TSLP / il-13 bispecific antibody and use thereof

Abstract

The present invention relates to a bispecific antibody against TSLP / IL-13 and the use thereof, and in particular, to antibodies against TSLP and IL-13 or antigen-binding fragments thereof, nucleic acid molecules encoding same, and preparation methods therefor. The antibodies against TSLP and IL-13 or the antigen-binding fragments thereof of the present invention exhibit a high specificity and affinity for TSLP and IL-13. The present invention further relates to the use of the antibodies or the antigen-binding fragments thereof in the treatment of diseases.

Description

Tslp / il-13 bispecific antibodies and uses thereof

[0001] This application claims the benefit of Chinese application No. 202411377000.3, filed on September 29, 2024, the content of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0002] The present invention is in the field of therapeutic antibodies, and more specifically, the present invention relates to an antibody against TSLP and IL-13; and to the use of said antibody in the treatment of diseases. BACKGROUND

[0003] Thymic stromal lymphopoietin (TSLP), an IL-7-like cytokine, has the effect of enhancing thymocyte proliferation, which can be divided into two types: short subtype (60 amino acids) and long subtype (159 amino acids), regulated by different promoters. The short subtype receptor is unknown; the long subtype TSLP binds to TSLPR with high affinity, and then recruits IL-7Rα to form a TSLP / TSLPR / IL-7Rα trimer. Human TSLP has 43% homology with mouse, and 90% homology with human and monkey. Its functions are: to initiate intracellular type 2 signals, activate JAK1 / 2 pathways, activate cell proliferation, anti-apoptosis, and airway inflammation; to activate DCs residing in tissues, which then migrate out of tissues, activate CD4+ T cells to transform into Th2 cells, and promote the formation of memory T cells. TSLP is mainly expressed in the lung, skin and intestine, and is also expressed in mast cells, smooth muscle cells, fibroblasts, dendritic cells, trophoblast cells and cancer or cancer-related cells. The short subtype TSLP is constitutively expressed and expressed in the skin, intestine, oral epithelium and salivary glands to maintain homeostasis, and is down-regulated under inflammatory conditions. While the long subtype TSLP is inducible and is up-regulated in inflammatory diseases such as atopic dermatitis (AD), allergic asthma (AA), etc. The mRNA expression of TSLPR is more extensive, expressed in heart, skeletal muscle, kidney and liver, DCs, T cells, B cells, mast cells, NKT cells, monocytes, and highly expressed in the bone marrow immune system, liver and gallbladder, etc. In terms of disease manifestations, the concentration and expression level of TSLP in the serum and airway epithelium of asthma patients are much higher than that of healthy individuals. In addition, primary human bronchial epithelial cells and primary lung endothelial cells of severe asthma patients also highly express TSLP.

[0004] ​Interleukin-13 (IL-13) is a cytokine of the chemokine family, produced by Th2 cells, with a molecular weight of about 10 KD, located on chromosome 5, closely linked to the IL-4 gene. Human IL-13 has 58% homology with mouse, and human IL-13 has 94% homology with monkey. The amino acid sequence of IL-13 molecule has 20% to 25% homology with IL-4, and also has many similarities with IL-4 in function. IL-13 receptor is a heterodimer composed of IL-13Rα-1 and IL-4Rα. IL-13 is a key regulator of inflammatory immune response, and plays a key role in asthma and parasitic immunity. IL-13 is mainly produced in B cells, monocytes, epithelial cells and smooth muscle cells, as well as activated CD4+ T cells and a small part of CD8+ T cells, mast cells, basophils, eosinophils and natural killer T cells. A large amount of data reports that IL-13 is expressed in the bronchial tissue, nasal lavage fluid and induced sputum of asthma patients. Under allergen stimulation, IL-13 mRNA and IL-13 protein expression are also detected in bronchoalveolar lavage fluid, confirming that the cytokine is produced in the lung when the respiratory tract is stimulated.

[0005] The current single-target drugs for AD and asthma on the market include (Xolair, Cinqair, Nucala, Fasenra, Dupixent, tralokinumab, Lebrikizumab), and the main research targets include IgE and IL-4, IL-5, IL-13, TSLP, OX40 and their corresponding receptors. At present, there is no bispecific antibody drug targeting TSLP and IL-13 on the market, therefore, it is urgent and necessary to develop an antibody targeting TSLP and IL-13 with higher specificity, lower toxicity and side effects, better clinical efficacy and more convenient administration, which will provide patients with more drug options. SUMMARY

[0006] The present application provides a high-affinity TSLP / IL-13 bispecific antibody, which can specifically recognize or bind to TSLP and IL-13, and inhibit or block the binding of TSLP to TSLP receptor and the binding of IL-13 to IL-13 receptor, thereby being able to down-regulate or block the activity of TSLP and IL-13 (for example, inhibiting or blocking the initiation of STAT-6 induced by TSLP and / or IL-13, inhibiting or blocking the secretion of cytokines induced by TSLP and / or IL-13).

[0007] Therefore, in one aspect, the present application provides a bispecific antibody comprising a first antigen binding domain that specifically binds to TSLP and a second antigen binding domain that specifically binds to IL-13.

[0008] In certain embodiments, the first antigen binding domain comprises a first light chain variable region (VL) and a first heavy chain variable region (VH), which first VL and first VH collectively form a domain capable of specifically binding to TSLP; and the second antigen binding domain comprises a second VL and a second VH, which second VL and second VH collectively form a domain capable of specifically binding to IL-13.

[0009] In certain embodiments, the first VL comprises a LCDR1, a LCDR2, and a LCDR3 contained in a VL as set forth in SEQ ID NO: 40 or 44; and / or, the first VH comprises a HCDR1, a HCDR2, and a HCDR3 contained in a VH as set forth in SEQ ID NO: 39 or 43.

[0010] wherein the CDRs are defined by the Kabat, Chothia, Abm, or IMGT numbering system.

[0011] In certain embodiments, the first VL comprises a LCDR1, a LCDR2, and a LCDR3 contained in a VL as set forth in SEQ ID NO: 40; and / or, the first VH comprises a HCDR1, a HCDR2, and a HCDR3 contained in a VH as set forth in SEQ ID NO: 39.

[0012] wherein the CDRs are defined by the Kabat, Chothia, Abm, or IMGT numbering system.

[0013] In certain embodiments, the first VL comprises a LCDR1, a LCDR2, and a LCDR3 contained in a VL as set forth in SEQ ID NO: 44; and / or, the first VH comprises a HCDR1, a HCDR2, and a HCDR3 contained in a VH as set forth in SEQ ID NO: 43.

[0014] wherein the CDRs are defined by the Kabat, Chothia, Abm, or IMGT numbering system.

[0015] In certain embodiments, the first VL comprises:

[0016] (i) a LCDR1 comprising a sequence as set forth in SEQ ID NO: 12, a LCDR2 comprising a sequence as set forth in SEQ ID NO: 13, and a LCDR3 comprising a sequence as set forth in SEQ ID NO: 14; wherein the CDRs are defined by the Kabat numbering system;

[0017] (ii) LCDR1 comprising a sequence as set forth in SEQ ID NO: 12, LCDR2 comprising a sequence as set forth in SEQ ID NO: 13, and LCDR3 comprising a sequence as set forth in SEQ ID NO: 14; wherein the CDRs are defined by the Chothia numbering system;

[0018] (iii) LCDR1 comprising a sequence as set forth in SEQ ID NO: 12, LCDR2 comprising a sequence as set forth in SEQ ID NO: 13, and LCDR3 comprising a sequence as set forth in SEQ ID NO: 14; wherein the CDRs are defined by the Abm numbering system; or,

[0019] (iv) LCDR1 comprising a sequence as set forth in SEQ ID NO: 22, LCDR2 comprising a sequence as set forth in SEQ ID NO: 23, and LCDR3 comprising a sequence as set forth in SEQ ID NO: 14; wherein the CDRs are defined by the IMGT numbering system.

[0020] In certain embodiments, the first VH comprises:

[0021] (i) HCDR1 comprising a sequence as set forth in SEQ ID NO: 17, HCDR2 comprising a sequence as set forth in SEQ ID NO: 18, and HCDR3 comprising a sequence as set forth in SEQ ID NO: 11; wherein the CDRs are defined by the Kabat numbering system;

[0022] (ii) HCDR1 comprising a sequence as set forth in SEQ ID NO: 9, HCDR2 comprising a sequence as set forth in SEQ ID NO: 10, and HCDR3 comprising a sequence as set forth in SEQ ID NO: 11; wherein the CDRs are defined by the Chothia numbering system;

[0023] (iii) HCDR1 comprising a sequence as set forth in SEQ ID NO: 15, HCDR2 comprising a sequence as set forth in SEQ ID NO: 16, and HCDR3 comprising a sequence as set forth in SEQ ID NO: 11; wherein the CDRs are defined by the Abm numbering system; or,

[0024] (iv) HCDR1 comprising a sequence as set forth in SEQ ID NO: 19, HCDR2 comprising a sequence as set forth in SEQ ID NO: 20, and HCDR3 comprising a sequence as set forth in SEQ ID NO: 21; wherein the CDRs are defined by the IMGT numbering system.

[0025] In certain embodiments of the bispecific antibodies of the application,

[0026] (a) the first VL comprises:

[0027] (a-i) LCDR1 comprising a sequence as shown in SEQ ID NO: 12, LCDR2 comprising a sequence as shown in SEQ ID NO: 13, and LCDR3 comprising a sequence as shown in SEQ ID NO: 14; wherein the CDRs are defined by the Kabat numbering system;

[0028] (a-ii) LCDR1 comprising a sequence as shown in SEQ ID NO: 12, LCDR2 comprising a sequence as shown in SEQ ID NO: 13, and LCDR3 comprising a sequence as shown in SEQ ID NO: 14; wherein the CDRs are defined by the Chothia numbering system;

[0029] (a-iii) LCDR1 comprising a sequence as shown in SEQ ID NO: 12, LCDR2 comprising a sequence as shown in SEQ ID NO: 13, and LCDR3 comprising a sequence as shown in SEQ ID NO: 14; wherein the CDRs are defined by the Abm numbering system; or,

[0030] (a-iv) LCDR1 comprising a sequence as shown in SEQ ID NO: 22, LCDR2 comprising a sequence as shown in SEQ ID NO: 23, and LCDR3 comprising a sequence as shown in SEQ ID NO: 14; wherein the CDRs are defined by the IMGT numbering system;

[0031] and / or,

[0032] (b) the first VH comprises:

[0033] (b-i) HCDR1 comprising a sequence as shown in SEQ ID NO: 17, HCDR2 comprising a sequence as shown in SEQ ID NO: 18, and HCDR3 comprising a sequence as shown in SEQ ID NO: 11; wherein the CDRs are defined by the Kabat numbering system;

[0034] (b-ii) HCDR1 comprising a sequence as shown in SEQ ID NO: 9, HCDR2 comprising a sequence as shown in SEQ ID NO: 10, and HCDR3 comprising a sequence as shown in SEQ ID NO: 11; wherein the CDRs are defined by the Chothia numbering system;

[0035] (b-iii) HCDR1 comprising a sequence as set forth in SEQ ID NO: 15, HCDR2 comprising a sequence as set forth in SEQ ID NO: 16, and HCDR3 comprising a sequence as set forth in SEQ ID NO: 11; wherein the CDRs are defined by the Abm numbering system; or,

[0036] (b-iv) HCDR1 comprising a sequence as set forth in SEQ ID NO: 19, HCDR2 comprising a sequence as set forth in SEQ ID NO: 20, and HCDR3 comprising a sequence as set forth in SEQ ID NO: 21; wherein the CDRs are defined by the IMGT numbering system.

[0037] In certain embodiments, (a) the first VL comprises: LCDR1 comprising a sequence as set forth in SEQ ID NO: 12, LCDR2 comprising a sequence as set forth in SEQ ID NO: 13, and LCDR3 comprising a sequence as set forth in SEQ ID NO: 14; and / or, (b) the first VH comprises: HCDR1 comprising a sequence as set forth in SEQ ID NO: 17, HCDR2 comprising a sequence as set forth in SEQ ID NO: 18, and HCDR3 comprising a sequence as set forth in SEQ ID NO: 11; wherein the CDRs are defined by the Kabat numbering system.

[0038] In certain embodiments, (a) the first VL comprises: LCDR1 comprising a sequence as set forth in SEQ ID NO: 12, LCDR2 comprising a sequence as set forth in SEQ ID NO: 13, and LCDR3 comprising a sequence as set forth in SEQ ID NO: 14; and / or, (b) the first VH comprises: HCDR1 comprising a sequence as set forth in SEQ ID NO: 9, HCDR2 comprising a sequence as set forth in SEQ ID NO: 10, and HCDR3 comprising a sequence as set forth in SEQ ID NO: 11; wherein the CDRs are defined by the Chothia numbering system.

[0039] In certain embodiments, (a) the first VL comprises: LCDR1 comprising a sequence as set forth in SEQ ID NO: 12, LCDR2 comprising a sequence as set forth in SEQ ID NO: 13, and LCDR3 comprising a sequence as set forth in SEQ ID NO: 14; and / or, (b) the first VH comprises: HCDR1 comprising a sequence as set forth in SEQ ID NO: 15, HCDR2 comprising a sequence as set forth in SEQ ID NO: 16, and HCDR3 comprising a sequence as set forth in SEQ ID NO: 11; wherein the CDRs are defined by the Abm numbering system.

[0040] In some embodiments, (a) the first VL comprises: a LCDR1 comprising a sequence as set forth in SEQ ID NO: 22, a LCDR2 comprising a sequence as set forth in SEQ ID NO: 23, and a LCDR3 comprising a sequence as set forth in SEQ ID NO: 14; and / or, (b) the first VH comprises: a HCDR1 comprising a sequence as set forth in SEQ ID NO: 19, a HCDR2 comprising a sequence as set forth in SEQ ID NO: 20, and a HCDR3 comprising a sequence as set forth in SEQ ID NO: 21; wherein the CDRs are defined by the IMGT numbering system.

[0041] In some embodiments, the first VL comprises an amino acid sequence as set forth in SEQ ID NO: 40 or 44, and / or the first VH comprises an amino acid sequence as set forth in SEQ ID NO: 39 or 43.

[0042] In some embodiments, the first VL comprises an amino acid sequence as set forth in SEQ ID NO: 40, and the first VH comprises an amino acid sequence as set forth in SEQ ID NO: 39; or the first VL comprises an amino acid sequence as set forth in SEQ ID NO: 44, and the first VH comprises an amino acid sequence as set forth in SEQ ID NO: 43.

[0043] In some embodiments, the second VL comprises a LCDR1, a LCDR2, and a LCDR3 contained in a VL as set forth in SEQ ID NO: 42; and / or the second VH comprises a HCDR1, a HCDR2, and a HCDR3 contained in a VH as set forth in SEQ ID NO: 41;

[0044] wherein the CDRs are defined by the Kabat, Chothia, Abm, or IMGT numbering system.

[0045] In some embodiments, the second VL comprises:

[0046] (i) a LCDR1 comprising a sequence as set forth in SEQ ID NO: 27, a LCDR2 comprising a sequence as set forth in SEQ ID NO: 28, and a LCDR3 comprising a sequence as set forth in SEQ ID NO: 29; wherein the CDRs are defined by the Kabat numbering system;

[0047] (ii) a LCDR1 comprising a sequence as set forth in SEQ ID NO: 27, a LCDR2 comprising a sequence as set forth in SEQ ID NO: 28, and a LCDR3 comprising a sequence as set forth in SEQ ID NO: 29; wherein the CDRs are defined by the Chothia numbering system;

[0048] (iii) LCDR1 comprising a sequence as set forth in SEQ ID NO: 27, LCDR2 comprising a sequence as set forth in SEQ ID NO: 28, and LCDR3 comprising a sequence as set forth in SEQ ID NO: 29; wherein the CDRs are defined by the Abm numbering system; or,

[0049] (iv) LCDR1 comprising a sequence as set forth in SEQ ID NO: 37, LCDR2 comprising a sequence as set forth in SEQ ID NO: 38, and LCDR3 comprising a sequence as set forth in SEQ ID NO: 29; wherein the CDRs are defined by the IMGT numbering system.

[0050] In certain embodiments, the second VH comprises:

[0051] (i) HCDR1 comprising a sequence as set forth in SEQ ID NO: 32, HCDR2 comprising a sequence as set forth in SEQ ID NO: 33, and HCDR3 comprising a sequence as set forth in SEQ ID NO: 26; wherein the CDRs are defined by the Kabat numbering system;

[0052] (ii) HCDR1 comprising a sequence as set forth in SEQ ID NO: 24, HCDR2 comprising a sequence as set forth in SEQ ID NO: 25, and HCDR3 comprising a sequence as set forth in SEQ ID NO: 26; wherein the CDRs are defined by the Chothia numbering system;

[0053] (iii) HCDR1 comprising a sequence as set forth in SEQ ID NO: 30, HCDR2 comprising a sequence as set forth in SEQ ID NO: 31, and HCDR3 comprising a sequence as set forth in SEQ ID NO: 26; wherein the CDRs are defined by the Abm numbering system; or,

[0054] (iv) HCDR1 comprising a sequence as set forth in SEQ ID NO: 34, HCDR2 comprising a sequence as set forth in SEQ ID NO: 35, and HCDR3 comprising a sequence as set forth in SEQ ID NO: 36; wherein the CDRs are defined by the IMGT numbering system.

[0055] In certain embodiments of the bispecific antibodies of the application,

[0056] (a) the second VL comprises:

[0057] (a-i) LCDR1 comprising a sequence as shown in SEQ ID NO: 27, LCDR2 comprising a sequence as shown in SEQ ID NO: 28, and LCDR3 comprising a sequence as shown in SEQ ID NO: 29; wherein the CDRs are defined by the Kabat numbering system;

[0058] (a-ii) LCDR1 comprising a sequence as shown in SEQ ID NO: 27, LCDR2 comprising a sequence as shown in SEQ ID NO: 28, and LCDR3 comprising a sequence as shown in SEQ ID NO: 29; wherein the CDRs are defined by the Chothia numbering system;

[0059] (a-iii) LCDR1 comprising a sequence as shown in SEQ ID NO: 27, LCDR2 comprising a sequence as shown in SEQ ID NO: 28, and LCDR3 comprising a sequence as shown in SEQ ID NO: 29; wherein the CDRs are defined by the Abm numbering system; or,

[0060] (a-iv) LCDR1 comprising a sequence as shown in SEQ ID NO: 37, LCDR2 comprising a sequence as shown in SEQ ID NO: 38, and LCDR3 comprising a sequence as shown in SEQ ID NO: 29; wherein the CDRs are defined by the IMGT numbering system;

[0061] and / or,

[0062] (b) the second VH comprises:

[0063] (b-i) HCDR1 comprising a sequence as shown in SEQ ID NO: 32, HCDR2 comprising a sequence as shown in SEQ ID NO: 33, and HCDR3 comprising a sequence as shown in SEQ ID NO: 26; wherein the CDRs are defined by the Kabat numbering system;

[0064] (b-ii) HCDR1 comprising a sequence as shown in SEQ ID NO: 24, HCDR2 comprising a sequence as shown in SEQ ID NO: 25, and HCDR3 comprising a sequence as shown in SEQ ID NO: 26; wherein the CDRs are defined by the Chothia numbering system;

[0065] (b-iii) HCDR1 comprising a sequence as shown in SEQ ID NO: 30, HCDR2 comprising a sequence as shown in SEQ ID NO: 31, and HCDR3 comprising a sequence as shown in SEQ ID NO: 26; wherein the CDRs are defined by the Abm numbering system; or,

[0066] (b-iv) HCDR1 comprising a sequence as set forth in SEQ ID NO: 34, HCDR2 comprising a sequence as set forth in SEQ ID NO: 35, and HCDR3 comprising a sequence as set forth in SEQ ID NO: 36; wherein the CDRs are defined by the IMGT numbering system.

