL-13 at a concentration of 1 μg / mL were prepared with coating solution, 100 μL per well was added to the enzyme-labeled plate, which was coated at 4°C overnight; after washing and blocking the coated enzyme-labeled plate, different concentrations of TSLP / IL-13 BsAb were added, the antibody was incubated at room temperature for 1 h, then washed, Anti-hFc-HRP secondary antibody solution (1:4000 dilution) was added, and incubated at room temperature for 1 h, 100 μL of prepared TMB developing solution was added to each well, and color development was carried out at 37°C for 5 min, then 50 μL of stop solution was added to each well; the absorbance at 450 nm was read by an enzyme-labeled instrument. Anti-KLH hIgG1 was used as a negative control. The data were processed by GraphPad Prism 6 software, the antibody concentration was used as the abscissa, the absorbance at 450 nm was used as the ordinate, the four-parameter regression calculation method was used for curve fitting, and the EC50 was calculated. The results are shown in Figure 60 and 61 .

[0699] Figure 60 It is shown that TSLP / IL-13 BsAb binds to human and cynomolgus monkey IL-13, with EC50 of 0.45 nM and 0.56 nM, respectively, and does not bind to rat and mouse IL-13. Figure 61 It is shown that the negative control Anti-KLH hIgG1 does not bind to human, cynomolgus monkey, rat and mouse IL-13.

[0700] Example 22, Study on the binding of TSLP / IL-13 BsAb to TSLP of different species

[0701] ELISA was used to coat human, cynomolgus monkey, mouse and rat TSLP recombinant proteins on a 96-well enzyme-labeled plate, and after blocking, different concentrations of TSLP / IL-13 BsAb stock solution were added for incubation, Anti-human Fc secondary antibody containing horseradish peroxidase (HRP) was used for labeling, TMB substrate was used for color development, and the absorbance at 450 nm was measured to calculate the half maximal effective concentration (EC50), so as to evaluate the binding activity of TSLP / IL-13 BsAb to human, cynomolgus monkey, mouse and rat TSLP.

[0702] Prepare 1 pg / mL human, cynomolgus monkey, rat and mouse TSLP with coating solution, respectively, and add 100 pL to enzyme-labeled plates, and incubate at 4°C overnight. After washing and blocking the coated enzyme-labeled plates, add different concentrations of TSLP / IL-13 BsAb, incubate the antibody at room temperature for 1 h, wash, add prepared Anti-hFc-HRP secondary antibody solution (1:4000 dilution), continue to incubate at room temperature for 1 h, add 100 pL of prepared TMB color developing solution to each well, develop color at 37°C for 5 min, and then add 50 pL of stop solution to each well. Read the absorbance at 450 nm wavelength by an enzyme-labeled instrument. Use Anti-KLH hIgG1 as a negative control. Process with GraphPad Prism 6 software, take the antibody concentration as the abscissa and the 450 nm absorbance as the ordinate, perform curve fitting by four-parameter regression calculation, and calculate the EC50. The results are shown in Figure 62 and 63

[0703] Figure 62 It is shown that TSLP / IL-13 BsAb binds to human and cynomolgus monkey TSLP, with EC50 of 0.05 nM and 0.11 nM, respectively, and does not bind to rat and mouse TSLP. Figure 63 It is shown that the negative control Anti-KLH hIgG1 does not bind to human, cynomolgus monkey, rat and mouse TSLP.

[0704] Example 23, Study on TSLP / IL-13 BsAb Blocking IL-13 / IL-13Rα1 Complex Binding to IL-4Rα

[0705] Using surface plasmon resonance technology (SPR / Biacore T200), IL-4Rα is immobilized on a CM5 chip through amino coupling, then the IL-13 / IL-13Rα1 complex is incubated with gradient-diluted TSLP / IL-13 BsAb, and then flows through the chip surface as an analyte, to evaluate the activity of TSLP / IL-13 BsAb blocking IL-13 / IL-13Rα1 complex binding to IL-4Rα by the binding signal of the analyte.

[0706] ​Select CM5 chip 4 channels, set the coupling amount 3000 RU, flow rate 10 μL / min, put in the coupling reagent (Amine Coupling Kit) and 30 μg / mL IL-4Rα, complete the coupling. Select 4-3 channels, put in the gradient dilution of TSLP / IL-13 BsAb and IL-13 / IL-13Rα1 mixed solution in turn, set the binding time 100 s, dissociation time 60 s, flow rate 30 μL / min, regeneration time 30 s, flow rate 30 μL / min. In the data analysis software Biacore T200 Evaluation Software, find the corresponding data file, Report Point select baseline to obtain the curve atlas; export to the final binding signal, use GraphPad Prism software for processing, take the sample to be tested as the abscissa, the resonance unit (RU) as the ordinate to plot. Use Anti-KLH hIgG1 as negative control. The results are shown in Figure 64 .