[0067] In certain embodiments, (a) the second VL comprises: LCDR1 comprising a sequence as set forth in SEQ ID NO: 27, LCDR2 comprising a sequence as set forth in SEQ ID NO: 28, and LCDR3 comprising a sequence as set forth in SEQ ID NO: 29; and / or, (b) the second VH comprises: HCDR1 comprising a sequence as set forth in SEQ ID NO: 32, HCDR2 comprising a sequence as set forth in SEQ ID NO: 33, and HCDR3 comprising a sequence as set forth in SEQ ID NO: 26; wherein the CDRs are defined by the Kabat numbering system.

[0068] In certain embodiments, (a) the second VL comprises: LCDR1 comprising a sequence as set forth in SEQ ID NO: 27, LCDR2 comprising a sequence as set forth in SEQ ID NO: 28, and LCDR3 comprising a sequence as set forth in SEQ ID NO: 29; and / or, (b) the second VH comprises: HCDR1 comprising a sequence as set forth in SEQ ID NO: 24, HCDR2 comprising a sequence as set forth in SEQ ID NO: 25, and HCDR3 comprising a sequence as set forth in SEQ ID NO: 26; wherein the CDRs are defined by the Chothia numbering system.

[0069] In certain embodiments, (a) the second VL comprises: LCDR1 comprising a sequence as set forth in SEQ ID NO: 27, LCDR2 comprising a sequence as set forth in SEQ ID NO: 28, and LCDR3 comprising a sequence as set forth in SEQ ID NO: 29; and / or, (b) the second VH comprises: HCDR1 comprising a sequence as set forth in SEQ ID NO: 30, HCDR2 comprising a sequence as set forth in SEQ ID NO: 31, and HCDR3 comprising a sequence as set forth in SEQ ID NO: 26; wherein the CDRs are defined by the Abm numbering system.

[0070] In certain embodiments, (a) the second VL comprises: a LCDR1 comprising a sequence as set forth in SEQ ID NO: 37, a LCDR2 comprising a sequence as set forth in SEQ ID NO: 38, and a LCDR3 comprising a sequence as set forth in SEQ ID NO: 29; and / or, (b) the second VH comprises: a HCDR1 comprising a sequence as set forth in SEQ ID NO: 34, a HCDR2 comprising a sequence as set forth in SEQ ID NO: 35, and a HCDR3 comprising a sequence as set forth in SEQ ID NO: 36; wherein the CDRs are defined by the IMGT numbering system.

[0071] In certain embodiments, the second VL comprises an amino acid sequence as set forth in SEQ ID NO: 42, and / or the second VH comprises an amino acid sequence as set forth in SEQ ID NO: 41.

[0072] In certain embodiments of the bispecific antibodies of the application:

[0073] (i) the first VL comprises a LCDR1, a LCDR2, and a LCDR3 contained within a VL as set forth in SEQ ID NO: 40 or 44, the first VH comprises a HCDR1, a HCDR2, and a HCDR3 contained within a VH as set forth in SEQ ID NO: 39 or 43; and

[0074] (ii) the second VL comprises a LCDR1, a LCDR2, and a LCDR3 contained within a VL as set forth in SEQ ID NO: 42, the second VH comprises a HCDR1, a HCDR2, and a HCDR3 contained within a VH as set forth in SEQ ID NO: 41,

[0075] wherein the CDRs are defined by the Kabat, Chothia, Abm, or IMGT numbering system.

[0076] In certain embodiments of the bispecific antibodies of the application:

[0077] (i) the first VL comprises a LCDR1, a LCDR2, and a LCDR3 contained within a VL as set forth in SEQ ID NO: 40, the first VH comprises a HCDR1, a HCDR2, and a HCDR3 contained within a VH as set forth in SEQ ID NO: 39; and

[0078] (ii) the second VL comprises a LCDR1, a LCDR2, and a LCDR3 contained within a VL as set forth in SEQ ID NO: 42, the second VH comprises a HCDR1, a HCDR2, and a HCDR3 contained within a VH as set forth in SEQ ID NO: 41,

[0079] wherein the CDRs are defined by the Kabat, Chothia, Abm, or IMGT numbering system.

[0080] In certain embodiments of the bispecific antibodies of the application:

[0081] (i) the first VL comprises a LCDR1, a LCDR2, and a LCDR3 contained within a VL as set forth in SEQ ID NO: 44, the first VH comprises a HCDR1, a HCDR2, and a HCDR3 contained within a VH as set forth in SEQ ID NO: 43; and

[0082] (ii) the second VL comprises a LCDR1, a LCDR2, and a LCDR3 contained within a VL as set forth in SEQ ID NO: 42, the second VH comprises a HCDR1, a HCDR2, and a HCDR3 contained within a VH as set forth in SEQ ID NO: 41,

[0083] wherein the CDRs are defined by the Kabat, Chothia, Abm, or IMGT numbering system.

[0084] In certain embodiments of the bispecific antibodies of the application:

[0085] (a) the first VL comprises:

[0086] (a-i) a LCDR1 comprising a sequence as set forth in SEQ ID NO: 12, a LCDR2 comprising a sequence as set forth in SEQ ID NO: 13, and a LCDR3 comprising a sequence as set forth in SEQ ID NO: 14; wherein the CDRs are defined by the Kabat numbering system;

[0087] (a-ii) a LCDR1 comprising a sequence as set forth in SEQ ID NO: 12, a LCDR2 comprising a sequence as set forth in SEQ ID NO: 13, and a LCDR3 comprising a sequence as set forth in SEQ ID NO: 14; wherein the CDRs are defined by the Chothia numbering system;

[0088] (a-iii) a LCDR1 comprising a sequence as set forth in SEQ ID NO: 12, a LCDR2 comprising a sequence as set forth in SEQ ID NO: 13, and a LCDR3 comprising a sequence as set forth in SEQ ID NO: 14; wherein the CDRs are defined by the Abm numbering system; or,

[0089] (a-iv) LCDR1 comprising a sequence as set forth in SEQ ID NO: 22, LCDR2 comprising a sequence as set forth in SEQ ID NO: 23, and LCDR3 comprising a sequence as set forth in SEQ ID NO: 14; wherein the CDRs are defined by the IMGT numbering system;

[0090] and,

[0091] (b) the first VH comprises:

[0092] (b-i) HCDR1 comprising a sequence as set forth in SEQ ID NO: 17, HCDR2 comprising a sequence as set forth in SEQ ID NO: 18, and HCDR3 comprising a sequence as set forth in SEQ ID NO: 11; wherein the CDRs are defined by the Kabat numbering system;

[0093] (b-ii) HCDR1 comprising a sequence as set forth in SEQ ID NO: 9, HCDR2 comprising a sequence as set forth in SEQ ID NO: 10, and HCDR3 comprising a sequence as set forth in SEQ ID NO: 11; wherein the CDRs are defined by the Chothia numbering system;

[0094] (b-iii) HCDR1 comprising a sequence as set forth in SEQ ID NO: 15, HCDR2 comprising a sequence as set forth in SEQ ID NO: 16, and HCDR3 comprising a sequence as set forth in SEQ ID NO: 11; wherein the CDRs are defined by the Abm numbering system; or,

[0095] (b-iv) HCDR1 comprising a sequence as set forth in SEQ ID NO: 19, HCDR2 comprising a sequence as set forth in SEQ ID NO: 20, and HCDR3 comprising a sequence as set forth in SEQ ID NO: 21; wherein the CDRs are defined by the IMGT numbering system;

[0096] and

[0097] (c) the second VL comprises:

[0098] (c-i) LCDR1 comprising a sequence as set forth in SEQ ID NO: 27, LCDR2 comprising a sequence as set forth in SEQ ID NO: 28, and LCDR3 comprising a sequence as set forth in SEQ ID NO: 29; wherein the CDRs are defined by the Kabat numbering system;

[0099] (c-ii) LCDR1 comprising a sequence as set forth in SEQ ID NO: 27, LCDR2 comprising a sequence as set forth in SEQ ID NO: 28, and LCDR3 comprising a sequence as set forth in SEQ ID NO: 29; wherein the CDRs are defined by the Chothia numbering system;

[0100] (c-iii) LCDR1 comprising a sequence as set forth in SEQ ID NO: 27, LCDR2 comprising a sequence as set forth in SEQ ID NO: 28, and LCDR3 comprising a sequence as set forth in SEQ ID NO: 29; wherein the CDRs are defined by the Abm numbering system; or,

[0101] (c-iv) LCDR1 comprising a sequence as set forth in SEQ ID NO: 37, LCDR2 comprising a sequence as set forth in SEQ ID NO: 38, and LCDR3 comprising a sequence as set forth in SEQ ID NO: 29; wherein the CDRs are defined by the IMGT numbering system;

[0102] and,

[0103] (d) the second VH comprises:

[0104] (d-i) HCDR1 comprising a sequence as set forth in SEQ ID NO: 32, HCDR2 comprising a sequence as set forth in SEQ ID NO: 33, and HCDR3 comprising a sequence as set forth in SEQ ID NO: 26; wherein the CDRs are defined by the Kabat numbering system;

[0105] (d-ii) HCDR1 comprising a sequence as set forth in SEQ ID NO: 24, HCDR2 comprising a sequence as set forth in SEQ ID NO: 25, and HCDR3 comprising a sequence as set forth in SEQ ID NO: 26; wherein the CDRs are defined by the Chothia numbering system;

[0106] (d-iii) HCDR1 comprising a sequence as set forth in SEQ ID NO: 30, HCDR2 comprising a sequence as set forth in SEQ ID NO: 31, and HCDR3 comprising a sequence as set forth in SEQ ID NO: 26; wherein the CDRs are defined by the Abm numbering system; or,

[0107] (d-iv) HCDR1 comprising a sequence as set forth in SEQ ID NO: 34, HCDR2 comprising a sequence as set forth in SEQ ID NO: 35, and HCDR3 comprising a sequence as set forth in SEQ ID NO: 36; wherein the CDRs are defined by the IMGT numbering system.

[0108] In certain embodiments, the first VL comprises: an LCDR1 comprising a sequence as set forth in SEQ ID NO: 12, an LCDR2 comprising a sequence as set forth in SEQ ID NO: 13, and an LCDR3 comprising a sequence as set forth in SEQ ID NO: 14; and, the first VH comprises: an HCDR1 comprising a sequence as set forth in SEQ ID NO: 17, an HCDR2 comprising a sequence as set forth in SEQ ID NO: 18, and an HCDR3 comprising a sequence as set forth in SEQ ID NO: 11; and, the second VL comprises: an LCDR1 comprising a sequence as set forth in SEQ ID NO: 27, an LCDR2 comprising a sequence as set forth in SEQ ID NO: 28, and an LCDR3 comprising a sequence as set forth in SEQ ID NO: 29; and, the second VH comprises: an HCDR1 comprising a sequence as set forth in SEQ ID NO: 32, an HCDR2 comprising a sequence as set forth in SEQ ID NO: 33, and an HCDR3 comprising a sequence as set forth in SEQ ID NO: 26; wherein the CDRs are defined by the Chothia numbering system.

[0109] In certain embodiments, the first VL comprises: an LCDR1 comprising a sequence as set forth in SEQ ID NO: 12, an LCDR2 comprising a sequence as set forth in SEQ ID NO: 13, and an LCDR3 comprising a sequence as set forth in SEQ ID NO: 14; and, the first VH comprises: an HCDR1 comprising a sequence as set forth in SEQ ID NO: 9, an HCDR2 comprising a sequence as set forth in SEQ ID NO: 10, and an HCDR3 comprising a sequence as set forth in SEQ ID NO: 11; and, the second VL comprises: an LCDR1 comprising a sequence as set forth in SEQ ID NO: 27, an LCDR2 comprising a sequence as set forth in SEQ ID NO: 28, and an LCDR3 comprising a sequence as set forth in SEQ ID NO: 29; and, the second VH comprises: an HCDR1 comprising a sequence as set forth in SEQ ID NO: 24, an HCDR2 comprising a sequence as set forth in SEQ ID NO: 25, and an HCDR3 comprising a sequence as set forth in SEQ ID NO: 26; wherein the CDRs are defined by the Chothia numbering system.

[0110] In certain embodiments, the first VL comprises: an LCDR1 comprising a sequence as set forth in SEQ ID NO: 12, an LCDR2 comprising a sequence as set forth in SEQ ID NO: 13, and an LCDR3 comprising a sequence as set forth in SEQ ID NO: 14; and, the first VH comprises: an HCDR1 comprising a sequence as set forth in SEQ ID NO: 15, an HCDR2 comprising a sequence as set forth in SEQ ID NO: 16, and an HCDR3 comprising a sequence as set forth in SEQ ID NO: 11; and, the second VL comprises: an LCDR1 comprising a sequence as set forth in SEQ ID NO: 27, an LCDR2 comprising a sequence as set forth in SEQ ID NO: 28, and an LCDR3 comprising a sequence as set forth in SEQ ID NO: 29; and, the second VH comprises: an HCDR1 comprising a sequence as set forth in SEQ ID NO: 30, an HCDR2 comprising a sequence as set forth in SEQ ID NO: 31, and an HCDR3 comprising a sequence as set forth in SEQ ID NO: 26; wherein the CDRs are defined by the Abm numbering system.

[0111] In certain embodiments, the first VL comprises: an LCDR1 comprising a sequence as set forth in SEQ ID NO: 22, an LCDR2 comprising a sequence as set forth in SEQ ID NO: 23, and an LCDR3 comprising a sequence as set forth in SEQ ID NO: 14; and, the first VH comprises: an HCDR1 comprising a sequence as set forth in SEQ ID NO: 19, an HCDR2 comprising a sequence as set forth in SEQ ID NO: 20, and an HCDR3 comprising a sequence as set forth in SEQ ID NO: 21; and, the second VL comprises: an LCDR1 comprising a sequence as set forth in SEQ ID NO: 37, an LCDR2 comprising a sequence as set forth in SEQ ID NO: 38, and an LCDR3 comprising a sequence as set forth in SEQ ID NO: 29; and, the second VH comprises: an HCDR1 comprising a sequence as set forth in SEQ ID NO: 34, an HCDR2 comprising a sequence as set forth in SEQ ID NO: 35, and an HCDR3 comprising a sequence as set forth in SEQ ID NO: 36; wherein the CDRs are defined by the IMGT numbering system.

[0112] In certain embodiments of the bispecific antibodies of the application:

[0113] (i) the first VL comprises an amino acid sequence as set forth in SEQ ID NO: 40 or 44, and / or the first VH comprises an amino acid sequence as set forth in SEQ ID NO: 39 or 43; and

[0114] (ii) the second VL comprises an amino acid sequence as shown in SEQ ID NO: 42 and / or the second VH comprises an amino acid sequence as shown in SEQ ID NO: 41.

[0115] In certain embodiments of the bispecific antibody of the application:

[0116] (i) the first VL comprises an amino acid sequence as shown in SEQ ID NO: 40 and / or the first VH comprises an amino acid sequence as shown in SEQ ID NO: 39; or the first VL comprises an amino acid sequence as shown in SEQ ID NO: 44 and / or the first VH comprises an amino acid sequence as shown in SEQ ID NO: 43; and

[0117] (ii) the second VL comprises an amino acid sequence as shown in SEQ ID NO: 42 and / or the second VH comprises an amino acid sequence as shown in SEQ ID NO: 41.

[0118] In certain embodiments, the first and the second antigen binding domain are each independently selected from a scFv, a Fab.

[0119] Fab-Fab

[0120] In certain embodiments, the first and the second antigen binding domain are a Fab, and the Fab of the first or the second antigen binding domain comprises a domain exchange in the format of a CrossMab.

[0121] Unless explicitly stated otherwise, a Fab domain can be arranged according to its native orientation or comprises a domain displacement or exchange (e.g. a domain exchange in the format of a Crossmab) that facilitates correct VH and VL pairing.

[0122] In certain embodiments, the domain exchange in the format of a CrossMab is selected from:

[0123] (i) CrossMab Fab : in a Fab, the antibody light chain CL-VL is exchanged with the antibody heavy chain CH1-VH;

[0124] (ii) CrossMab VH-VL : in a Fab, the VL and VH of the antibody are exchanged;

[0125] (iii) CrossMab CH1-CL : in a Fab, the CH and CL of the antibody are exchanged.

[0126] CrossMab is a method to prevent light chain mispairing, see US patent US9266967B2, which is incorporated by reference in its entirety. This method mainly takes advantage of the principle that the same parts of an antibody repel each other, i.e., VH and VH repel each other, CH1 and CH1 repel each other, CL and CL repel each other, and VL and VL repel each other. The purpose of preventing light chain mispairing is achieved by regionally swapping the heavy and light chains of one Fab in a bispecific antibody (e.g., swapping the light chain CL-VL with the heavy chain CH1-VH, swapping the VL and VH, or swapping the CH and CL).

[0127] In certain embodiments, the bispecific antibody further comprises an Fc domain, the Fc domain comprising a first and a second Fc domain monomer, and the first and second Fc domain monomers each independently comprise one or more modifications of amino acids that are capable of promoting heterodimerization of the first and second Fc domain monomers.

[0128] In certain embodiments, the Fc domain comprises a first Fc domain monomer comprising an amino acid modification capable of forming a knob structure and a second Fc domain monomer comprising an amino acid modification capable of forming a hole structure, wherein the hole structure is capable of pairing with the knob structure to form a heterodimeric Fc domain.

[0129] As will be readily understood by one skilled in the art, the hole structure is capable of pairing with the knob structure to form a "knob in hole structure" that can be used to reduce heavy chain mispairing in a bispecific antibody. The "knob" and "hole" structures can be introduced by mutating the corresponding positions in the CH3 domains of the two Fc domain monomers to form a specific interaction interface between the two Fc domain monomers (see Ridgway et al., Protein Eng., 9:617-621 (1996); WO 2006 / 028936; which are incorporated by reference in their entireties). Amino acid substitutions that can be used to form the "knob" structure include, but are not limited to, S354C and T366W, wherein the numbering is according to the Eu numbering scheme. Amino acid substitutions that can be used to form the "hole" structure include, but are not limited to, Y349C, T366S, L368A, and Y407V, wherein the numbering is according to the Eu numbering scheme. Due to the mutual attraction between the "knob" and "hole" structures and the mutual repulsion between "knob" and "knob" structures, the knob in hole structure is effective in reducing heavy chain mispairing in a bispecific antibody.

[0130] In certain embodiments, the Fc domain monomer is derived from an Fc domain of a human immunoglobulin and includes modifications that are capable of forming a knob structure or a hole structure. In certain embodiments, the human immunoglobulin is IgGl, IgG2, IgG3, or IgG4. In certain embodiments, the Fc domain monomer (e.g., the first Fc domain monomer) includes one or more modifications that form a knob structure, wherein the one or more modifications include S354C and / or T366W amino acid substitutions, wherein numbering is according to the Eu numbering scheme. In certain embodiments, the Fc domain monomer (e.g., the first Fc domain monomer) includes S354C and T366W amino acid substitutions, wherein numbering is according to the Eu numbering scheme. In certain embodiments, the Fc domain monomer (e.g., the second Fc domain monomer) includes one or more modifications that form a hole structure, wherein the one or more modifications include Y349C, T366S, L368A, and / or Y407V amino acid substitutions, wherein numbering is according to the Eu numbering scheme. In certain embodiments, the Fc domain monomer (e.g., the second Fc domain monomer) includes Y349C, T366S, L368A, and Y407V amino acid substitutions, wherein numbering is according to the Eu numbering scheme.

[0131] In certain embodiments, the first Fc domain monomer and / or the second Fc domain monomer is derived from a human immunoglobulin (e.g., IgGl, IgG2, IgG3, or IgG4).

[0132] In certain embodiments, the CH2 domain (residues 231-340 of human IgGl, numbered according to the Eu numbering system), the CH3 domain (residues 341-447 of human IgGl, numbered according to the Eu numbering system), and / or the hinge region (residues 216-230, numbered according to the Eu numbering system) of the first Fc domain monomer and / or the second Fc domain monomer is introduced into one, two, or more mutations (e.g., amino acid substitutions) to alter one or more functional properties of the antibody, e.g., serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity.

[0133] In certain embodiments, the CH2 domain, the CH3 domain, and / or the hinge region of the first Fc domain monomer and / or the second Fc domain monomer is introduced into one, two, or more mutations (e.g., amino acid substitutions) to reduce or ablate effector function of the Fc region.

[0134] In certain embodiments, the first and / or second Fc domain monomer has altered (e.g., enhanced or reduced or ablated) effector function (e.g., ADCC, CDC, ADCP activity) and / or prolonged half-life (e.g., enhanced FcRn binding activity) compared to the wild-type Fc domain monomer from which it is derived.

[0135] In certain embodiments, the first and / or second Fc domain monomer is derived from human immunoglobulin IgGl, and the first and / or second Fc domain monomer comprises the substitution mutation N434A. In certain embodiments, the mutated amino acid position is defined by the EU numbering system.

[0136] In certain embodiments, the first Fc domain monomer comprises the amino acid sequence of SEQ ID NO: 45, and the second Fc domain monomer comprises the amino acid sequence of SEQ ID NO: 46.

[0137] In certain embodiments, the first Fc domain monomer (e.g., the first Fc domain monomer comprising the amino acid sequence of SEQ ID NO: 45) lacks a C-terminal lysine, and / or the second Fc domain monomer (e.g., the second Fc domain monomer comprising the amino acid sequence of SEQ ID NO: 46) lacks a C-terminal lysine.

[0138] In certain embodiments, the first and second antigen binding domains are each linked to one of the first and second Fc domain monomers.

[0139] In certain embodiments, the first antigen binding domain is linked to the first Fc domain monomer, and the second antigen binding domain is linked to the second Fc domain monomer; or the first antigen binding domain is linked to the second Fc domain monomer, and the second antigen binding domain is linked to the first Fc domain monomer.

[0140] Fab (CrossMab)-Fab

[0141] In certain embodiments, the first and second antigen binding domains are Fabs, and the Fab of the first antigen binding domain comprises a domain swap in the format of a CrossMab.

[0142] In certain embodiments, the Fab of the first antigen binding domain comprises a domain swap of VH and VL in the format of a CrossMab VH-VL In certain embodiments, the Fab of the first antigen binding domain comprises a domain swap of VH and VL in the format of a CrossMab

[0143] In certain embodiments, the Fab of the first antigen binding domain comprises a CrossMab CH1-CL domain swapping of CH1 and CL in the format.

[0144] In certain embodiments, the bispecific antibody comprises a peptide chain A, a peptide chain B, a peptide chain C, and a peptide chain D; wherein the peptide chain A comprises the first VL and a heavy chain CH1 region, the peptide chain B comprises the first VH, a light chain constant region, a hinge region, and the first Fc domain monomer (or the second Fc domain monomer), the peptide chain C comprises the second VH, a heavy chain CH1 region, a hinge region, the second Fc domain monomer (or the first Fc domain monomer), and the peptide chain D comprises the second VL and a light chain constant region.

[0145] In certain embodiments, the peptide chain A comprises, from N-terminus to C- terminus, the first VL and a heavy chain CH1 region, the peptide chain B comprises, from N- terminus to C-terminus, the first VH, a light chain constant region, a hinge region, and the first Fc domain monomer (or the second Fc domain monomer), the peptide chain C comprises, from N-terminus to C-terminus, the second VH, a heavy chain CH1 region, a hinge region, the second Fc domain monomer (or the first Fc domain monomer), and / or, the peptide chain D comprises, from N-terminus to C-terminus, the second VL and a light chain constant region.

[0146] In certain embodiments, each adjacent domains of the peptide chain A are optionally connected or not connected by a linker, each adjacent domains of the peptide chain B are optionally connected or not connected by a linker, each adjacent domains of the peptide chain C are optionally connected or not connected by a linker, and / or, each adjacent domains of the peptide chain D are optionally connected or not connected by a linker.

[0147] In certain embodiments, the linkers are each independently the same or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers). In certain embodiments, the peptide linkers are each independently selected from a peptide linker comprising one or more glycine (G) and / or serine (S), for example, a peptide linker having the structure of (GGGGS)n n (SEQ ID NO: 64), wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, the peptide linkers are each independently comprises an amino acid sequence as set forth in SEQ ID NO: 48, 50, or 51.

[0148] In certain embodiments, the first VL comprised by the peptide chain A is connected to the heavy chain CH1 region by a peptide linker comprising one or more serine (S). In certain embodiments, the first VL comprised by the peptide chain A is connected to the heavy chain CH1 region by a peptide linker comprising two serine (S) residues.

[0149] In certain embodiments, the N-terminal glutamic acid or glutamine of the first VH is cyclized to form pyroglutamic acid or a pyroglutamate, the N-terminal glutamic acid or glutamine of the first VL is cyclized to form pyroglutamic acid or a pyroglutamate, the N-terminal glutamic acid or glutamine of the second VH is cyclized to form pyroglutamic acid or a pyroglutamate, and / or, the N-terminal glutamic acid or glutamine of the second VL is cyclized to form pyroglutamic acid or a pyroglutamate.

[0150] In certain embodiments of the bispecific antibody of the application:

[0151] (i) the N-terminal glutamine of the first VH comprising an amino acid sequence as set forth in SEQ ID NO: 39 or 43 is cyclized to form pyroglutamic acid or a pyroglutamate;

[0152] (ii) the N-terminal glutamic acid of the first VL comprising an amino acid sequence as set forth in SEQ ID NO: 40 or 44 is cyclized to form pyroglutamic acid or a pyroglutamate; and / or,

[0153] (iii) the N-terminal glutamic acid of the second VH comprising an amino acid sequence as set forth in SEQ ID NO: 41 is cyclized to form pyroglutamic acid or a pyroglutamate.

[0154] As known to the person skilled in the art, pyroglutamic acid is the conjugate acid of pyroglutamate and is in equilibrium with pyroglutamate in solution.

[0155] In certain embodiments, the light chain constant region comprises an amino acid sequence as set forth in SEQ ID NO: 53 or 63, the heavy chain CH1 region comprises an amino acid sequence as set forth in SEQ ID NO: 52, and / or, the hinge region comprises an amino acid sequence as set forth in SEQ ID NO: 49.

[0156] In certain embodiments, the peptide chain A comprises an amino acid sequence as set forth in SEQ ID NO: 7, the peptide chain B comprises an amino acid sequence as set forth in SEQ ID NO: 6, the peptide chain C comprises an amino acid sequence as set forth in SEQ ID NO: 5, and / or, the peptide chain D comprises an amino acid sequence as set forth in SEQ ID NO: 4.

[0157] In certain embodiments, the bispecific antibody possesses one or more of the features selected from the group consisting of:

[0158] (1) the N-terminal glutamic acid or glutamine of the peptide chain A undergoes cyclization to form pyroglutamic acid or pyroglutamate (e.g., the N-terminal glutamic acid of the peptide chain A comprising the amino acid sequence set forth in SEQ ID NO: 7 undergoes cyclization to form pyroglutamic acid or pyroglutamate);

[0159] (2) the N-terminal glutamic acid or glutamine of the peptide chain B undergoes cyclization to form pyroglutamic acid or pyroglutamate; and / or, the C-terminal lysine of the peptide chain B is lacking (e.g., the N-terminal glutamine of the peptide chain B comprising the amino acid sequence set forth in SEQ ID NO: 6 undergoes cyclization to form pyroglutamic acid or pyroglutamate and / or the C-terminal lysine thereof is lacking);

[0160] (3) the N-terminal glutamic acid or glutamine of the peptide chain C undergoes cyclization to form pyroglutamic acid or pyroglutamate; and / or, the C-terminal lysine of the peptide chain C is lacking (e.g., the N-terminal glutamic acid of the peptide chain C comprising the amino acid sequence set forth in SEQ ID NO: 5 undergoes cyclization to form pyroglutamic acid or pyroglutamate and / or the C-terminal lysine thereof is lacking);

[0161] (4) the N-terminal glutamic acid or glutamine of the peptide chain D undergoes cyclization to form pyroglutamic acid or pyroglutamate.

[0162] In certain embodiments, the bispecific antibody comprises:

[0163] (a) a peptide chain A comprising the amino acid sequence set forth in SEQ ID NO: 7, a peptide chain B comprising the amino acid sequence set forth in SEQ ID NO: 6, a peptide chain C comprising the amino acid sequence set forth in any one of SEQ ID NOs: 54-56, and / or, a peptide chain D comprising the amino acid sequence set forth in SEQ ID NO: 4;

[0164] (b) a peptide chain A comprising the amino acid sequence set forth in SEQ ID NO: 7, a peptide chain B comprising the amino acid sequence set forth in any one of SEQ ID NOs: 57-59, a peptide chain C comprising the amino acid sequence set forth in SEQ ID NO: 5, and / or, a peptide chain D comprising the amino acid sequence set forth in SEQ ID NO: 4; or,

[0165] (c) a peptide chain A comprising the amino acid sequence set forth in SEQ ID NO: 7, a peptide chain B comprising the amino acid sequence set forth in any one of SEQ ID NOs: 57-59, a peptide chain C comprising the amino acid sequence set forth in any one of SEQ ID NOs: 54-56, and / or, a peptide chain D comprising the amino acid sequence set forth in SEQ ID NO: 4.

[0166] Fab-Fab (CrossMab)

[0167] In certain embodiments, the first and the second antigen binding domain are Fabs, and the Fab of the second antigen binding domain comprises a domain swap of the CHI and the CL in the format of a CrossMab.

[0168] In certain embodiments, the Fab of the second antigen binding domain comprises a domain swap of the CHI and the CL in the format of a CrossMab VH-VL In certain embodiments, the Fab of the second antigen binding domain comprises a domain swap of the CHI and the CL in the format of a CrossMab

[0169] In certain embodiments, the Fab of the second antigen binding domain comprises a domain swap of the CHI and the CL in the format of a CrossMab CH1-CL In certain embodiments, the Fab of the second antigen binding domain comprises a domain swap of the CHI and the CL in the format of a CrossMab

[0170] In certain embodiments, the bispecific antibody comprises a peptide chain A, a peptide chain B, a peptide chain C, and a peptide chain D; wherein the peptide chain A comprises the first VL and a light chain constant region, the peptide chain B comprises the first VH, a heavy chain CHI region, a hinge region, and the first Fc domain monomer (or the second Fc domain monomer), the peptide chain C comprises the second VH, a light chain constant region, a hinge region, the second Fc domain monomer (or the first Fc domain monomer), and the peptide chain D comprises the second VL and a heavy chain CHI region.

[0171] In certain embodiments, the peptide chain A comprises from N-terminal to C-terminal the first VL and a light chain constant region, the peptide chain B comprises from N-terminal to C-terminal the first VH, a heavy chain CHI region, a hinge region, and the first Fc domain monomer (or the second Fc domain monomer), the peptide chain C comprises from N-terminal to C-terminal the second VH, a light chain constant region, a hinge region, the second Fc domain monomer (or the first Fc domain monomer), and / or the peptide chain D comprises from N-terminal to C-terminal the second VL and a heavy chain CHI region.

[0172] In certain embodiments, each adjacent domains of the peptide chain A are optionally connected by or without a linker, each adjacent domains of the peptide chain B are optionally connected by or without a linker, each adjacent domains of the peptide chain C are optionally connected by or without a linker, and / or each adjacent domains of the peptide chain D are optionally connected by or without a linker.

[0173] In certain embodiments, the linkers are each independently the same or different peptide linkers (e.g., a rigid peptide linker or a flexible peptide linker). In certain embodiments, the peptide linkers are each independently selected from a peptide linker comprising one or more glycine (G) and / or serine (S), for example, having the sequence of (GGGGS) npeptide linker of the structure set forth in SEQ ID NO: 64, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, the peptide linkers each independently comprise an amino acid sequence as set forth in SEQ ID NO: 48, 50, or 51.

[0174] In certain embodiments, the second VL and the heavy chain CH1 region comprised by the peptide chain D are connected by a peptide linker comprising one or more serine (S). In certain embodiments, the second VL and the heavy chain CH1 region comprised by the peptide chain D are connected by a peptide linker comprising two serine (S) residues.

[0175] In certain embodiments, the N-terminal glutamic acid or glutamine of the first VH is cyclized to form pyroglutamic acid or a pyroglutamate, the N-terminal glutamic acid or glutamine of the first VL is cyclized to form pyroglutamic acid or a pyroglutamate, the N-terminal glutamic acid or glutamine of the second VH is cyclized to form pyroglutamic acid or a pyroglutamate, and / or, the N-terminal glutamic acid or glutamine of the second VL is cyclized to form pyroglutamic acid or a pyroglutamate.

[0176] In certain embodiments of the bispecific antibody of the application:

[0177] (i) the N-terminal glutamine of the first VH comprising an amino acid sequence as set forth in SEQ ID NO: 39 or 43 is cyclized to form pyroglutamic acid or a pyroglutamate;

[0178] (ii) the N-terminal glutamic acid of the first VL comprising an amino acid sequence as set forth in SEQ ID NO: 40 or 44 is cyclized to form pyroglutamic acid or a pyroglutamate; and / or,

[0179] (iii) the N-terminal glutamic acid of the second VH comprising an amino acid sequence as set forth in SEQ ID NO: 41 is cyclized to form pyroglutamic acid or a pyroglutamate.

[0180] In certain embodiments, the light chain constant region comprises an amino acid sequence as set forth in SEQ ID NO: 53 or 63, the heavy chain CH1 region comprises an amino acid sequence as set forth in SEQ ID NO: 52, and / or, the hinge region comprises an amino acid sequence as set forth in SEQ ID NO: 49.

[0181] In certain embodiments, the bispecific antibody possesses one or more of the features selected from:

[0182] (1) the N-terminal glutamic acid or glutamine of the peptide chain A is cyclized to form pyroglutamic acid or a pyroglutamate;

[0183] (2) the N-terminal glutamic acid or glutamine of the peptide chain B undergoes cyclization to form pyroglutamic acid or pyroglutamate; and / or, the C-terminal lysine of the peptide chain B is lacking;

[0184] (3) the N-terminal glutamic acid or glutamine of the peptide chain C undergoes cyclization to form pyroglutamic acid or pyroglutamate; and / or, the C-terminal lysine of the peptide chain C is lacking;

[0185] (4) the N-terminal glutamic acid or glutamine of the peptide chain D undergoes cyclization to form pyroglutamic acid or pyroglutamate.

[0186] Fab / scFv

[0187] In certain embodiments, the first antigen binding domain is a scFv and the second antigen binding domain is a Fab; or, the first antigen binding domain is a Fab and the second antigen binding domain is a scFv.

[0188] scFv-Fab

[0189] In certain embodiments, the first antigen binding domain is a scFv and the second antigen binding domain is a Fab.

[0190] In certain embodiments, the bispecific antibody comprises a peptide chain I and a peptide chain II; wherein, the peptide chain I comprises the second VL and a light chain constant region, and the peptide chain II comprises: the first VHand the first VL, the second VHand a heavy chain CH1 region, a hinge region, and an Fc domain monomer.

[0191] In certain embodiments, the peptide chain I comprises, from N-terminal to C-terminal, the second VL and a light chain constant region, and / or, the peptide chain II comprises, from N-terminal to C-terminal: (i) the first VHand the first VL, the second VHand a heavy chain CH1 region, a hinge region, and an Fc domain monomer; or, (ii) the first VL, the first VHand the second VHand a heavy chain CH1 region, a hinge region, and an Fc domain monomer.

[0192] In certain embodiments, each adjacent domain of the peptide chain I is optionally connected by or without a linker, and / or, each adjacent domain of the peptide chain II is optionally connected by or without a linker.

[0193] In certain embodiments, each of the linkers is independently the same or different peptide linker (e.g., a rigid peptide linker or a flexible peptide linker). In certain embodiments, each of the peptide linkers is independently selected from a peptide linker comprising one or more glycine (G) and / or serine (S), e.g., with (GGGGS) npeptide linker of the structure set forth in SEQ ID NO: 64, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, the peptide linkers each independently comprise an amino acid sequence as set forth in SEQ ID NO: 48, 50, or 51.

[0194] In certain embodiments, the first VH and the first VL are capable of forming an intrachain disulfide bond linkage between them.

[0195] In certain embodiments, the first VL comprises a cysteine residue at a position corresponding to position 100 of SEQ ID NO: 40, and / or the first VH comprises a cysteine residue at a position corresponding to position 44 of SEQ ID NO: 39.

[0196] In certain embodiments, the first VL comprises an amino acid sequence as set forth in SEQ ID NO: 44, and the first VH comprises an amino acid sequence as set forth in SEQ ID NO: 43.

[0197] In certain embodiments, the N-terminal glutamic acid or glutamine of the peptide chain I undergoes cyclization to form pyroglutamic acid or pyroglutamate, and / or the N-terminal glutamic acid or glutamine of the peptide chain II undergoes cyclization to form pyroglutamic acid or pyroglutamate.

[0198] In certain embodiments, the first VH is located at the N-terminus of the peptide chain II, and the N-terminal glutamic acid or glutamine of the first VH undergoes cyclization to form pyroglutamic acid or pyroglutamate (e.g., a first VH comprising an amino acid sequence as set forth in SEQ ID NO: 43 or 39 is located at the N-terminus of the peptide chain II, and the N-terminal glutamine thereof undergoes cyclization to form pyroglutamic acid or pyroglutamate); or,

[0199] the first VL is located at the N-terminus of the peptide chain II, and the N-terminal glutamic acid or glutamine of the first VL undergoes cyclization to form pyroglutamic acid or pyroglutamate (e.g., a first VL comprising an amino acid sequence as set forth in SEQ ID NO: 44 or 40 is located at the N-terminus of the peptide chain II, and the N-terminal glutamic acid thereof undergoes cyclization to form pyroglutamic acid or pyroglutamate).

[0200] In certain embodiments, the Fc domain monomer is derived from a human immunoglobulin (e.g., IgGl, IgG2, IgG3, or IgG4).

[0201] In certain embodiments, the CH2 domain (residues 231-340 of human IgGl, numbered according to the Eu numbering system), the CH3 domain (residues 341-447 of human IgGl, numbered according to the Eu numbering system), and / or the hinge region (residues 216-230, numbered according to the Eu numbering system) of the Fc domain monomer is introduced with one, two or more mutations (e.g., amino acid substitutions) to alter one or more functional properties of the antibody, e.g., serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity.

[0202] In certain embodiments, the CH2 domain, the CH3 domain, and / or the hinge region of the Fc domain monomer is introduced with one, two or more mutations (e.g., amino acid substitutions) to reduce or ablate effector function of the Fc region.

[0203] In certain embodiments, the Fc domain monomer is selected from the Fc domain monomer of a wild-type human immunoglobulin (e.g., IgGl) or a variant thereof (e.g., an Fc domain monomer comprising a mutation or chemical modification); wherein the variant has altered (e.g., enhanced or reduced or ablated) effector function (e.g., ADCC, CDC, ADCP activity) and / or prolonged half-life (e.g., enhanced FcRn binding activity) compared to the wild-type Fc domain monomer from which it is derived.

[0204] In certain embodiments, the Fc domain monomer is selected from the Fc domain monomer of a wild-type human immunoglobulin IgGl or a variant thereof; wherein the variant comprises the substitution mutation N434A compared to the Fc domain monomer of wild-type human immunoglobulin IgGl. In certain embodiments, the mutated amino acid position is defined by the EU numbering system.