[0707] Figure 64 It is shown that TSLP / IL-13 BsAb effectively blocks the binding of IL-13 / IL-13Rα1 complex to IL-4Rα, and the negative control Anti-KLH hIgG1 has no blocking effect.

[0708] Example 24, Study on the Blocking Activity of TSLP / IL-13 BsAb to TSLP Binding to TSLP Receptor (TSLPR)

[0709] Using enzyme-linked immunosorbent assay (ELISA), coat TSLPR-hFc recombinant protein on a 96-well enzyme-labeled plate, after blocking, add the mixed solution of TSLP / IL-13 BsAb and TSLP-mFc recombinant protein in a volume ratio of 1:1, develop by horseradish peroxidase (HRP) labeled anti-mFc secondary antibody, measure the absorbance at a wavelength of 450 nm, calculate the half-effective inhibition concentration (IC50) of TSLP / IL-13 BsAb, and thus evaluate the activity of TSLP / IL-13 BsAb in blocking the binding of TSLP to TSLPR.

[0710] TSLPR-hFc was diluted to 1 μg / mL with coating solution, 100 μL per well was added to the enzyme-labeled plate, which was coated at 4°C overnight; after washing and blocking the coated enzyme-labeled plate, 100 μL of the mixed solution of diluted TSLP / IL-13 BsAb stock solution, negative control Anti-KLH hIgG1 and 200 ng / mL TSLP-mFc was added to each well, two wells were prepared for each dilution, and incubated at room temperature for 1 h; after washing, 100 μL of prepared Anti-mFc-HRP secondary antibody solution (1:4000 dilution) was added to each well, and incubated at room temperature for 1 h; 100 μL of prepared TMB developing solution was added to each well, and developed at 37°C for 5 min in the dark; 50 μL of stop solution was added to each well; the absorbance at 450 nm was read by an enzyme-labeled instrument. The data were processed by GraphPad Prism 6 software, the antibody concentration was taken as the abscissa, the absorbance at 450 nm was taken as the ordinate, the four-parameter regression calculation was used for curve fitting, and the IC50 was calculated. The results are shown in Figure 65

[0711] Figure 65 It is shown that TSLP / IL-13 BsAb effectively blocks the binding of TSLP to TSLPR, and the IC50 is 7.26 nM; the negative control Anti-KLH hIgG1 has no blocking activity.

[0712] Example 25, Study on the blocking activity of TSLP / IL-13 BsAb to TSLP binding to cell surface TSLP receptor (TSLPR)

[0713] Flow cytometry was used to pre-incubate different concentrations of TSLP / IL-13 BsAb stock solution and TSLP-mFc fusion protein, then the mixed solution was incubated with target cells expressing TSLPR, the binding of TSLP to cell surface TSLPR was detected, and then the half-effective inhibition concentration (IC50) was calculated to evaluate the blocking activity of TSLP / IL-13 BsAb to TSLP binding to cell surface TSLPR.

[0714] BA / F3-TSLPR-IL7Rα cells were cultured in cell culture medium at 37°C, 5% CO2. The cells were collected by centrifugation for 10 min, and the cell density was adjusted to 1 mL containing 1×10 7 The cells were blocked in blocking solution for 30 min on ice. At the same time, antibody diluent and TSLP solution were prepared, 60 μL of antibody diluent was mixed with 60 μL of TSLP solution, and incubated at 37°C for 30 min. The cell density was adjusted to 1 mL containing 5×10 5 ​Cells were centrifuged and the supernatant was discarded. 100 μL of antibody-TSLP mixture was added to each well and the cells were incubated at 4°C for 60 min. After incubation, the cells were washed with 200 μL of flow buffer. The cells were labeled with 50 μL of APC secondary antibody dilution solution per well. After incubation on ice for 20 min, the cells were washed. 50 μL of PI solution was added to each well, and the cells were incubated on ice for 5 min. After washing, 50 μL of PBS was added to resuspend the cells. The mean fluorescence intensity of the cells was read using a flow cytometer, and the results were recorded. The fluorescence value of the PI-negative cell population was analyzed, and the data were processed using GraphPad Prism 6 software. The antibody concentration was used as the horizontal coordinate, and the mean fluorescence intensity of APC was used as the vertical coordinate. The IC50 was calculated using a four-parameter regression analysis. Anti-KLH hIgG1 was used as a negative control. The results are shown in Figure 66

[0715] Figure 66 It was shown that the TSLP / IL-13 BsAb effectively blocked the binding of TSLP to the cell surface TSLPR, with an IC50 of 1.46 nM. The negative control Anti-KLH hIgG1 had no significant blocking activity.

[0716] Although the preferred embodiments of the present application have been shown and described herein, it should be apparent to those skilled in the art that many variations, modifications, and substitutions can be made thereto without departing from the scope of the application. It is to be understood that various alternatives to the embodiments described herein can be employed. The following claims are intended to define the scope of the application and to encompass all methods and structures that fall within the scope of the claims and their equivalents.​

Claims

1. A bispecific antibody or antigen-binding fragment thereof, comprising a first antigen-binding region that binds to IL-13 and a second antigen-binding region that binds to TSLP, wherein the first antigen-binding region comprises a first light chain variable region (VL1) and a first heavy chain variable region (VH1), and the second antigen-binding region comprises a second light chain variable region (VL2) and a second heavy chain variable region (VH2), wherein The VL1 comprises LCDR 1-3 having amino acid sequences as shown in SEQ ID NOs: 21-23, respectively. The VH1 comprises HCDR 1 having an amino acid sequence as shown in SEQ ID NO: 24, HCDR 2 having an amino acid sequence as shown in SEQ ID NO: 25 or 26, and HCDR 3 having an amino acid sequence as shown in SEQ ID NO: 27, The VL2 comprises LCDR 1-3 having amino acid sequences as shown in SEQ ID NOs: 35-37, respectively, and The VH2 comprises HCDRs 1-3 having the amino acid sequences shown in SEQ ID NOs: 30-32, respectively.

2. The bispecific antibody or antigen-binding fragment thereof according to claim 1, wherein The VL1 comprises LCDR 1-3 having amino acid sequences as shown in SEQ ID NOs: 21-23, respectively. The VH1 comprises HCDRs 1-3 having amino acid sequences shown in SEQ ID NOs: 24, 25 and 27, respectively. The VL2 comprises LCDR 1-3 having amino acid sequences as shown in SEQ ID NOs: 35-37, respectively, and The VH2 comprises HCDRs 1-3 having the amino acid sequences shown in SEQ ID NOs: 30-32, respectively.

3. The bispecific antibody or antigen-binding fragment thereof according to claim 1, wherein The VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to any one of SEQ ID NOs: 1, 3, 46, 53, The VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to any one of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 52, The VL2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 33 or 51, and The VH2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 28 or 50.

4. The bispecific antibody or antigen-binding fragment thereof according to claim 3, wherein The VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to any one of SEQ ID NOs: 1, 3, 46, 53, The VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to any one of SEQ ID NOs: 5, 7, 9, 11, 13, 52, The VL2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 33, and The VH2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:

28.

5. The bispecific antibody or antigen-binding fragment thereof according to claim 4, wherein (i) the VL1 comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 53, and the VH1 comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 52; or (ii) the VL1 comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, and the VH1 comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9; or (iii) the VL1 comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, and the VH1 comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 5; or (iv) the VL1 comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, and the VH1 comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 7; or (v) the VL1 comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1, and the VH1 comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11; or (vi) the VL1 comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1, and the VH1 comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 13; or (vii) the VL1 comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:46, and the VH1 comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:5; or (viii) the VL1 comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:46, and the VH1 comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:7; or (ix) the VL1 comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 46, and the VH1 comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9; or (x) the VL1 comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 46, and the VH1 comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11; or (xi) the VL1 comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:3, and the VH1 comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:5; or (xii) the VL1 comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:3, and the VH1 comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:7; or (xiii) the VL1 comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 3, and the VH1 comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 9; or (xiv) the VL1 comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:3, and the VH1 comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:

11.