[0205] In certain embodiments, the Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO: 47.

[0206] In certain embodiments, the Fc domain monomer (e.g., the Fc domain monomer comprising the amino acid sequence set forth in SEQ ID NO: 47) lacks a C-terminal lysine.

[0207] In certain embodiments, the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 53 or 63, the heavy chain CH1 region comprises the amino acid sequence set forth in SEQ ID NO: 52, and / or the hinge region comprises the amino acid sequence set forth in SEQ ID NO: 49.

[0208] In certain embodiments, the peptide chain I comprises an amino acid sequence as set forth in SEQ ID NO: 4, and / or, the peptide chain II comprises an amino acid sequence as set forth in SEQ ID NO: 8.

[0209] In certain embodiments, the N-terminal glutamic acid or glutamine of the peptide chain II undergoes cyclization to form pyroglutamic acid or pyroglutamate; and / or, the C-terminal lysine of the peptide chain II is lacking (e.g., the N-terminal glutamine of the peptide chain II comprising an amino acid sequence as set forth in SEQ ID NO: 8 undergoes cyclization to form pyroglutamic acid or pyroglutamate and / or its C-terminal lysine is lacking); and / or,

[0210] the N-terminal glutamic acid or glutamine of the peptide chain I undergoes cyclization to form pyroglutamic acid or pyroglutamate.

[0211] In certain embodiments, the peptide chain I comprises an amino acid sequence as set forth in SEQ ID NO: 4, and / or, the peptide chain II comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 60-62.

[0212] In certain embodiments, the bispecific antibody comprises two of the peptide chain I and two of the peptide chain II.

[0213] In certain embodiments, the two peptide chains I comprised by the bispecific antibody are the same or different, and / or, the two peptide chains II comprised by the bispecific antibody are the same or different.

[0214] In certain embodiments, the bispecific antibody comprises two of the same peptide chain I and two of the same peptide chain II.

[0215] In certain embodiments, the bispecific antibody comprises:

[0216] (a) a first peptide chain I comprising an amino acid sequence as set forth in SEQ ID NO: 4;

[0217] (b) a second peptide chain I comprising an amino acid sequence as set forth in SEQ ID NO: 4;

[0218] (c) a first peptide chain II comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 8, 60-62; and,

[0219] (d) a second peptide chain II comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 8, 60-62.

[0220] Fab-scFv

[0221] In certain embodiments, the second antigen binding domain is an scFv and the first antigen binding domain is a Fab.

[0222] In certain embodiments, the bispecific antibody comprises a peptide chain I and a peptide chain II; wherein the peptide chain I comprises the first VL and a light chain constant region, and the peptide chain II comprises: the second VH, the second VL, the first VH, a heavy chain CH1 region, a hinge region, and an Fc domain monomer.

[0223] In certain embodiments, the peptide chain I comprises, from N-terminus to C- terminus, the first VL and a light chain constant region, and / or the peptide chain II comprises, from N-terminus to C-terminus: (i) the second VH, the second VL, the first VH, a heavy chain CH1 region, a hinge region, and an Fc domain monomer; or (ii) the second VL, the second VH, the first VH, a heavy chain CH1 region, a hinge region, and an Fc domain monomer.

[0224] In certain embodiments, each adjacent domain of the peptide chain I is optionally connected by or without a linker, and / or each adjacent domain of the peptide chain II is optionally connected by or without a linker.

[0225] In certain embodiments, each of the linkers is independently the same or different peptide linker (e.g., a rigid peptide linker or a flexible peptide linker). In certain embodiments, each of the peptide linkers is independently selected from a peptide linker comprising one or more glycine (G) and / or serine (S), for example, a peptide linker having the structure of (GGGGS)n n (SEQ ID NO: 64), wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In certain embodiments, each of the peptide linkers independently comprises an amino acid sequence as set forth in SEQ ID NO: 48, 50, or 51.

[0226] In certain embodiments, an intrachain disulfide bond linkage between the second VH and the second VL is capable of being formed.

[0227] In certain embodiments, the N-terminal glutamic acid or glutamine of the peptide chain I undergoes cyclization to form pyroglutamic acid or pyroglutamate, and / or the N-terminal glutamic acid or glutamine of the peptide chain II undergoes cyclization to form pyroglutamic acid or pyroglutamate.

[0228] In certain embodiments, the second VH is located at the N-terminus of the peptide chain II, and the N-terminal glutamic acid or glutamine of the second VH undergoes cyclization to form pyroglutamic acid or pyroglutamate (e.g., the second VH comprising an amino acid sequence as set forth in SEQ ID NO: 41 is located at the N-terminus of the peptide chain II, and the N-terminal glutamine thereof undergoes cyclization to form pyroglutamic acid or pyroglutamate); or,

[0229] the second VL is located at the N-terminus of the peptide chain II, and the N-terminal glutamic acid or glutamine of the second VL undergoes cyclization to form pyroglutamic acid or pyroglutamate.

[0230] In certain embodiments, the Fc domain monomer is derived from a human immunoglobulin (e.g., IgGl, IgG2, IgG3, or IgG4).

[0231] In certain embodiments, the CH2 domain (residues 231-340 of human IgGl, numbered according to the Eu numbering system), the CH3 domain (residues 341-447 of human IgGl, numbered according to the Eu numbering system), and / or the hinge region (residues 216-230, numbered according to the Eu numbering system) of the Fc domain monomer is introduced with one, two, or more mutations (e.g., amino acid substitutions) to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity.

[0232] In certain embodiments, the CH2 domain, the CH3 domain, and / or the hinge region of the Fc domain monomer is introduced with one, two, or more mutations (e.g., amino acid substitutions) to reduce or ablate the effector function of the Fc region.

[0233] In certain embodiments, the Fc domain monomer is selected from a Fc domain monomer of a wild-type human immunoglobulin (e.g., IgGl) or a variant thereof (e.g., a Fc domain monomer comprising a mutation or a chemical modification); wherein the variant has altered (e.g., enhanced or reduced or ablated) effector function (e.g., ADCC, CDC, ADCP activity) and / or prolonged half-life (e.g., enhanced FcRn binding activity) as compared to the wild-type Fc domain monomer from which it is derived.

[0234] In certain embodiments, the Fc domain monomer is selected from a Fc domain monomer of a wild-type human immunoglobulin IgGl or a variant thereof; wherein the variant comprises the substitution mutation N434A as compared to the Fc domain monomer of wild-type human immunoglobulin IgGl. In certain embodiments, the mutated amino acid position is defined by the EU numbering system.

[0235] In certain embodiments, the Fc domain monomer comprises an amino acid sequence as set forth in SEQ ID NO: 47.

[0236] In certain embodiments, the C-terminal lysine of the Fc domain monomer (e.g., the Fc domain monomer comprising an amino acid sequence as set forth in SEQ ID NO: 47) is lacking.

[0237] In certain embodiments, the light chain constant region comprises an amino acid sequence as set forth in SEQ ID NO: 53 or 63, the heavy chain CH1 region comprises an amino acid sequence as set forth in SEQ ID NO: 52, and / or, the hinge region comprises an amino acid sequence as set forth in SEQ ID NO: 49.

[0238] In certain embodiments, the N-terminal glutamic acid or glutamine of the peptide chain II is cyclized to form pyroglutamic acid or pyroglutamate; and / or, the C-terminal lysine of the peptide chain II is lacking; and / or,

[0239] the N-terminal glutamic acid or glutamine of the peptide chain I is cyclized to form pyroglutamic acid or pyroglutamate.

[0240] In certain embodiments, the bispecific antibody comprises two of the peptide chain I and two of the peptide chain II.

[0241] In certain embodiments, the two peptide chains I comprised by the bispecific antibody are the same or different, and / or, the two peptide chains II comprised by the bispecific antibody are the same or different.

[0242] In certain embodiments, the bispecific antibody comprises two of the same peptide chain I and two of the same peptide chain II.

[0243] In certain embodiments, the bispecific antibody comprises the first peptide chain I, the second peptide chain I, the first peptide chain II, and the second peptide chain II.

[0244] As will be readily understood by one skilled in the art, cyclization of the N-terminal glutamic acid or glutamine of an antibody peptide chain to form pyroglutamic acid (salt) is a common post-translational modification of antibodies, and pyroglutamic acid is the conjugate acid of pyroglutamate and is in equilibrium with pyroglutamate in solution. Thus, in certain embodiments of the bispecific antibodies of the present application, the N-termini of the different peptide chains comprised by the bispecific antibody can each independently have or lack pyroglutamate modification (e.g., the N-terminal glutamic acid or glutamine of the different peptide chains comprised by the bispecific antibody are each independently converted or not converted to pyroglutamate).

[0245] For example, the N-terminal glutamic acid or glutamine of each of the peptide chain A, the peptide chain B, the peptide chain C, and the peptide chain D comprised by the bispecific antibody is independently converted or not converted to pyroglutamic acid (salt).

[0246] For example, the N-terminal glutamic acid or glutamine of each of the peptide chain I and the peptide chain II (e.g., two peptide chains I and two peptide chains II) comprised by the bispecific antibody is independently converted or not converted to pyroglutamic acid (salt).

[0247] In certain embodiments, the present application provides compositions comprising the bispecific antibodies disclosed herein, wherein each of the bispecific antibodies can independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine, and / or comprise an N-terminal glutamine or glutamic acid, an N-terminal amino acid cyclized to pyroglutamic acid, or an N-terminal amino acid cyclized to pyroglutamic acid salt.

[0248] In certain embodiments, the bispecific antibodies disclosed herein include post-translational modifications thereof (e.g., C-terminal lysine clipping in the heavy chain, conversion of N-terminal glutamine or glutamic acid to pyroglutamic acid or pyroglutamic acid salt in the heavy chain and / or light chain), which can occur upon recombinant expression in a host cell (e.g., a CHO cell) or during purification / storage.

[0249] In certain embodiments, the bispecific antibodies possess one or more features selected from the group consisting of:

[0250] (a) inhibits or blocks the binding of TSLP (e.g., human and / or monkey TSLP) to TSLPR (e.g., inhibits or blocks the binding of TSLP to TSLPR / IL-7Rα);

[0251] (b) inhibits or blocks the binding of IL-13 (e.g., human and / or monkey IL-13) to IL-13 receptor (e.g., inhibits or blocks the binding of IL-13 to IL-13Rα-1 / IL-4Rα);

[0252] (c) downregulates or abrogates the activity of TSLP (e.g., human and / or monkey TSLP);

[0253] (d) downregulates or abrogates the activity of IL-13 (e.g., human and / or monkey IL-13);

[0254] (e) downregulates or blocks the expression of OX40L;

[0255] (f) inhibits or blocks the activation and / or proliferation of mast cells, DCs, NKT cells induced by TSLP (e.g., human and / or monkey TSLP) and / or IL-13 (e.g., human and / or monkey IL-13);

[0256] (g) inhibits or blocks TSLP (e.g., human and / or monkey TSLP) and / or IL-13 (e.g., human and / or monkey IL-13) induced STAT-6 activation;

[0257] (h) inhibits or blocks TSLP (e.g., human and / or monkey TSLP) and / or IL-13 (e.g., human and / or monkey IL-13) induced secretion of cytokines such as CCL26, CCL17, CCL22, IL-4, IL-13 or IL-5.

[0258] Preparation of antibodies

[0259] The bispecific antibodies of the present application can be prepared in a variety of ways known in the art, for example, by genetic engineering recombinant techniques. For example, DNA molecules encoding the peptide chains of the bispecific antibodies of the present application are obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into expression vectors, which are then transfected into host cells. The transfected host cells are then cultured under specific conditions and the bispecific antibodies of the present application are expressed.

[0260] In another aspect, the present application provides an isolated nucleic acid molecule or a set of nucleic acid molecules comprising a nucleotide sequence encoding the bispecific antibodies as described above. According to the codon degeneracy in the art, in certain embodiments, the nucleotide sequence is one that can be substituted according to the codon degeneracy. In certain embodiments, the nucleotide sequence is codon-optimized.

[0261] It is readily understood that the bispecific antibodies of the present application can consist of one or more polypeptide chains, and the isolated nucleic acid molecule or a set of nucleic acid molecules encoding the bispecific antibodies of the present application is not limited in the number of nucleic acid molecule chains.

[0262] In certain embodiments, the bispecific antibodies of the present application comprise a peptide chain I and a peptide chain II as described above, and the isolated nucleic acid molecule or a set of nucleic acid molecules comprises a first nucleotide sequence encoding the peptide chain I and a second nucleotide sequence encoding the peptide chain II of the bispecific antibodies of the present application, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different nucleic acid molecules.

[0263] In certain embodiments, the bispecific antibodies of the present application comprise a peptide chain A, a peptide chain B, a peptide chain C and a peptide chain D as described above, and the isolated nucleic acid molecule or a set of nucleic acid molecules comprises a first nucleotide sequence encoding the peptide chain A, a second nucleotide sequence encoding the peptide chain B, a third nucleotide sequence encoding the peptide chain C and a fourth nucleotide sequence encoding the peptide chain D of the bispecific antibodies of the present application, wherein the first nucleotide sequence, the second nucleotide sequence, the third nucleotide sequence and the fourth nucleotide sequence are present on the same or different nucleic acid molecules from each other.

[0264] In another aspect, the present application provides a vector comprising the isolated nucleic acid molecule or the set of nucleic acid molecules as described above.

[0265] In certain embodiments, the vector is a cloning vector or an expression vector.

[0266] In certain embodiments, the vector of the present application is, for example, a plasmid, a cosmid, a phage, a lentivirus, and the like. In certain embodiments, the vector is capable of expressing the antibody or antigen-binding fragment thereof of the present application in vivo in a subject, for example, a mammal, for example, a human.

[0267] It is readily understood that the isolated nucleic acid molecule or the set of nucleic acid molecules as described above can be present in the vector in any form. For example, when the isolated nucleic acid molecule or the set of nucleic acid molecules comprises a plurality of nucleotide sequences encoding different peptide chains, the plurality of nucleotide sequences can be located in the same vector or in different vectors. The orientation, relative position, and linkage of the plurality of nucleotide coding sequences on the vector are not limited.

[0268] In certain embodiments, the bispecific antibody of the present application comprises the peptide chain I and the peptide chain II as described above, and the vector comprises a first nucleotide sequence encoding the peptide chain I and a second nucleotide sequence encoding the peptide chain II of the bispecific antibody of the present application, wherein the first nucleotide sequence and the second nucleotide sequence are present on the same or different vector molecules. When the first nucleotide sequence and the second nucleotide sequence are present on different vector molecules, the vector of the present application comprises a first vector comprising the first nucleotide sequence and a second vector comprising the second nucleotide sequence.

[0269] In certain embodiments, the bispecific antibody of the present application comprises the peptide chain A, the peptide chain B, the peptide chain C, and the peptide chain D as described above, and the vector comprises a first nucleotide sequence encoding the peptide chain A, a second nucleotide sequence encoding the peptide chain B, a third nucleotide sequence encoding the peptide chain C, and a fourth nucleotide sequence encoding the peptide chain D of the bispecific antibody of the present application, wherein the first nucleotide sequence, the second nucleotide sequence, the third nucleotide sequence, and the fourth nucleotide sequence are present on the same or different vector molecules from each other.

[0270] In another aspect, the present application provides a host cell comprising the isolated nucleic acid molecule or the set of nucleic acid molecules as described above, or the vector.

[0271] The host cell can be a eukaryotic cell (e.g., a mammalian cell, an insect cell, a yeast cell) or a prokaryotic cell (e.g., E. coli). Suitable eukaryotic cells include, but are not limited to, NS0 cells, Vero cells, Hela cells, COS cells, CHO cells, ExpiCHO cells, HEK293 cells, Expi293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells. In certain embodiments, the host cell of the application is a mammalian cell, e.g., CHO (e.g., CHO-EBNA, CHO-K1, CHO-S, CHO DXB11, ExpiCHO, CHO DG44).

[0272] In another aspect, the application provides a method of making a bispecific antibody as described above, comprising culturing a host cell as described above under conditions that allow expression of the bispecific antibody, and recovering the bispecific antibody from the cultured host cell

[0273] Derivatized antibodies

[0274] The bispecific antibodies of the application can be derivatized, e.g., linked to another molecule (e.g., another polypeptide or protein). In general, derivatization (e.g., labeling) of the bispecific antibodies will not adversely affect their binding to TSLP and IL-13, particularly human TSLP and human IL-13. Thus, the bispecific antibodies of the application are intended to include such derivatized forms. For example, the bispecific antibodies of the application can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as a detectable agent, a pharmaceutical agent, and / or a protein or polypeptide that can mediate conjugation of the bispecific antibody to another molecule, such as an avidin or polyhistidine tag.

[0275] As one of the derivatives of the antibody, the application provides a conjugate comprising an antibody or antigen-binding fragment thereof of the application and a conjugating moiety.

[0276] In certain embodiments, the conjugating moiety is selected from a therapeutic agent.

[0277] In certain embodiments, the conjugating moiety is selected from a substance that can improve the biological properties of the antibody, e.g., increase the serum half-life, e.g., can be a chemical group, such as polyethylene glycol (PEG), methyl or ethyl, or a sugar group.

[0278] Therapeutic applications

[0279] In another aspect, the present application provides a pharmaceutical composition comprising the bispecific antibody, or the isolated nucleic acid molecule or set of nucleic acid molecules, or the vector, or the host cell, or the conjugate, as described above, and a pharmaceutically acceptable carrier and / or excipient.

[0280] In certain embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent.

[0281] In certain embodiments, the additional pharmaceutically active agent is selected from the group consisting of: immunosuppressants, bronchodilators, other antagonists or antibodies of cytokines or cytokine receptors, antibiotics, radiation therapy, leukotriene antagonists, PDE4 inhibitors, antihistamines or antitussive drugs.

[0282] In certain embodiments, the bispecific antibody and the additional pharmaceutically active agent are provided as separate components or as a mixture.

[0283] In certain embodiments, the pharmaceutical composition is for use in preventing and / or treating and / or adjuvant therapy of a disease associated with TSLP and / or IL-13 in a subject.

[0284] In certain embodiments, the disease is characterized by elevated and / or excessive TSLP and / or IL-13 expression and activity.

[0285] In certain embodiments, the disease is an allergic inflammatory or autoimmune disease.

[0286] In certain embodiments, the disease is selected from the group consisting of: atopic dermatitis (AD), asthma, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis, allergic conjunctivitis, allergic rhinitis (AR), Netherton syndrome, eosinophilic esophagitis (EOE), food allergy, allergic diarrhea, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis (ABPA), allergic fungal sinusitis, rheumatoid arthritis, systemic sclerosis, keloids, ulcerative colitis, chronic rhinosinusitis (CRS) and nasal polyps, chronic eosinophilic pneumonia, eosinophilic bronchitis, Churg-Strauss syndrome, hypereosinophilic syndrome, eosinophilic granulomatosis with polyangiitis, inflammatory bowel disease, urticaria, systemic mastocytosis, cutaneous mastocytosis, recurrent spontaneous idiopathic angioedema, diabetes mellitus, myasthenia gravis, gastritis, pemphigus, primary biliary cirrhosis, multiple sclerosis, lupus, colitis, rheumatoid, psoriasis and thyroid disease.

[0287] In certain embodiments, the pharmaceutical composition is for use in eliciting at least one of the following biological activities in a subject:

[0288] (a) inhibits or blocks the binding of TSLP to TSLPR (e.g., inhibits or blocks the binding of TSLP to TSLPR / IL-7Rα);

[0289] (b) inhibits or blocks the binding of IL-13 to IL-13 receptor (e.g., inhibits or blocks the binding of IL-13 to IL-13Rα-1 / IL-4Rα);

[0290] (c) down-regulates or abrogates the activity of TSLP;

[0291] (d) down-regulates or abrogates the activity of IL-13;

[0292] (e) down-regulates or blocks the expression of OX40L;

[0293] (f) inhibits or blocks TSLP and / or IL-13 induced activation and / or proliferation of mast cells, DCs, NKT cells;

[0294] (g) inhibits or blocks TSLP and / or IL-13 induced STAT-6 activation;

[0295] (h) inhibits or blocks TSLP and / or IL-13 induced secretion of cytokines such as CCL26, CCL17, CCL22, IL-4, IL-13 or IL-5.

[0296] In another aspect, the present application provides the use of a bispecific antibody, or an isolated nucleic acid molecule or set of nucleic acid molecules, or a vector, or a host cell, or a conjugate, or a pharmaceutical composition as described above in the manufacture of a medicament for preventing and / or treating and / or co-treating a disease associated with TSLP and / or IL-13 in a subject.

[0297] In certain embodiments, the disease is characterized by elevated TSLP and / or IL-13 expression and / or excessive TSLP and / or IL-13 activity.

[0298] In certain embodiments, the disease is an allergic inflammatory or autoimmune disease.

[0299] In certain embodiments, the disease is selected from the group consisting of atopic dermatitis (AD), asthma, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis, allergic conjunctivitis, allergic rhinitis (AR), Netherton syndrome, eosinophilic esophagitis (EOE), food allergy, allergic diarrhea, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis (ABPA), allergic fungal sinusitis, rheumatoid arthritis, systemic sclerosis, scarring, ulcerative colitis, chronic rhinosinusitis (CRS) and nasal polyps, chronic eosinophilic pneumonia, eosinophilic bronchitis, Churg-Strauss syndrome, hypereosinophilic syndrome, eosinophilic granulomatosis with polyangiitis, inflammatory bowel disease, urticaria, systemic mastocytosis, cutaneous mastocytosis, recurrent spontaneous idiopathic angioedema, diabetes mellitus, myasthenia gravis, gastritis, pemphigus, primary biliary cirrhosis, multiple sclerosis, lupus, colitis, rheumatoid, psoriasis, and thyroid disease.

[0300] In certain embodiments, the bispecific antibody, isolated nucleic acid molecule or set of nucleic acid molecules, vector, host cell, conjugate, or pharmaceutical composition is administered in combination with, e.g. simultaneously, separately or sequentially, another pharmaceutically active agent.

[0301] In certain embodiments, the other pharmaceutically active agent is selected from the group consisting of: immunosuppressants, bronchodilators, other antagonists or antibodies of cytokines or cytokine receptors, antibiotics, radiation therapy, leukotriene antagonists, PDE4 inhibitors, antihistamines or antitussive drugs.

[0302] In another aspect, the present application provides the use of a bispecific antibody, or an isolated nucleic acid molecule or set of nucleic acid molecules, or a vector, or a host cell, or a conjugate, or a pharmaceutical composition as described above in the treatment of a disease.

[0303] In certain embodiments, the disease is characterized by elevated expression of TSLP and / or IL-13 and / or excessive TSLP and / or IL-13 activity.

[0304] In certain embodiments, the disease is an allergic inflammatory or autoimmune disease.

[0305] In certain embodiments, the disease is selected from the group consisting of atopic dermatitis (AD), asthma, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis, allergic conjunctivitis, allergic rhinitis (AR), Netherton syndrome, eosinophilic esophagitis (EOE), food allergy, allergic diarrhea, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis (ABPA), allergic fungal sinusitis, rheumatoid arthritis, systemic sclerosis, scarring, ulcerative colitis, chronic rhinosinusitis (CRS) and nasal polyps, chronic eosinophilic pneumonia, eosinophilic bronchitis, Churg-Strauss syndrome, hypereosinophilic syndrome, eosinophilic granulomatosis with polyangiitis, inflammatory bowel disease, urticaria, systemic mastocytosis, cutaneous mastocytosis, recurrent spontaneous idiopathic angioedema, diabetes mellitus, myasthenia gravis, gastritis, pemphigus, primary biliary cirrhosis, multiple sclerosis, lupus, colitis, rheumatoid, psoriasis, and thyroid disease.

[0306] In certain embodiments, the bispecific antibody, isolated nucleic acid molecule or set of nucleic acid molecules, vector, host cell, conjugate, or pharmaceutical composition is administered in combination with, e.g. simultaneously, separately or sequentially, another pharmaceutically active agent.

[0307] In certain embodiments, the other pharmaceutically active agent is selected from the group consisting of: immunosuppressants, bronchodilators, other antagonists or antibodies of cytokines or cytokine receptors, antibiotics, radiation therapy, leukotriene antagonists, PDE4 inhibitors, antihistamines or antitussive drugs.

[0308] In another aspect, the present application provides a method for preventing and / or treating and / or adjuvant therapy of a disease associated with TSLP and / or IL-13 in a subject, said method comprising administering to a subject in need thereof an effective amount of a bispecific antibody, or an isolated nucleic acid molecule or set of nucleic acid molecules, or a vector, or a host cell, or a conjugate, or a pharmaceutical composition as described above.

[0309] In certain embodiments, the disease is characterized by elevated expression of TSLP and / or IL-13 and / or excessive TSLP and / or IL-13 activity.

[0310] In certain embodiments, the disease is an allergic inflammatory or autoimmune disease.

[0311] In certain embodiments, the disease is selected from the group consisting of atopic dermatitis (AD), asthma, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis, allergic conjunctivitis, allergic rhinitis (AR), Netherton syndrome, eosinophilic esophagitis (EOE), food allergy, allergic diarrhea, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis (ABPA), allergic fungal sinusitis, rheumatoid arthritis, systemic sclerosis, keloids, ulcerative colitis, chronic rhinosinusitis (CRS) and nasal polyps, chronic eosinophilic pneumonia, eosinophilic bronchitis, Churg-Strauss syndrome, hypereosinophilic syndrome, eosinophilic granulomatosis with polyangiitis, inflammatory bowel disease, urticaria, systemic mastocytosis, cutaneous mastocytosis, recurrent spontaneous idiopathic angioedema, diabetes mellitus, myasthenia gravis, gastritis, pemphigus, primary biliary cirrhosis, multiple sclerosis, lupus, colitis, rheumatoid, psoriasis, and thyroid disease.

[0312] In certain embodiments, the method further comprises administering to the subject an additional pharmaceutically active agent, for example, simultaneously, separately or sequentially.

[0313] In certain embodiments, the additional pharmaceutically active agent is selected from the group consisting of: immunosuppressants, bronchodilators, other antagonists or antibodies of cytokines or cytokine receptors, antibiotics, radiation therapy, leukotriene antagonists, PDE4 inhibitors, antihistamines or anti-tussive drugs.

[0314] The bispecific antibodies of the present application, the pharmaceutical compositions of the present application, can be formulated into any dosage form known in the medical arts, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injectable solutions, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, and the like. The preferred dosage form depends on the intended mode of administration and therapeutic use. The bispecific antibodies or pharmaceutical compositions of the present application should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such injections can be sterile injectable solutions. For example, sterile injectable solutions can be prepared by incorporating the antibody of the present application in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, including, but not limited to, pH adjusting agents, surfactants, adjuvants, ion strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof, followed by sterilization by filtration. In addition, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. In certain embodiments, the bispecific antibodies of the present application, the pharmaceutical compositions of the present application, are formulated in unit dosage form, e.g., in ampoules, or in multi-dose containers.

[0315] In addition, the bispecific antibody of the present application can be present in a pharmaceutical composition in unit dosage form to facilitate administration.

[0316] The bispecific antibody, pharmaceutical composition of the present application can be administered by any suitable method known in the art, including but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrafoliar, intracellular reticulum, inguinal, intravesical, local (e.g., powder, salve or drops), or nasal routes. However, for many therapeutic uses, the preferred route / means of administration is parenteral administration (e.g., intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). The skilled artisan will appreciate that the route and / or means of administration will vary depending on the intended purpose. In a part of preferred embodiments, the bispecific antibody, pharmaceutical composition of the present application is administered by intravenous infusion or injection.

[0317] The pharmaceutical composition of the present application can include a "therapeutically effective amount" of the bispecific antibody of the present application. A "therapeutically effective amount" means an amount sufficient to cure or at least partially arrest the disease and its complications in an individual already suffering from the disease. The therapeutically effective amount of the bispecific antibody of the present application can vary depending on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, body weight and gender, the mode of administration of the drug, and other treatments concurrently administered, and the like.

[0318] In the present application, the administration regimen can be adjusted to obtain the best intended response (e.g., therapeutic response). For example, it can be administered in a single dose, it can be administered multiple times over a period of time, or it can be proportionally reduced or increased in dose depending on the urgency of the treatment situation.

[0319] In the present application, the subject can be a mammal, for example, a human.

[0320] All technical features disclosed in the present specification, or steps in all disclosed methods or processes, can be combined in any manner unless mutually exclusive technical features and / or steps are excluded.

[0321] Abbreviations

[0322] CDR Complementarity determining region in immunoglobulin variable region

[0323] FR Antibody framework region: amino acid residues in antibody variable region other than CDR residues

[0324] HC Antibody heavy chain

[0325] LC Antibody light chain

[0326] VH Antibody heavy chain variable region

[0327] VL antibody light chain variable region

[0328] IgG immunoglobulin G

[0329] IMGT numbering system based on The international ImMunoGeneTics information system (IMGT) initiated by Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003. (IMGT)) initiated by Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003.

[0330] TSLP thymic stromal lymphopoietin

[0331] AbM CDR definition method derived from the work of Martin (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272).

[0332] Kabat immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

[0333] Chothia immunoglobulin numbering system proposed by Chothia et al. that identifies the boundaries of CDR regions based on the location of structural loops (see, e.g., Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883).

[0334] mAb monoclonal antibody

[0335] EC 50 concentration that produces 50% stimulation

[0336] IC 50 Concentration for 50% inhibition

[0337] ELISA Enzyme-linked immunosorbent assay

[0338] PCR Polymerase chain reaction

[0339] HRP Horseradish peroxidase

[0340] HSA Human serum albumin

[0341] K D Equilibrium dissociation constant

[0342] Ka Association rate constant

[0343] Kd Dissociation rate constant

[0344] AD Atopic dermatitis

[0345] AA Allergic asthma

[0346] IL-13 Interleukin-13

[0347] FcRn Neonatal Fc receptor

[0348] CDR-H1 Complementarity determining region 1 in the immunoglobulin heavy chain variable region, also known as HCDR1

[0349] CDR-H2 Complementarity determining region 2 in the immunoglobulin heavy chain variable region, also known as HCDR2

[0350] CDR-H3 Complementarity determining region 3 in the immunoglobulin heavy chain variable region, also known as HCDR3

[0351] CDR-L1 Complementarity determining region 1 in the immunoglobulin light chain variable region, also known as LCDR1

[0352] CDR-L2 Complementarity determining region 2 in the immunoglobulin light chain variable region, also known as LCDR2

[0353] CDR-L3 Complementarity determining region 3 in the immunoglobulin light chain variable region, also known as LCDR3

[0354] Terminology definitions

[0355] In the present application, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless otherwise indicated. Also, the viral, biochemical, immunological laboratory procedures used in the present application are those well known in the art. Furthermore, the following definitions are provided in order to better define the present application and to provide guidance to those skilled in the art in the practice of the present application.

[0356] When the terms "for example," "e.g.," "for instance," "such as," or "including" are used in the detailed description and / or the appended claims, they are intended to be construed as "by way of illustration," rather than "by way of limitation." The phrase "example and / or embodiment" indicates that the example or embodiment is one, among others, that is possible.

[0357] The terms "a" and "an" and "the" and similar referents in the context of describing the application (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0358] As used herein, the term "antibody" is used in the broadest sense, and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity. For example, immunoglobulin molecules can be comprised of two pairs of polypeptide chains (each pair having one light (LC) and one heavy (HC) chain). The antibody light chains can be classified as kappa (kappa) and lambda (lambda) light chains. The heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define a

[0359] The term "antibody" also includes embodiments in which the heavy chain constant region comprises a C-terminal lysine, or lacks a C-terminal lysine or a C-terminal glycine- lysine dipeptide. The term also includes embodiments in which the N-terminal amino acid of the variable region of the antibody has cyclized to pyroglutamic acid or a pyroglutamate salt. Thus, in a composition comprising antibodies of the application, the various antibodies therein can independently comprise a C-terminal lysine, lack a C-terminal lysine, lack a C-terminal glycine-lysine, and / or comprise an N-terminal glutamine or glutamic acid or an N-terminal amino acid cyclized to pyroglutamic acid or a pyroglutamate salt.

[0360] In this document, unless the context clearly indicates otherwise, the term "antibody" includes not only intact antibodies, but also antigen-binding fragments of antibodies.

[0361] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The precise boundaries of these amino acid residues can be defined according to various numbering systems known in the art, e.g., as defined in accordance with the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272), the MacCallum numbering system (MacCallum et al., (1996) J Mol Biol 262:732-745, see also, e.g., Martin A. "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann and Duibel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001)), the AHo numbering system (Honegger and Pluckthun, A., J. Mol. Biol. 309:657-670 (2001)), or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003). For a given antibody, one of skill in the art will readily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to those skilled in the art (see, e.g., Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0362] In the present application, the CDRs contained by the antibodies of the present application or antigen binding fragments thereof can be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained by the antibodies of the present application or antigen binding fragments thereof are determined by the Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering system.

[0363] As used herein, the term "framework region" or "FR" residues refer to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.

[0364] The term "antibody" is not limited by any particular method of producing the antibody. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be an antibody of different isotype, e.g., an IgG (e.g., an IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibody.

[0365] As used herein, the term "bispecific antibody" refers to an antibody having binding specificity for two different antigens (or epitopes), which comprises two antibodies having binding specificity for different antigens (or epitopes). The antigen binding domains are capable of binding two different binding sites and / or target molecules. Each of the antigen binding domains comprised by the bispecific antibody can each independently be selected from a full-length antibody (e.g., an IgG antibody) or an antigen binding fragment thereof (e.g., Fv, Fab, scFab, or scFv). In some cases, each of the antigen binding domains is connected by a peptide linker.

[0366] As used herein, the term "Fv fragment" means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. The Fv fragment is generally considered the minimum antibody fragment that can form a complete antigen binding site. It is thought that the six CDRs confer the antigen-binding specificity of an antibody. However, even a single variable domain (e.g., an Fd fragment, which contains only three CDRs specific for an antigen) is capable of recognizing and binding antigen, although at a lower affinity than the entire binding site.

[0367] As used herein, the term "Fc domain" means an antibody fragment formed by disulfide bonds between the second constant region (CH2), the third constant region (CH3) of the first heavy chain and the second, third constant regions of the second heavy chain of an antibody. The Fc fragment of an antibody has a variety of different functions, but is not involved in antigen binding.

[0368] As used herein, the term "Fc domain monomer" (e.g., first Fc domain monomer, second Fc domain monomer) means an antibody heavy chain fragment comprising the second, third constant regions of an antibody heavy chain formed by a single peptide chain, and optionally comprising a hinge region.

[0369] In certain embodiments, when the term "Fc domain monomer" (e.g., first Fc domain monomer, second Fc domain monomer) is used to describe a feature of the bispecific antibodies of the application, the term will be interpreted as comprising or not comprising a hinge region. In certain embodiments, when the term "Fc domain monomer" (e.g., first Fc domain monomer, second Fc domain monomer) is used to describe a feature of the bispecific antibodies of the application, the term will be interpreted as not comprising a hinge region.

[0370] As used herein, the term "scFv" refers to a single polypeptide chain comprising a VL and a VH domain, wherein the VL and VH are connected by a linker. Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art peptide linkers consist of repeating GGGGS amino acid sequences (SEQ ID NO: 50) or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4(SEQ ID NO: 48) can be used, but variants thereof can also be used. In some cases, a disulfide bond can also be present between the VH and VL of the scFv.

[0371] As used herein, the term "Fab fragment" means an antibody fragment consisting of VL, VH, CL and CHI domains, which typically consists of one peptide chain comprising VL and CL and another peptide chain comprising VH and CHI, however the skilled person understands that the Fab domain can be arranged according to the natural orientation described above, but can also comprise domain permutations or swaps that facilitate correct VH and VL pairing (e.g. in the form of Crossmab).

[0372] As used herein, the terms "monoclonal antibody", "monoclonal", "mAb" have the same meaning and are used interchangeably to refer to one antibody or one fragment of an antibody from a population of highly homogenous antibody molecules, i.e. a population of antibody molecules that are exactly the same except for natural mutations that can spontaneously occur. A monoclonal antibody has high specificity for a single epitope on an antigen. A polyclonal antibody, in contrast to a monoclonal antibody, typically comprises at least 2 or more different antibodies, which typically recognize different epitopes on the antigen. Furthermore, the modifier "monoclonal" indicates only that the antibody is obtained from a population of highly homogenous antibody molecules, and is not to be construed as requiring production of the antibody by any particular method.

[0373] As used herein, the term "bispecific antibody" or "BsAb" refers to an antibody having two different binding domains that enable the bispecific antibody to bind to two different antigens or two different epitopes simultaneously (e.g. two different epitopes present on the same antigen, two different epitopes derived from different antigens, respectively).

[0374] As used herein, the term "CrossMab" refers to a construction method for bispecific antibodies that correctly pairs light chains with their cognate heavy chains by swapping heavy and light chain domains within the antigen-binding fragment (Fab) of one half of the bispecific antibody. This "crossover" preserves antigen-binding affinity but makes the two arms different so that light chain mispairing does not occur. The three possible "CrossMab" formats are: CrossMab Fab , which refers to a crossover or position exchange of the complete VH-CH1 and VL-CL domains of one half of the bispecific antibody; CrossMab VH-VL , which refers to a crossover or position exchange of only the VH and VL domains of one half of the bispecific antibody; and CrossMab CH1-CL , which refers to a crossover or position exchange of the CH1 and CL domains within the Fab domain of one half of the bispecific antibody. CrossMab antibodies have been described or claimed in WO2009080252, WO2009080253, WO2009080251, WO2009080254, WO2010136172, WO2010145792, and WO2013026831. The term "CrossMab" antibody is well known in the art; see, e.g., Brinkmann and Kontermann, MAbs 9(2): 182-212 (2017); Kontermann and Brinkmann, Drug Discovery Today 20(7):838-846 (2015); Schaefer et al., PNAS, 108 11187-1191 (2011); Klein et al., MAbs 8(6):1010-1020 (2016); and Klein et al., MAbs 4(6):653-663 (2012).

[0375] As used herein, the term "specifically binds" refers to a nonrandom binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed. The strength or affinity of a specific binding interaction can be determined by the equilibrium dissociation constant (KD) or half maximal effective concentration (EC 50 ) of the interaction.

[0376] The specific binding properties between two molecules can be determined using methods well known in the art. One method involves measuring the rate of formation and dissociation of antigen-binding site / antigen complexes. The "association rate constant" (k a or k on ) and the "dissociation rate constant" (k dis or k offBoth can be calculated from concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187). dis / k on The ratio is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59:439-473). KD and k can be measured by any effective method. on and k dis The dissociation constant can be measured using bioluminescence interferometry (e.g., the ForteBio Octet method) in some implementations. Alternatively, surface plasmon resonance techniques (e.g., Biacore) or Kinexa can be used.

[0377] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0378] Expression and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Typically, in cloning vectors, this sequence is the one that enables the vector to replicate independently of the host chromosomal DNA, and it includes an origin of replication or an autonomous replication sequence. As used herein, the term "expression vector" refers to a vector containing recombinant polynucleotides that include expression regulatory sequences operatively linked to the nucleotide sequence to be expressed. Expression vectors contain sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or an in vitro expression system. Expression vectors include all those known in the art, such as entrapment, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0379] As used herein, the term "host cell" refers to a cell that can be used for the introduction of a vector, including but not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblast cells, NS0 cells, Vero cells, Hela cells, COS cells, CHO cells (e.g., CHO-K1, CHO-S, CHO DXB11, ExpiCHO, CHO DG44 cells), ExpiCHO cells, HEK293 cells, Expi293 cells, BHK cells, and MDCKII cells.