6. The bispecific antibody or antigen-binding fragment thereof according to claim 5, wherein (i) the VL1 comprises the amino acid sequence shown in SEQ ID NO: 53 and the VH1 comprises the amino acid sequence shown in SEQ ID NO: 52; or (ii) the VL1 comprises the amino acid sequence shown in SEQ ID NO: 1 and the VH1 comprises the amino acid sequence shown in SEQ ID NO: 9; or (iii) the VL1 comprises the amino acid sequence shown in SEQ ID NO: 1 and the VH1 comprises the amino acid sequence shown in SEQ ID NO: 5; or (iv) the VL1 comprises the amino acid sequence shown in SEQ ID NO: 1 and the VH1 comprises the amino acid sequence shown in SEQ ID NO: 7; or (v) the VL1 comprises the amino acid sequence shown in SEQ ID NO: 1 and the VH1 comprises the amino acid sequence shown in SEQ ID NO: 11; or (vi) the VL1 comprises the amino acid sequence shown in SEQ ID NO: 1 and the VH1 comprises the amino acid sequence shown in SEQ ID NO: 13; and The VL2 comprises the amino acid sequence shown in SEQ ID NO: 33, and the VH2 comprises the amino acid sequence shown in SEQ ID NO:

28.

7. The bispecific antibody or antigen-binding fragment thereof according to claim 6, wherein the antibody comprises a first heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 42, a first light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 44, a second heavy chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 38, A second light chain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:

40.

8. The bispecific antibody or antigen-binding fragment thereof according to claim 7, wherein the antibody comprises a first heavy chain having the amino acid sequence shown in SEQ ID NO: 42, a first light chain having the amino acid sequence shown in SEQ ID NO: 44, a second heavy chain having the amino acid sequence shown in SEQ ID NO: 38, and a second light chain having the amino acid sequence shown in SEQ ID NO:

40.

9. An antibody or antigen-binding fragment thereof that specifically binds to IL-13, comprising a light chain variable region (VL) and a heavy chain variable region (VH), wherein The VL comprises an amino acid sequence selected from any one of SEQ ID NOs: 1, 3, 46, and 53, and The VH comprises an amino acid sequence selected from any one of SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, and 52.

10. The antibody or antigen-binding fragment thereof according to claim 9, wherein (i) the VL comprises the amino acid sequence shown in SEQ ID NO: 53, and the VH comprises the amino acid sequence shown in SEQ ID NO: 52; or (ii) the VL comprises the amino acid sequence shown in SEQ ID NO: 1, and the VH comprises the amino acid sequence shown in SEQ ID NO: 9; or (iii) the VL comprises the amino acid sequence shown in SEQ ID NO: 1, and the VH comprises the amino acid sequence shown in SEQ ID NO: 5; or (iv) the VL comprises the amino acid sequence shown in SEQ ID NO: 1, and the VH comprises the amino acid sequence shown in SEQ ID NO: 7; or (v) the VL comprises the amino acid sequence shown in SEQ ID NO: 1, and the VH comprises the amino acid sequence shown in SEQ ID NO: 11; or (vi) the VL comprises the amino acid sequence shown in SEQ ID NO: 1, and the VH comprises the amino acid sequence shown in SEQ ID NO: 13; or (vii) the VL comprises the amino acid sequence shown in SEQ ID NO: 46, and the VH comprises the amino acid sequence shown in SEQ ID NO: 5; or (viii) the VL comprises the amino acid sequence set forth in SEQ ID NO: 46, and the VH comprises the amino acid sequence set forth in SEQ ID NO: 7; or (iv) the VL comprises the amino acid sequence shown in SEQ ID NO: 46, and the VH comprises the amino acid sequence shown in SEQ ID NO: 9; or (x) the VL comprises the amino acid sequence shown in SEQ ID NO: 46, and the VH comprises the amino acid sequence shown in SEQ ID NO: 11; or (xi) the VL comprises the amino acid sequence shown in SEQ ID NO: 3, and the VH comprises the amino acid sequence shown in SEQ ID NO: 5; or (xii) the VL comprises the amino acid sequence shown in SEQ ID NO: 3, and the VH comprises the amino acid sequence shown in SEQ ID NO: 7; or (xiii) the VL comprises the amino acid sequence shown in SEQ ID NO: 3, and the VH comprises the amino acid sequence shown in SEQ ID NO: 9; or (xiv) the VL comprises the amino acid sequence shown in SEQ ID NO: 3, and the VH comprises the amino acid sequence shown in SEQ ID NO:

11.