[0380] As used herein, the term "identity" is used in reference to the matching of sequences between two polypeptides or between two nucleic acids. When a position in each of two sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percentage of identity" between two sequences is a function of the number of matching positions shared by the sequences divided by the number of positions compared x 100. For example, if 6 of 10 positions in two sequences are matched then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 of 6 positions are matched). Typically, the comparison is made over the length of the sequences being compared, after aligning the two sequences to produce maximum identity. Such alignment can be achieved by methods such as that of Needleman et al. (1970) J. Mol. Biol. 48:443-453, conveniently by using computer programs such as the Align program (DNAstar, Inc.). Percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) as integrated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, percentage identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J MoI Biol. 48:444-453 (1970)) as implemented in the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a gap length weight of 1, 2, 3, 4, 5, or 6.

[0381] The writing of the twenty conventional amino acids referred to herein follows the conventional usage. See, e.g., Immunology - A Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the present application, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also in the present application, amino acids are generally represented by the one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

[0382] As used herein, the term "pharmaceutically acceptable carriers and / or excipients" refers to carriers and / or excipients that are compatible, in pharmacology and / or physiology, with the subject and the active ingredient, which are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancers, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives. For example, pH adjusting agents include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic or non-ionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents to maintain osmotic pressure include, but are not limited to, sugars, NaCl, and the like. Agents to delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like. Stabilizers have the meaning generally understood by those skilled in the art, which are capable of stabilizing the desired activity of the active ingredient in the drug, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolysate), and the like.

[0383] As used herein, the term "prevent" refers to an approach taken to stop or delay the occurrence of a disease or disorder or symptoms (e.g., atopic dermatitis, asthma, chronic obstructive pulmonary disease) in a subject. As used herein, the term "treat" refers to an approach taken to obtain a beneficial or desired clinical result. For the purposes of this application, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilization (i.e., not worsening) of the state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Moreover, "treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0384] As used herein, the term "subject" refers to a mammal, such as a primate, e.g., a human. In certain embodiments, the subject (e.g., human) has atopic dermatitis, asthma, or chronic obstructive pulmonary disease, or is at risk of having the aforementioned diseases.

[0385] As used herein, the term "effective amount" refers to an amount that is sufficient to achieve or at least partially achieve a desired effect. For example, an effective amount for preventing a disease (e.g., atopic dermatitis, asthma, chronic obstructive pulmonary disease) refers to an amount that is sufficient to prevent, stop, or delay the occurrence of the disease (e.g., atopic dermatitis, asthma, chronic obstructive pulmonary disease); an effective amount for treating a disease refers to an amount that is sufficient to cure or at least partially arrest the existing disease or complications. Determining such an effective amount is well within the capability of those skilled in the art. For example, an amount effective for therapeutic purposes will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient, e.g., age, weight, and gender, the mode of administration of the drug, and other therapies that the patient is undergoing, etc.

[0386] As used herein, the term "effector function" refers to those biological activities attributable to an antibody Fc region (a native sequence Fc region or an amino acid sequence variant Fc region) and which are associated with the isotype of an antibody. Examples of antibody effector functions include but are not limited to: Fc receptor binding affinity, antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), cell surface receptor (e.g., B cell receptor) binding, B cell activation, cytokine secretion, half-life / clearance rate of antibodies and antigen-antibody complexes, etc. Methods of altering the effector function of an antibody are known in the art, e.g., by introducing mutations into the Fc region.

[0387] As used herein, the term "antibody-dependent cell-mediated cytotoxicity (ADCC)" refers to a form of cytotoxicity in which Ig binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, or macrophages) that then bind the antigen-coated target cell and kill the target cell by secreting cytotoxins.

[0388] In the present context, "combination" includes therapies that can be administered separately, e.g. separately formulated (e.g. can be provided in a kit), and therapies that can be administered together in a single formulation (i.e. "co-formulation"). In certain embodiments, the bispecific antibodies of the present application can be administered sequentially. In other embodiments, the bispecific antibodies can be administered simultaneously. The bispecific antibodies of the present application can be used in combination with at least one other (active) agent in any manner.

[0389] In the present context, unless specified otherwise herein or manifestly contradicted by context, "A, B, and / or C" or a similar expression is to be understood as "A, B, C, or any combination of these", e.g. as any one selected from the group consisting of A, B, C, A and B, A and C, B and C, A and B and C.

[0390] Embodiments of the application will be described in greater detail below with reference to the accompanying drawings and examples, but a person skilled in the art will understand that the following drawings and examples serve only to illustrate the application, and not to limit the scope thereof. Various objects and advantageous aspects of the application will become apparent to the person skilled in the art from the following detailed description of the preferred embodiments in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0391] Figure 1A: Bispecific antibody Bs-1 structure.

[0392] Figure IB: Bispecific antibody Bs-2 structure.

[0393] Figure 2: Antibodies inhibit human IL-13 to promote TF1 cell proliferation.

[0394] Figure 3: Antibodies inhibit human TSLP to promote Ba / F3-hTSLPR-hIL7Ra overexpressing cell line proliferation.

[0395] Figure 4: Antibodies inhibit IL-13 induced STAT-6 activation in 293F- hIL13R / IL4Ra-Luc cells.

[0396] Figure 5: Antibodies inhibit TSLP induced STAT-6 activation in 293F- hTSLPR / IL7Ra-Luc cells.

[0397] Figure 6A: Antibodies inhibit monkey TSLP-promoted proliferation of Ba / F3-hTSLPR- hIL7Ra overexpressing cell line.

[0398] Figure 6B: Antibodies inhibit monkey IL-13-induced STAT-6 activation in 293F- hIL13R / IL4Ra-Luc cells.

[0399] Figure 7: Antibodies inhibit human IL-13-induced CCL26 release from A549 cells in vitro.

[0400] Figure 8: Antibodies inhibit human PBMCs to secrete chemokine CCL17.

[0401] Figure 9A: Antibodies block TSLP+IL-13 synergistic induction of CCL17 secretion in human PBMCs.

[0402] Figure 9B: Antibodies block TSLP+IL-13 synergistic induction of CCL17 secretion in human PBMCs.

[0403] Sequence Information

[0404] Information of the sequences involved in the present application is described in the following table: DETAILED DESCRIPTION

[0405] The present application will now be described with reference to the following examples, which are intended to be illustrative, but not limiting, of the present application.

[0406] Unless otherwise indicated, the molecular biology and immunological techniques utilized in the present application are performed according to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, and F. M. Ausubel et al., Short Protocols in Molecular Biology, 3rd Ed., John Wiley & Sons, Inc., 1995. Those skilled in the art know that the examples describe the present application in a manner that is illustrative, but not intended to limit the scope of the application as claimed.

[0407] Example 1: Preparation of Anti-Human TSLP / IL-13 Bispecific Antibodies

[0408] 1.1 Structure and Sequences of Bispecific Antibodies

[0409] According to the published patents CN114729037A and CN101141980B, the sequences of the anti-TSLP antibody mAb-1 and the anti-IL-13 antibody mAb-2 are determined, respectively. Specifically, the heavy chain sequence of the antibody mAb-1 is SEQ ID NO: 1; the light chain sequence is SEQ ID NO: 2; the heavy chain sequence of the antibody mAb-2 is SEQ ID NO: 3; and the light chain sequence is SEQ ID NO: 4.

[0410] Based on the sequences of the anti-TSLP antibody mAb-1 and the anti-IL-13 antibody mAb-2, the bispecific antibodies Bs-1 and Bs-2 provided by the present application, which can simultaneously bind to TSLP and IL-13, adopt “1+1 IgG crossmab” and “2+2 scFv-IgG” structures, respectively. The specific description is as follows.

[0411] Bispecific antibody Bs-1

[0412] The bispecific antibody Bs-1 adopts the “1+1 IgG crossmab” antibody structure, as shown in FIG. 1A, which is composed of 4 chains, including 2 different heavy chains and two different light chains. Point mutations of Knob-into-hole are introduced into the CH3 domains of the two heavy chains to prevent mispairing of the two heavy chains, and further introduce N434A mutations for extending the half-life. Specifically, the amino acid mutations used to form the “knob” structure in Bs-1 are S354C and T366W; the amino acid mutations used to form the “hole” structure are Y349C, T366S, L368A and Y407V. Among them, the amino acid positions of the above-mentioned mutations are according to the EU numbering. The CH1 and CL domains in the IL-13 binding Fab retain the original antibody sequence (Fab with VH-CH1 and VL-CL structure), while the CH1 and CL domains in the TSLP binding Fab are exchanged to obtain Fab with VH-CL and VL-CH1 structure, in order to facilitate the correct assembly of the two different light chains. The bispecific antibody Bs-1 includes 4 polypeptide chains, which are heavy chain 1 (SEQ ID NO: 5), light chain 1 (SEQ ID NO: 4), heavy chain 2 (SEQ ID NO: 6), and light chain 2 (SEQ ID NO: 7).

[0413] Bispecific antibody Bs-2

[0414] Bs-2 is a bispecific antibody with a "2+2 scFv-IgG" structure, as shown in Figure 1B, which is composed of 4 polypeptide chains, in which the heavy chain 1 and the heavy chain 2 sequences are the same, and the light chain 1 and the light chain 2 sequences are the same. Specifically, the light chain 1 / light chain 2 of Bs-2 is composed of the light chain of mAb-2, and the sequence is Seq ID NO: 4. The heavy chain 1 / heavy chain 2 sequence of Bs-2 is shown in SEQ ID NO: 8, which is formed by scFv of mAb-1 antibody VH, VL, connected at the N-terminus of mAb-2 VH, linked by a linker (SEQ ID NO: 48), and the scFv structure in the VH, VL region respectively contains point mutations of VH-G44C, VL-G100C to enable two cysteines to form disulfide bonds, increasing the stability of the scFv. In order to prolong the half-life of the antibody, a mutation of N434A was introduced at the 434 site (Eu Numbering) of the two heavy chains.

[0415] 1.2 Gene synthesis and bispecific antibody expression

[0416] According to the sequences of the above monoclonal antibodies and bispecific antibodies, we commissioned Jinshui to conduct codon optimization and gene synthesis. Then, the antibody heavy and light chain coding nucleotide sequences were synthesized and cloned into pTT5 vectors, respectively. After plasmid extraction, the antibody heavy and light chain corresponding pTT5 plasmids were transfected into CHOS-EBNA cells at the same time. After centrifugation to collect the cell supernatant, protein A (MabSelect SuRe, GE) was used for protein purification to obtain mAb-1, mAb-2, Bs-1 and Bs-2 antibodies.

[0417] Example 2: Detection of TSLP / IL-13 bispecific antibody affinity to TSLP

[0418] The affinity of Bs-1, Bs-2 bispecific antibody to human TSLP (Acrobiosystems, Catalog No. TSP-H52Hb) and monkey TSLP (Acrobiosystems, Catalog No. TSP-C52H4) antigen was determined using a ForteBio Octet molecular interaction instrument (Pall life sciences). The specific method is as follows: the antibody to be tested was diluted to 5 μg / ml with PBST (0.02% Tween-20), and the human TSLP and monkey TSLP protein gradient was diluted to 200 nM, 100 nM, 50 nM, 25 nM, 12.50 nM, 6.25 nM, 3.125 nM, 0 nM, then the antibody to be tested was captured with Protein A Sensor for 60 s in PBST solution, after equilibration for 60 s in PBST, it was combined with 2 antigen proteins for 120 s, then dissociated for 180 s, global fitting was used to analyze the results, and the affinity constant was obtained. The results are shown in Table 1, which show that Bs-1, Bs-2 bind strongly to human and monkey TSLP, and the cross-species affinity difference is small.

[0419] Table 1: Detection of protein binding ability of antibody

[0420] Example 3: Affinity of TSLP / IL-13 bispecific antibody to IL-13

[0421] The affinity of Bs-1, Bs-2 bispecific antibody to human IL-13 (Sino Biological, Catalog No. 10369-HNAC) and monkey IL-13 (Sino Biological, Catalog No. 11057-CNAH) antigen was determined using a ForteBio Octet molecular interaction instrument. The specific method is as follows: the antibody to be tested was diluted to 5 μg / ml with PBST, and the human IL-13 and monkey IL-13 protein gradient was diluted to 200 nM, 100 nM, 50 nM, 25 nM, 12.50 nM, 6.25 nM, 3.125 nM, 0 nM, then the antibody to be tested was captured with Protein A Sensor probe for 60 s in PBST solution, after equilibration for 60 s in PBST, it was combined with human and monkey antigen proteins for 120 s, then dissociated for 180 s, global fitting was used to analyze the results, and the affinity constant was obtained. The results are shown in Table 2, which show that Bs-1, Bs-2 bind strongly to human and monkey TSLP, and the cross-species affinity difference is small.

[0422] Table 2: Detection of protein binding ability of antibody

[0423] Example 4: Dual antibody inhibits human IL-13 promoting TF1 cell proliferation

[0424] To determine the blocking effect of the dual antibody on hIL-13 promoting TF1 cell (Coelacanth, item number CBP60808) proliferation, the TF1 was centrifuged to remove the culture medium one day before the experiment, washed once with 1640 culture medium, resuspended with 1640 culture medium without GM-CSF, and incubated in an incubator for 24 h. The antibody was started at 20 μg / mL, 3-fold dilution, 11 gradients, and 100 μL of the diluted antibody was added to each well of a 96-well cell plate (Thermo\167425). The antigen hIL-13 was diluted to 32 ng / mL with 1640+10% FBS medium, and 50 μL was added to the 96-well cell plate containing 100 μL of the antibody. The cells starved overnight the day before were counted to measure the viability, and 50 μL of cells were added to each well, with a cell count of 15000 per well. After mixing, the cells were incubated at 37°C for 72 h, 20 μL of CCK8 (Suzhou Rayan\QDY-003-D) was added to each well, mixed well, and after about 3 h, the OD 450nm absorption value was read with a microplate reader. The raw data were imported into data analysis software for four-parameter fitting to calculate the IC 50 value. The results are shown in Figure 2 and Table 3, and Bs-1 and Bs-2 antibodies have a strong inhibitory effect on hIL-13 promoting TF1 proliferation.

[0425] Table 3: Inhibition of antibody on hIL-13 promoting TF1 cell proliferation

[0426] Example 5: Dual antibody inhibits human TSLP promoting Ba / F3-hTSLPR-hIL7Rα overexpression cell line proliferation

[0427] To determine the blocking effect of the dual antibody on hTSLP promoting Ba / F3-hTSLPR-hIL7Rα cell (Coelacanth) proliferation, the Ba / F3-hTSLPR-hIL7Rα cells were starved one day before the experiment. The specific steps are as follows: the cells were centrifuged to remove the culture medium, washed once with 1640 culture medium, resuspended with 1640 culture medium without growth factor IL-3, and incubated in a 37°C incubator for 24 h. The antibody was started at 30 μg / mL, 3-fold dilution, 11 gradients, and 100 μL of the diluted antibody was added to each well of a 96-well cell plate. The antigen hTSLP was diluted to 0.6 ng / mL with 1640+10% FBS medium, and 50 μL was added to the 96-well cell plate. The cells starved overnight were counted to measure the viability, and 50 μL of cells were also added to each well, with a cell count of 15000. After mixing, the cells were incubated at 37°C for 72 h, 20 μL of CCK8 was added to each well, and the OD 450nmAbsorbance. The raw data were imported into data analysis software for four-parameter fitting to calculate IC 50 Values. The results are shown in Figure 3 and Table 4. Bs-1 and Bs-2 antibodies have a strong inhibitory effect on hTSLP-induced proliferation of Ba / F3 cells.

[0428] Table 4: Inhibition of hTSLP-induced proliferation of Ba / F3-hTSLPR-hIL7Ra cells by antibodies

[0429] Example 6: Dual antibody inhibition of IL-13-induced STAT-6 activation in 293F- hIL13R / IL4Ra-Luc cells

[0430] 293F-hIL13R / IL4Ra-Luc cells (Nanjing Keye, Cat. No. CBP74110) are obtained by stably transfecting HEK293F cells with receptor genes, STAT6 genes and LUC reporter genes. Upon stimulation by IL-13 antigen, the signal pathway is activated, thereby activating STAT6 and activating the expression of luciferase. Bio-Lite reagent (Novozyme / DD1201-03) is used as a substrate to emit a fluorescent signal. 293F-hIL13R / IL4Ra-Luc cells were plated at 3.5 x 10 4 cells / 80 μL / well the day before the experiment in DMEM containing 10% fetal bovine serum. On the day of the experiment, the antibody was diluted 11 times at a 3-fold dilution starting at 150 μg / mL, 10 μL / well. The antigen concentration was 1.5 ng / mL, 10 μL / well. The antigen, the antibody diluted in gradient, and the cells were mixed and incubated for 20 h. Then 20 μL of Bio-Lite reagent was added to each well, and the mixture was mixed for 2-3 min on a shaker. The fluorescence signal value was read on a microplate reader, and the results were imported into data analysis software to calculate IC 50 Values. The results are shown in Figure 4 and Table 5. Bs-1 and Bs-2 have a strong inhibitory effect on IL-13-induced STAT-6 activation.

[0431] Table 5: Inhibition of IL-13-induced signal transduction by antibodies

[0432] Example 7: Dual antibody inhibition of TSLP-induced STAT-6 activation in 293F- hTSLPR / IL7Ra-Luc cells

[0433] 293F-hTSLPR / IL7Ra-Luc cells (Coastal proteins, Cat#XCC22) were obtained by stably transfecting HEK293F cells with receptor gene, STAT6 gene and LUC reporter gene. Upon TSLP antigen stimulation, the signal pathway is activated, starting STAT 6, activating the expression of luciferase, using Bio-Lite reagent as substrate, which can emit a fluorescent signal. 293F-hTSLPR / IL7Ra-Luc cells were plated at 4x10 4 cells / 100μL / well the day before with 10% DMEM. On the day of the experiment, the antibody was started at 50μg / mL, 3-fold dilution of 11 concentrations, 50μL / well. The antigen concentration was 12ng / mL, 50μL / well. The antigen, the antibody diluted in gradient, and the cells were mixed and incubated for 20h, then 20μL Bio-Lite reagent was added to each well, shaken and mixed, and then the fluorescence signal value was read using an enzyme-labeled instrument. The results were imported into data analysis software to calculate the IC 50 value. The results are shown in Figure 5 and Table 6, Bs-1, Bs-2 have a strong inhibitory effect on the activation of STAT-6 induced by TSLP.

[0434] Table 6: Inhibition of TSLP signaling pathway by antibodies

[0435] Example 8: Species cross-reactivity

[0436] To explore whether there is a difference in the blocking effect of antibodies on monkey TSLP and monkey IL-13, monkey TSLP and IL-13 were tested on Ba / F3-hTSLPRa-hIL7Rα, 293F-hIL13R / IL4Ra-Luc, respectively.

[0437] The method for detecting the blocking activity of the double antibody on monkey TSLP is as follows: the concentration of monkey TSLP is 2ng / mL, the starting concentration of the antibody is 20μg / mL, the antibody is diluted by 3 times, and there are 11 concentration gradients. Mix monkey TSLP, antibody diluted in gradient, and Ba / F3-hTSLPRa-hIL7Rα cells (1.5x10 4 cells / well), incubate for 72h, then add 20μL CCK8 to each well, and read the OD 450nm absorption value using an enzyme-labeled instrument. The raw data were imported into data analysis software for four-parameter curve fitting to calculate the IC 50 value. The results are shown in Figure 6A and Table 7.