11. The antibody or antigen-binding fragment thereof according to claim 10, wherein (i) the VL comprises the amino acid sequence shown in SEQ ID NO: 53 and the VH comprises the amino acid sequence shown in SEQ ID NO: 52; or (ii) the VL comprises the amino acid sequence shown in SEQ ID NO: 1 and the VH comprises the amino acid sequence shown in SEQ ID NO: 9; or (iii) the VL comprises the amino acid sequence shown in SEQ ID NO: 1 and the VH comprises the amino acid sequence shown in SEQ ID NO: 5; or (iv) the VL comprises the amino acid sequence shown in SEQ ID NO: 1 and the VH comprises the amino acid sequence shown in SEQ ID NO: 7; or (v) the VL comprises the amino acid sequence shown in SEQ ID NO: 1 and the VH comprises the amino acid sequence shown in SEQ ID NO: 11; or (vi) the VL comprises the amino acid sequence shown in SEQ ID NO: 1 and the VH comprises the amino acid sequence shown in SEQ ID NO:

13.

12. The antibody or antigen-binding fragment thereof according to any one of claims 9 to 11, wherein the antibody is of an isotype selected from the group consisting of IgG, IgA, IgM, IgE and IgD.

13. The antibody or antigen-binding fragment thereof according to any one of claims 9 to 12, wherein the antibody is of a subclass selected from IgG1, IgG2, IgG3 and IgG4.

14. The antibody or antigen-binding fragment thereof according to any one of claims 9 to 13, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, Fv, scFv and ds-scFv.

15. The antibody or antigen-binding fragment thereof according to any one of claims 9 to 14, wherein the antibody is a monoclonal antibody, a bispecific antibody or a multispecific antibody. 16 . The antibody or antigen-binding fragment thereof according to claim 15 , wherein the antibody is a bispecific antibody, further comprising a second antigen-binding region that binds to a second antigen.

17. A nucleic acid comprising a nucleotide sequence encoding the bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 or the antibody or antigen-binding fragment thereof according to any one of claims 9 to 16.

18. The nucleic acid according to claim 17, comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to any one of SEQ ID NOs: 2, 4, 56, a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to any one of SEQ ID NOs: 6, 8, 10, 12, 14, 16, 18, 20, 55, a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 29, and A nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:

34.

19. The nucleic acid according to claim 17, comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 39, a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 41, a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 43, and A nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:

45.

20. A vector comprising the nucleic acid according to any one of claims 17 to 19.

21. A host cell comprising the nucleic acid according to any one of claims 17 to 19 or the vector according to claim 20.

22. A pharmaceutical composition comprising (i) the bispecific antibody or antigen-binding fragment thereof according to any one of claims 1-8, or the antibody or antigen-binding fragment thereof according to any one of claims 9-16; and (ii) a pharmaceutically acceptable carrier or excipient.

23. The pharmaceutical composition of claim 22, further comprising a second therapeutic agent.

24. The pharmaceutical composition of claim 23, wherein the second therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent, and a small molecule drug.

25. A conjugate comprising the bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or the antibody or antigen-binding fragment thereof according to any one of claims 9 to 16, and a chemical moiety conjugated thereto.

26. The conjugate of claim 25, wherein the chemical moiety is selected from the group consisting of a therapeutic agent, a detectable moiety, and an immunostimulatory molecule.

27. A method for treating a type 2 inflammatory response-related disease in a subject, comprising administering to the subject an effective amount of the bispecific antibody or antigen-binding fragment thereof according to any one of claims 1-8, the antibody or antigen-binding fragment thereof according to any one of claims 9-16, the pharmaceutical composition according to any one of claims 22-24, or the conjugate according to claim 25 or 26.

28. The method of claim 27, wherein the disease is an IL-13 and / or TSLP mediated disease (eg, inflammatory diseases and autoimmune diseases).

29. The method of claim 28, wherein the disease is selected from atopic dermatitis, allergic inflammation (e.g., allergic rhinitis, allergic sinusitis, allergic conjunctivitis), asthma, urticaria, chronic obstructive pulmonary disease, eosinophilic esophagitis, rheumatoid arthritis, multiple sclerosis, idiopathic pulmonary fibrosis, eczema (e.g., hand eczema, asthma eczema), acidic granulocytic gastroenteritis, Crohn's disease, systemic sclerosis, ulcerative colitis, chronic rhinosinusitis with nasal polyps.

30. The method of any one of claims 27-29, further comprising administering to the subject a second therapeutic agent.

31. The method of claim 30, wherein the second therapeutic agent is selected from the group consisting of an antibody, a chemotherapeutic agent, and a small molecule drug.

32. A method for inhibiting IgE antibody production in a subject, comprising administering to the subject an effective amount of the bispecific antibody or antigen-binding fragment thereof according to any one of claims 1-8, the antibody or antigen-binding fragment thereof according to any one of claims 9-16, the pharmaceutical composition according to any one of claims 22-24, or the conjugate according to claim 25 or 26.

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