[0438] The blocking activity of the double antibodies on monkey IL-13 was detected as follows: the final concentration of monkey IL-13 was 5 ng / mL, 10 μL / well. The initial concentration of the antibody was 150 μg / mL, and the antibody was diluted by 3 times, a total of 11 concentration gradients, 10 μL / well. The monkey IL-13, the antibody diluted in gradient, and the 293F-hIL13R / IL4Ra-Luc cells (3.5 x 10 4 cells / 80 μL / well) were mixed and incubated for 20 h, then 20 μL of Bio-Lite reagent was added to each well, shaken and mixed, and the fluorescence signal value was read using an enzyme-labeled instrument. The results were imported into data analysis software to calculate the IC 50 value. The results are shown in Figure 6B and Table 8.

[0439] The results show that Bs-1 and Bs-2 have strong inhibitory effect on the initiation of STAT-6 induced by TSLP or IL-13.

[0440] Table 7: Inhibition of antibody on the proliferation of Ba / F3-hTSLPR-hIL7Ra cells

[0441] Table 8: Inhibition of antibody on the transmission of IL-13 terminal signal pathway

[0442] Example 9: Inhibition of antibody on human IL-13 induced release of CCL26 from A549 cells in vitro

[0443] A549 cells (small cell lung cancer cells) will secrete eosinophil chemotactic factor, such as CCL26, under the stimulation of IL-13 and other stimulating factors. Chemotactic factor CC ligand 26 (CCL26) is a small molecular weight cytokine belonging to the CC chemotactic factor family, also known as Eotaxin-3, macrophage inflammatory protein 4a (MIP-4a), thymus stromal chemotactic factor 1 (TSC-1) and IMAC. It is expressed in various tissues, including heart, lung and ovary, as well as IL-4 stimulated endothelial cells. Eotaxin-3 produces chemotaxis to eosinophils and basophils by binding to the chemotactic factor receptor CCR3 on the cell surface. Eotaxin-3 and Eotaxin-1, Eotaxin-2 selectively activate CCR3. The interaction of Eotaxin-3-CCR3 plays an important role in allergic diseases such as atopic dermatitis and bronchial asthma.

[0444] In order to detect the blocking effect of the antibody on IL-13 induced CCL26 factor stimulation, A549 cells were used for the experiment. The specific implementation method is as follows: A549 cells were seeded in 96-well plates at a concentration of 4 x 10 4Inoculate cells with 100 μL / well overnight. The next day, serially dilute the antibody (starting at 80 μg / mL, 3-fold dilution, 10 concentration gradients, 50 μL each). Add 50 μL of antigen (8 ng / mL). Mix the antigen, serially diluted antibody, and cells. Add 30 μM of human serum albumin (HSA), incubate at 37°C for 44 h, then add 100 μg / mL of heparin sodium and incubate for another 4 h. Collect the supernatant. Quantify CCL26 using a Human Eotaxin 3 ELISA Kit (Thermo / EH171RB). Analyze the results using data analysis software, import the data, and calculate the IC50. 50 The results are shown in Figure 7 and Table 9. The results show that Bs-1 and Bs-2 have a strong inhibitory effect on blocking the secretion of CCL26 by A549 cells.

[0445] Table 9: Double anti-inflammatory drugs inhibit CCL26 secretion in A549 cells

[0446] Example 10: Inhibition of CCL17 secretion by TSLP-induced PBMCs by antibody in vitro

[0447] CCL17, a member of the CC chemokine family, is a basic 94-amino acid precursor protein that, through cleavage, produces a 71-amino acid mature secretory protein. It is primarily expressed at high levels in the thymus, with low levels also observed in the lungs, colon, and small intestine. Transient expression of CCL17 also occurs in stimulated peripheral blood mononuclear cells. CCL17 specifically binds to and induces T cell chemotaxis, playing a crucial role, particularly in type 2 immune responses. Furthermore, CCL17 plays a key role in recruiting CCR4-positive Th2 lymphocytes, participating in Th2 cell transport in eosinophil-related diseases (such as AA and AD), and may be involved in tumor cell transport, such as in certain T-cell lymphomas. CCL17 is also considered a homeostatic and inducible neuromodulatory chemokine, maintaining the typical highly branched morphology of hippocampal microglia under homeostatic conditions and promoting microglia morphological adaptation to acute LPS-induced neuroinflammation. Under TSLP stimulation, PBMCs increase CCL17 secretion, exacerbating inflammatory risk, etc. To verify the blocking function of the bispecific antibody against the interaction between TSLP and PBMCs, PBMCs (peripheral blood mononuclear cells, CELI, catalog number XFB-HP025B) were charged at 2.5 × 10⁻⁶. 5Cells were plated at 2.5x10 cells / well / 100 μL in 96-well plates. Antibodies were diluted in 3-fold serial dilutions starting at 40 μg / mL in a volume of 50 μL. Antigen was added at a concentration of 1.2 ng / mL in a volume of 50 μL. After incubation for 48 h at 37 °C, the supernatant was collected and CCL17 was quantified using the TARC / CCL17 Human ELISA Kit (Thermo / EHCCL17). Numerical results were analyzed in data analysis software, and data were imported into data analysis software to calculate IC 50 Results are shown in Figure 8 and Table 10. The results show that Bs-1 and Bs-2 have a strong inhibitory effect on the secretion of CCL17 by PBMC cells.

[0448] Table 10: Dual antibody inhibition of CCL17 secretion by PBMC cells

[0449] Example 11: Antibodies block TSLP and IL-13 synergistic induction of CCL17 secretion in human PBMC in vitro

[0450] Type 2 immunity is the type of immunity that the body uses to defend against worms and venom, and the immune cells involved include ILC2, TC2, Th2, which induce mast cell, eosinophil / basophil activation and B cell IgE production through the production of cytokines to fight infection. Generally, the body's immune system will coordinate type 2 immunity / inflammation stability to prevent life-threatening infection. But under the driving of some abnormal factors, the adjustment function of the immune system is impaired, leading to the occurrence of type 2 inflammatory diseases. Common type 2 inflammatory diseases include: atopic dermatitis (AD, also known as specific dermatitis), chronic prurigo (CPG), chronic urticaria (CU), asthma, chronic sinusitis with nasal polyps (CRSwNP), eosinophilic gastrointestinal disease, allergic rhinitis, etc. TSLP and IL-13 synergistically increase the risk of AD and asthma and other diseases. To evaluate and compare the inhibitory ability of dual antibodies and single antibodies on the synergistic effect of TSLP and IL-13 in vitro, PBMC were plated at 2.5x10 5 Cells were plated at 2.5x10 cells / well / 100 μL in 96-well plates. Antibodies were diluted in 3-fold serial dilutions starting at 40 μg / mL in a volume of 50 μL. Antigen was added at a concentration of 1.2 ng / mL in a volume of 50 μL. After incubation for 48 h at 37 °C, the supernatant was collected and CCL17 was quantified using the TARC / CCL17 Human ELISA Kit (Thermo / EHCCL17). Numerical results were analyzed in data analysis software, and data were imported into data analysis software to calculate IC 50The results are shown in FIGS. 9A-B, where FIG. 9A shows the effect of Bs-1, Bs-2, mAb-1 and mAb-2 antibodies on the inhibition of CCL17 secretion by PBMC cells in the presence of 2 ng / mL of both TSLP and IL-13; and FIG. 9B shows the effect of Bs-1, Bs-2, mAb-1 and mAb-2 antibodies on the inhibition of CCL17 secretion by PBMC cells in the presence of 0.2 ng / mL of TSLP and 0.5 ng / mL of IL-13. The results show that Bs-1 and Bs-2 have a strong inhibitory effect on the secretion of CCL17 by PBMC cells stimulated by TSLP and IL-13 in synergy, while the control antibody mAb-1 has only a partial inhibitory effect, which is weaker than that of Bs-1 and Bs-2. mAb-2 shows almost no inhibitory effect when stimulated by TSLP and IL-13 at the same concentrations. When the concentration of TSLP is reduced to 0.2 ng / mL and the concentration of IL-13 is reduced to 0.5 ng / mL, mAb-2 shows a significant inhibitory effect, but the effect is still weaker than that of Bs-1 and Bs-2. These results show the significant advantage of bispecific antibodies in inhibiting chemokines.

[0451] Example 12: Binding activity of antibodies to neonatal receptor FcRn

[0452] The FcRn (neonatal Fc receptor) heavy chain is encoded by the Fc gamma receptor and FCGRT gene located on chromosome 19q13.35. FcRn recycles IgG by binding to the Fc region of IgG at acidic pH in the early body and releasing IgG at neutral pH by exocytosis on the cell surface. Therefore, the ability of antibodies to bind to FcRn indirectly reflects the length of the half-life of antibodies in the human body.

[0453] Specific detection method steps: The affinity of hIL-13 antigen to TSLP / IL-13 bispecific antibody was determined using ForteBio Octet molecular interaction instrument (Pall life sciences). FcRn was diluted to 2.3 μg / ml (pH 6.0) with PBST (0.02% Tween-20), and FcRn was adsorbed to the needle with a probe. The gradient of the antibody to be tested was diluted to 200 nM, 100 nM, 50 nM, 25 nM, 12.50 nM, 6.25 nM, 3.125 nM, 0 nM (pH 6.0), and the probe was regenerated with PBST (pH 7.4). FcRn was captured in solution for 60 s with a SA Sensor (Pall life sciences), followed by binding to the antibody to be tested for 60 s, and then dissociation for 60 s. Global fitting was used to analyze the results to obtain the affinity constant. The results are shown in Table 11, which show that Bs-1 and Bs-2 have a high affinity for FcRn.

[0454] Table 11: Affinity of bispecifics to FcRn

[0455] While the specific embodiments of the application have been described in detail, those skilled in the art will appreciate that various modifications and alterations to the details can be made within the scope of the application as disclosed in the teachings of all patents and publications cited herein, and it is intended to cover all such modifications and alterations. The entire contents of the above-identified patents and publications are hereby incorporated by reference.

Claims

A bispecific antibody comprising a first antigen binding domain that specifically binds to TSLP and a second antigen binding domain that specifically binds to IL-13. The bispecific antibody of claim 1, wherein, The first antigen binding domain comprises a first light chain variable region (VL) and a first heavy chain variable region (VH), which together form a domain capable of specifically binding to TSLP; and the second antigen binding domain comprises a second VL and a second VH, which together form a domain capable of specifically binding to IL-13. The bispecific antibody of claim 1 or 2, wherein, The first VL comprises a LCDR1, a LCDR2 and a LCDR3 contained in a VL as set forth in SEQ ID NO: 40 or 44; and / or, the first VH comprises a HCDR1, a HCDR2 and a HCDR3 contained in a VH as set forth in SEQ ID NO: 39 or 43; wherein the CDRs are defined by the Kabat, Chothia, Abm or IMGT numbering system. The bispecific antibody of any one of claims 1-3, wherein: (a) the first VL comprises: (a-i) a LCDR1 comprising a sequence as set forth in SEQ ID NO: 12, a LCDR2 comprising a sequence as set forth in SEQ ID NO: 13, and a LCDR3 comprising a sequence as set forth in SEQ ID NO: 14; wherein the CDRs are defined by the Kabat numbering system; (a-ii) a LCDR1 comprising a sequence as set forth in SEQ ID NO: 12, a LCDR2 comprising a sequence as set forth in SEQ ID NO: 13, and a LCDR3 comprising a sequence as set forth in SEQ ID NO: 14; wherein the CDRs are defined by the Chothia numbering system; (a-iii) a LCDR1 comprising a sequence as set forth in SEQ ID NO: 12, a LCDR2 comprising a sequence as set forth in SEQ ID NO: 13, and a LCDR3 comprising a sequence as set forth in SEQ ID NO: 14; wherein the CDRs are defined by the Abm numbering system; or, (a-iv) a LCDR1 comprising a sequence as set forth in SEQ ID NO: 22, a LCDR2 comprising a sequence as set forth in SEQ ID NO: 23, and a LCDR3 comprising a sequence as set forth in SEQ ID NO: 14; wherein the CDRs are defined by the IMGT numbering system; and / or, (b) the first VH comprises: (b-i) a HCDR1 comprising a sequence as set forth in SEQ ID NO: 17, a HCDR2 comprising a sequence as set forth in SEQ ID NO: 18, and a HCDR3 comprising a sequence as set forth in SEQ ID NO: 11; wherein the CDRs are defined by the Kabat numbering system; (b-ii) HCDR1 comprising a sequence as set forth in SEQ ID NO: 9, HCDR2 comprising a sequence as set forth in SEQ ID NO: 10, and HCDR3 comprising a sequence as set forth in SEQ ID NO: 11; wherein the CDRs are defined by the Chothia numbering system; (b-iii) HCDR1 comprising a sequence as set forth in SEQ ID NO: 15, HCDR2 comprising a sequence as set forth in SEQ ID NO: 16, and HCDR3 comprising a sequence as set forth in SEQ ID NO: 11; wherein the CDRs are defined by the Abm numbering system; or, (b-iv) HCDR1 comprising a sequence as set forth in SEQ ID NO: 19, HCDR2 comprising a sequence as set forth in SEQ ID NO: 20, and HCDR3 comprising a sequence as set forth in SEQ ID NO: 21; wherein the CDRs are defined by the IMGT numbering system. The bispecific antibody of any one of claims 1-4, wherein, the first VL comprises an amino acid sequence as set forth in SEQ ID NO: 40 or 44, and / or the first VH comprises an amino acid sequence as set forth in SEQ ID NO: 39 or 43; Preferably, the first VL comprises an amino acid sequence as set forth in SEQ ID NO: 40, and the first VH comprises an amino acid sequence as set forth in SEQ ID NO: 39; or the first VL comprises an amino acid sequence as set forth in SEQ ID NO: 44, and the first VH comprises an amino acid sequence as set forth in SEQ ID NO:

43. The bispecific antibody of any one of claims 1-5, wherein, the second VL comprises LCDR1, LCDR2, and LCDR3 contained in a VL as set forth in SEQ ID NO: 42; and / or the second VH comprises HCDR1, HCDR2, and HCDR3 contained in a VH as set forth in SEQ ID NO: 41; wherein the CDRs are defined by the Kabat, Chothia, Abm, or IMGT numbering system. The bispecific antibody of any one of claims 1-6, wherein: (a) the second VL comprises: (a-i) LCDR1 comprising a sequence as set forth in SEQ ID NO: 27, LCDR2 comprising a sequence as set forth in SEQ ID NO: 28, and LCDR3 comprising a sequence as set forth in SEQ ID NO: 29; wherein the CDRs are defined by the Kabat numbering system; (a-ii) LCDR1 comprising a sequence as set forth in SEQ ID NO: 27, LCDR2 comprising a sequence as set forth in SEQ ID NO: 28, and LCDR3 comprising a sequence as set forth in SEQ ID NO: 29; wherein the CDRs are defined by the Chothia numbering system; (a-iii) LCDR1 comprising a sequence as set forth in SEQ ID NO: 27, LCDR2 comprising a sequence as set forth in SEQ ID NO: 28, and LCDR3 comprising a sequence as set forth in SEQ ID NO: 29; wherein the CDRs are defined by the Abm numbering system; or, (a-iv) LCDR1 comprising a sequence as set forth in SEQ ID NO: 37, LCDR2 comprising a sequence as set forth in SEQ ID NO: 38, and LCDR3 comprising a sequence as set forth in SEQ ID NO: 29; wherein the CDRs are defined by the IMGT numbering system; and / or, (b) the second VH comprises: (b-i) HCDR1 comprising a sequence as set forth in SEQ ID NO: 32, HCDR2 comprising a sequence as set forth in SEQ ID NO: 33, and HCDR3 comprising a sequence as set forth in SEQ ID NO: 26; wherein the CDRs are defined by the Kabat numbering system; (b-ii) HCDR1 comprising a sequence as set forth in SEQ ID NO: 24, HCDR2 comprising a sequence as set forth in SEQ ID NO: 25, and HCDR3 comprising a sequence as set forth in SEQ ID NO: 26; wherein the CDRs are defined by the Chothia numbering system; (b-iii) HCDR1 comprising a sequence as set forth in SEQ ID NO: 30, HCDR2 comprising a sequence as set forth in SEQ ID NO: 31, and HCDR3 comprising a sequence as set forth in SEQ ID NO: 26; wherein the CDRs are defined by the Abm numbering system; or, (b-iv) HCDR1 comprising a sequence as set forth in SEQ ID NO: 34, HCDR2 comprising a sequence as set forth in SEQ ID NO: 35, and HCDR3 comprising a sequence as set forth in SEQ ID NO: 36; wherein the CDRs are defined by the IMGT numbering system. The bispecific antibody of any one of claims 1-7, wherein, the second VL comprises an amino acid sequence as set forth in SEQ ID NO: 42, and / or the second VH comprises an amino acid sequence as set forth in SEQ ID NO:

41. The bispecific antibody of any one of claims 1-8, wherein, (i) the first VL comprises LCDR1, LCDR2, and LCDR3 contained in a VL as set forth in SEQ ID NO: 40 or 44, and the first VH comprises HCDR1, HCDR2, and HCDR3 contained in a VH as set forth in SEQ ID NO: 39 or 43; and (ii) the second VL comprises LCDR1, LCDR2, and LCDR3 contained in a VL as set forth in SEQ ID NO: 42, and the second VH comprises HCDR1, HCDR2, and HCDR3 contained in a VH as set forth in SEQ ID NO: 41, wherein the CDRs are defined by the Kabat, Chothia, Abm, or IMGT numbering system. The bispecific antibody of any one of claims 1-9, wherein, (a) the first VL comprises: (a-i) LCDR1 comprising the sequence set forth in SEQ ID NO: 12, LCDR2 comprising the sequence set forth in SEQ ID NO: 13, and LCDR3 comprising the sequence set forth in SEQ ID NO: 14; wherein the CDRs are defined by the Kabat numbering system; (a-ii) LCDR1 comprising the sequence set forth in SEQ ID NO: 12, LCDR2 comprising the sequence set forth in SEQ ID NO: 13, and LCDR3 comprising the sequence set forth in SEQ ID NO: 14; wherein the CDRs are defined by the Chothia numbering system; (a-iii) LCDR1 comprising the sequence set forth in SEQ ID NO: 12, LCDR2 comprising the sequence set forth in SEQ ID NO: 13, and LCDR3 comprising the sequence set forth in SEQ ID NO: 14; wherein the CDRs are defined by the Abm numbering system; or, (a-iv) LCDR1 comprising the sequence set forth in SEQ ID NO: 22, LCDR2 comprising the sequence set forth in SEQ ID NO: 23, and LCDR3 comprising the sequence set forth in SEQ ID NO: 14; wherein the CDRs are defined by the IMGT numbering system; and, (b) the first VH comprises: (b-i) HCDR1 comprising the sequence set forth in SEQ ID NO: 17, HCDR2 comprising the sequence set forth in SEQ ID NO: 18, and HCDR3 comprising the sequence set forth in SEQ ID NO: 11; wherein the CDRs are defined by the Kabat numbering system; (b-ii) HCDR1 comprising the sequence set forth in SEQ ID NO: 9, HCDR2 comprising the sequence set forth in SEQ ID NO: 10, and HCDR3 comprising the sequence set forth in SEQ ID NO: 11; wherein the CDRs are defined by the Chothia numbering system; (b-iii) HCDR1 comprising the sequence set forth in SEQ ID NO: 15, HCDR2 comprising the sequence set forth in SEQ ID NO: 16, and HCDR3 comprising the sequence set forth in SEQ ID NO: 11; wherein the CDRs are defined by the Abm numbering system; or, (b-iv) HCDR1 comprising the sequence set forth in SEQ ID NO: 19, HCDR2 comprising the sequence set forth in SEQ ID NO: 20, and HCDR3 comprising the sequence set forth in SEQ ID NO: 21; wherein the CDRs are defined by the IMGT numbering system; and (c) the second VL comprises: (c-i) LCDR1 comprising a sequence as set forth in SEQ ID NO: 27, LCDR2 comprising a sequence as set forth in SEQ ID NO: 28, and LCDR3 comprising a sequence as set forth in SEQ ID NO: 29; wherein the CDRs are defined by the Kabat numbering system; (c-ii) LCDR1 comprising a sequence as set forth in SEQ ID NO: 27, LCDR2 comprising a sequence as set forth in SEQ ID NO: 28, and LCDR3 comprising a sequence as set forth in SEQ ID NO: 29; wherein the CDRs are defined by the Chothia numbering system; (c-iii) LCDR1 comprising a sequence as set forth in SEQ ID NO: 27, LCDR2 comprising a sequence as set forth in SEQ ID NO: 28, and LCDR3 comprising a sequence as set forth in SEQ ID NO: 29; wherein the CDRs are defined by the Abm numbering system; or, (c-iv) LCDR1 comprising a sequence as set forth in SEQ ID NO: 37, LCDR2 comprising a sequence as set forth in SEQ ID NO: 38, and LCDR3 comprising a sequence as set forth in SEQ ID NO: 29; wherein the CDRs are defined by the IMGT numbering system; and, (d) the second VH comprises: (d-i) HCDR1 comprising a sequence as set forth in SEQ ID NO: 32, HCDR2 comprising a sequence as set forth in SEQ ID NO: 33, and HCDR3 comprising a sequence as set forth in SEQ ID NO: 26; wherein the CDRs are defined by the Kabat numbering system; (d-ii) HCDR1 comprising a sequence as set forth in SEQ ID NO: 24, HCDR2 comprising a sequence as set forth in SEQ ID NO: 25, and HCDR3 comprising a sequence as set forth in SEQ ID NO: 26; wherein the CDRs are defined by the Chothia numbering system; (d-iii) HCDR1 comprising a sequence as set forth in SEQ ID NO: 30, HCDR2 comprising a sequence as set forth in SEQ ID NO: 31, and HCDR3 comprising a sequence as set forth in SEQ ID NO: 26; wherein the CDRs are defined by the Abm numbering system; or, (d-iv) HCDR1 comprising a sequence as set forth in SEQ ID NO: 34, HCDR2 comprising a sequence as set forth in SEQ ID NO: 35, and HCDR3 comprising a sequence as set forth in SEQ ID NO: 36; wherein the CDRs are defined by the IMGT numbering system. The bispecific antibody of any of claims 1-10, wherein, (i) the first VL comprises an amino acid sequence as set forth in SEQ ID NO: 40 or 44, and / or the first VH comprises an amino acid sequence as set forth in SEQ ID NO: 39 or 43; and (ii) the second VL comprises an amino acid sequence as set forth in SEQ ID NO: 41 or 45, and / or the second VH comprises an amino acid sequence as set forth in SEQ ID NO: 42 or 46. (ii) the second VL comprises an amino acid sequence as shown in SEQ ID NO: 42 and / or the second VH comprises an amino acid sequence as shown in SEQ ID NO: 41 ; Preferably, in the bispecific antibody: (i) the first VL comprises an amino acid sequence as shown in SEQ ID NO: 40 and / or the first VH comprises an amino acid sequence as shown in SEQ ID NO: 39; or, the first VL comprises an amino acid sequence as shown in SEQ ID NO: 44 and / or the first VH comprises an amino acid sequence as shown in SEQ ID NO: 43; and (ii) the second VL comprises an amino acid sequence as shown in SEQ ID NO: 42 and / or the second VH comprises an amino acid sequence as shown in SEQ ID NO:

41. The bispecific antibody of any one of claims 1-11, wherein, The first and second antigen binding domains are each independently selected from scFv, Fab. The bispecific antibody of claim 12, wherein, The first and second antigen binding domains are Fabs, and the Fab of the first or second antigen binding domain comprises a domain swap in the format of a CrossMab. The bispecific antibody of claim 13, further comprising a Fc domain, the Fc domain comprising first and second Fc domain monomers, and the first and second Fc domain monomers each independently comprise one or more modifications of amino acids that promote heterodimerization of the first and second Fc domain monomers. The bispecific antibody of claim 14, wherein, The Fc domain comprises a first Fc domain monomer comprising a modification of an amino acid that forms a knob structure and a second Fc domain monomer comprising a modification of an amino acid that forms a hole structure, wherein the hole structure pairs with the knob structure to form a heterodimeric Fc domain. The bispecific antibody of claim 14 or 15, wherein, The first and / or second Fc domain monomers are derived from a human immunoglobulin (e.g., IgGl, IgG2, IgG3, or IgG4); Preferably, the first and / or second Fc domain monomers have altered (e.g., enhanced or reduced or ablated) effector function (e.g., ADCC, CDC, ADCP activity) and / or extended half-life (e.g., enhanced FcRn binding activity) compared to the wild-type Fc domain monomer from which they are derived; Preferably, the first and / or second Fc domain monomers are derived from human immunoglobulin IgGl, and the first and / or second Fc domain monomers comprise the substitution mutation N434A. The bispecific antibody of any one of claims 14-16, wherein, The first Fc domain monomer comprises an amino acid sequence as shown in SEQ ID NO: 45 and the second Fc domain monomer comprises an amino acid sequence as shown in SEQ ID NO:

46. The bispecific antibody of any one of claims 14-17, wherein, the C-terminal lysine of the first Fc domain monomer (e.g., the first Fc domain monomer comprising an amino acid sequence as set forth in SEQ ID NO: 45) is lacking, and / or the C-terminal lysine of the second Fc domain monomer (e.g., the second Fc domain monomer comprising an amino acid sequence as set forth in SEQ ID NO: 46) is lacking. The bispecific antibody of any one of claims 14-18, wherein, the first antigen binding domain and the second antigen binding domain are each linked to one of the first and second Fc domain monomers; Preferably, the first antigen binding domain is linked to the first Fc domain monomer, and the second antigen binding domain is linked to the second Fc domain monomer; or, the first antigen binding domain is linked to the second Fc domain monomer, and the second antigen binding domain is linked to the first Fc domain monomer. The bispecific antibody of any one of claims 14-19, wherein, the first antigen binding domain and the second antigen binding domain are each Fab, and the Fab of the first antigen binding domain comprises a domain swap in the format of a CrossMab. The bispecific antibody of claim 20, wherein, the bispecific antibody comprises a peptide chain A, a peptide chain B, a peptide chain C, and a peptide chain D; wherein the peptide chain A comprises the first VL and a heavy chain CH1 region, the peptide chain B comprises the first VH, a light chain constant region, a hinge region, and the first Fc domain monomer (or the second Fc domain monomer), the peptide chain C comprises the second VH, a heavy chain CH1 region, a hinge region, the second Fc domain monomer (or the first Fc domain monomer), and the peptide chain D comprises the second VL and a light chain constant region; Preferably, the peptide chain A comprises the first VL and a heavy chain CH1 region from N-terminus to C-terminus, the peptide chain B comprises the first VH, a light chain constant region, a hinge region, and the first Fc domain monomer (or the second Fc domain monomer) from N-terminus to C-terminus, the peptide chain C comprises the second VH, a heavy chain CH1 region, a hinge region, the second Fc domain monomer (or the first Fc domain monomer) from N-terminus to C-terminus, and / or the peptide chain D comprises the second VL and a light chain constant region from N-terminus to C-terminus. The bispecific antibody of claim 21, wherein, each adjacent domains of the peptide chain A are optionally connected or not connected by a linker, each adjacent domains of the peptide chain B are optionally connected or not connected by a linker, each adjacent domains of the peptide chain C are optionally connected or not connected by a linker, and / or each adjacent domains of the peptide chain D are optionally connected or not connected by a linker; Preferably, the linkers are each independently the same or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers); preferably, the peptide linkers are each independently selected from a peptide linker comprising one or more glycine (G) and / or serine (S), e.g., having the structure of (GGGGS)n n (GGGGS)n, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; preferably, the peptide linkers are each independently comprise an amino acid sequence as set forth in SEQ ID NO: 48, 50, or 51. The bispecific antibody of claim 21 or 22, wherein, the N-terminal glutamic acid or glutamine of the first VH is cyclized to form pyroglutamic acid or pyroglutamate, the N-terminal glutamic acid or glutamine of the first VL is cyclized to form pyroglutamic acid or pyroglutamate, the N-terminal glutamic acid or glutamine of the second VH is cyclized to form pyroglutamic acid or pyroglutamate, and / or the N-terminal glutamic acid or glutamine of the second VL is cyclized to form pyroglutamic acid or pyroglutamate; for example, in the bispecific antibody: the first antigen binding domain and the second antigen binding domain are each Fab, and the Fab of the first antigen binding domain comprises a domain swap in the format of a CrossMab. (i) the N-terminal glutamine of the first VH comprising an amino acid sequence as set forth in SEQ ID NO: 39 or 43 undergoes cyclization to form pyroglutamic acid or a pyroglutamate salt; (ii) the N-terminal glutamic acid of the first VL comprising an amino acid sequence as set forth in SEQ ID NO: 40 or 44 undergoes cyclization to form pyroglutamic acid or a pyroglutamate salt; and / or, (iii) the N-terminal glutamic acid of the second VH comprising an amino acid sequence as set forth in SEQ ID NO: 41 undergoes cyclization to form pyroglutamic acid or a pyroglutamate salt. The bispecific antibody of any one of claims 21-23, wherein, the light chain constant region comprises an amino acid sequence as set forth in SEQ ID NO: 53 or 63, the heavy chain CH1 region comprises an amino acid sequence as set forth in SEQ ID NO: 52, and / or, the hinge region comprises an amino acid sequence as set forth in SEQ ID NO:

49. The bispecific antibody of any one of claims 21-24, wherein, the peptide chain A comprises an amino acid sequence as set forth in SEQ ID NO: 7, the peptide chain B comprises an amino acid sequence as set forth in SEQ ID NO: 6, the peptide chain C comprises an amino acid sequence as set forth in SEQ ID NO: 5, and / or, the peptide chain D comprises an amino acid sequence as set forth in SEQ ID NO:

4. the bispecific antibody of any one of claims 21-25, which possesses one or more features selected from the group consisting of: (1) the N-terminal glutamic acid or glutamine of the peptide chain A undergoes cyclization to form pyroglutamic acid or a pyroglutamate salt (e.g., the N-terminal glutamic acid of the peptide chain A comprising an amino acid sequence as set forth in SEQ ID NO: 7 undergoes cyclization to form pyroglutamic acid or a pyroglutamate salt); (2) the N-terminal glutamic acid or glutamine of the peptide chain B undergoes cyclization to form pyroglutamic acid or pyroglutamate; and / or, the C-terminal lysine of the peptide chain B is lacking (e.g., the N-terminal glutamine of the peptide chain B comprising an amino acid sequence as set forth in SEQ ID NO: 6 undergoes cyclization to form pyroglutamic acid or a pyroglutamate salt and / or the C-terminal lysine thereof is lacking); (3) the N-terminal glutamic acid or glutamine of the peptide chain C undergoes cyclization to form pyroglutamic acid or a pyroglutamate salt; and / or, the C-terminal lysine of the peptide chain C is lacking (e.g., the N-terminal glutamic acid of the peptide chain C comprising an amino acid sequence as set forth in SEQ ID NO: 5 undergoes cyclization to form pyroglutamic acid or a pyroglutamate salt and / or the C-terminal lysine thereof is lacking); (4) the N-terminal glutamic acid or glutamine of the peptide chain D undergoes cyclization to form pyroglutamic acid or a pyroglutamate salt; Preferably, the bispecific antibody comprises: (a) the peptide chain A comprising an amino acid sequence as set forth in SEQ ID NO: 7, the peptide chain B comprising an amino acid sequence as set forth in SEQ ID NO: 6, the peptide chain C comprising an amino acid sequence as set forth in any one of SEQ ID NO: 54-56, and / or, the peptide chain D comprising an amino acid sequence as set forth in SEQ ID NO: 4; (b) the peptide chain A comprises an amino acid sequence as set forth in SEQ ID NO: 7, the peptide chain B comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 57-59, the peptide chain C comprises an amino acid sequence as set forth in SEQ ID NO: 5, and / or, the peptide chain D comprises an amino acid sequence as set forth in SEQ ID NO: 4; or, (c) the peptide chain A comprises an amino acid sequence as set forth in SEQ ID NO: 7, the peptide chain B comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 57-59, the peptide chain C comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 54-56, and / or, the peptide chain D comprises an amino acid sequence as set forth in SEQ ID NO:

4. The bispecific antibody of claim 12, wherein, The first antigen binding domain is a scFv, and the second antigen binding domain is a Fab; or, the first antigen binding domain is a Fab, and the second antigen binding domain is a scFv. The bispecific antibody of claim 27, wherein, The first antigen binding domain is a scFv. The bispecific antibody of claim 28, wherein, The bispecific antibody comprises a peptide chain I and a peptide chain II; wherein, the peptide chain I comprises the second VL and a light chain constant region, and the peptide chain II comprises: the first VHand the first VL, the second VHand a heavy chain CH1 region, a hinge region and a Fc domain monomer; Preferably, the peptide chain I comprises the second VL and a light chain constant region from N-terminus to C-terminus, and / or, the peptide chain II comprises: (i) the first VHand the first VL, the second VHand a heavy chain CH1 region, a hinge region and a Fc domain monomer; or, (ii) the first VL, the first VHand the second VHand a heavy chain CH1 region, a hinge region and a Fc domain monomer, from N-terminus to C-terminus. The bispecific antibody of claim 29, wherein, Each adjacent domain of the peptide chain I is optionally connected by or without a linker, and / or, each adjacent domain of the peptide chain II is optionally connected by or without a linker; Preferably, the linkers are each independently the same or different peptide linkers (e.g., rigid peptide linkers or flexible peptide linkers); preferably, the peptide linkers are each independently selected from a peptide linker comprising one or more glycine (G) and / or serine (S), e.g., having the structure of (GGGGS)n n (GGGGS)n, wherein n is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; preferably, the peptide linkers are each independently comprise an amino acid sequence as set forth in SEQ ID NO: 48, 50, or 51. The bispecific antibody of claim 29 or 30, wherein, A disulfide bond connection within a chain can be formed between the first VHand the first VL; Preferably, the amino acid residue at the position corresponding to position 100 of SEQ ID NO: 40 in the first VL is a cysteine residue, and / or, the amino acid residue at the position corresponding to position 44 of SEQ ID NO: 39 in the first VHis a cysteine residue; Preferably, the first VL comprises an amino acid sequence as set forth in SEQ ID NO: 44, and the first VHis an amino acid sequence as set forth in SEQ ID NO:

43. The bispecific antibody of any one of claims 29-31, wherein, The N-terminal glutamic acid or glutamine of the peptide chain I undergoes cyclization to form pyroglutamic acid or pyroglutamate, and / or, the N-terminal glutamic acid or glutamine of the peptide chain II undergoes cyclization to form pyroglutamic acid or pyroglutamate; For example, the first VL is located at the N-terminus of the peptide chain II, and the N-terminal glutamic acid or glutamine of the first VL undergoes cyclization to form pyroglutamic acid or pyroglutamate (e.g., the first VL comprising the amino acid sequence set forth in SEQ ID NO: 44 or 40 is located at the N-terminus of the peptide chain II, and the N-terminal glutamic acid thereof undergoes cyclization to form pyroglutamic acid or pyroglutamate). For example, the first VL is located at the N-terminus of the peptide chain II, and the N-terminal glutamic acid or glutamine of the first VL undergoes cyclization to form pyroglutamic acid or pyroglutamate (e.g., the first VL comprising the amino acid sequence set forth in SEQ ID NO: 44 or 40 is located at the N-terminus of the peptide chain II, and the N-terminal glutamic acid thereof undergoes cyclization to form pyroglutamic acid or pyroglutamate). The bispecific antibody of any one of claims 29-32, wherein, The Fc domain monomer is derived from a human immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4); Preferably, the Fc domain monomer is selected from the Fc domain monomer of a wild-type human immunoglobulin (e.g., IgG1) or a variant thereof (e.g., an Fc domain monomer comprising a mutation or a chemical modification); wherein the variant has an altered (e.g., enhanced or reduced or ablated) effector function (e.g., ADCC, CDC, ADCP activity) and / or an extended half-life (e.g., enhanced FcRn binding activity) as compared to the wild-type Fc domain monomer from which it is derived; Preferably, the Fc domain monomer is selected from the Fc domain monomer of a wild-type human immunoglobulin IgG1 or a variant thereof; wherein the variant comprises the substitution mutation N434A as compared to the Fc domain monomer of a wild-type human immunoglobulin IgG1. Preferably, the Fc domain monomer comprises the amino acid sequence set forth in SEQ ID NO:

47. The bispecific antibody of any one of claims 29-33, wherein, The C-terminal lysine of the Fc domain monomer (e.g., the Fc domain monomer comprising the amino acid sequence set forth in SEQ ID NO: 47) is lacking. The bispecific antibody of any one of claims 29-34, wherein, The light chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 53 or 63, the heavy chain CH1 region comprises the amino acid sequence set forth in SEQ ID NO: 52, and / or the hinge region comprises the amino acid sequence set forth in SEQ ID NO:

49. The bispecific antibody of any one of claims 29-35, wherein, The peptide chain I comprises the amino acid sequence set forth in SEQ ID NO: 4, and / or the peptide chain II comprises the amino acid sequence set forth in SEQ ID NO:

8. The bispecific antibody of any one of claims 29-36, wherein The N-terminal glutamic acid or glutamine of the peptide chain II undergoes cyclization to form pyroglutamic acid or pyroglutamate; and / or, the C-terminal lysine of the peptide chain II is lacking (e.g., the N-terminal glutamine of the peptide chain II comprising the amino acid sequence set forth in SEQ ID NO: 8 undergoes cyclization to form pyroglutamic acid or pyroglutamate and / or the C-terminal lysine thereof is lacking); and / or, the N-terminal glutamic acid or glutamine of the peptide chain I undergoes cyclization to form pyroglutamic acid or pyroglutamate; Preferably, the peptide chain I comprises an amino acid sequence as set forth in SEQ ID NO: 4, and / or, the peptide chain II comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 60-62. The bispecific antibody of any one of claims 29-37, which comprises two of the peptide chain I and two of the peptide chain II; Preferably, the two peptide chains I comprised by the bispecific antibody are identical or different, and / or, the two peptide chains II comprised by the bispecific antibody are identical or different; Preferably, the bispecific antibody comprises two identical peptide chains I and two identical peptide chains II. The bispecific antibody of any one of claims 12, 27-38, which comprises: (a) a first peptide chain I comprising an amino acid sequence as set forth in SEQ ID NO: 4; (b) a second peptide chain I comprising an amino acid sequence as set forth in SEQ ID NO: 4; (c) a first peptide chain II comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 8, 60-62; and, (d) a second peptide chain II comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 8, 60-62. The bispecific antibody of any one of claims 1-39, which possesses one or more features selected from the group consisting of: (a) inhibits or blocks the binding of TSLP to TSLPR (e.g., inhibits or blocks the binding of TSLP to TSLPR / IL-7Rα); (b) inhibits or blocks the binding of IL-13 to IL-13 receptor (e.g., inhibits or blocks the binding of IL-13 to IL-13Rα-1 / IL-4Rα); (c) down-regulates or abrogates the activity of TSLP; (d) down-regulates or abrogates the activity of IL-13; (e) down-regulates or blocks the expression of OX40L; (f) inhibits or blocks the activation and / or proliferation of mast cells, DCs, NKT cells induced by TSLP and / or IL-13; (g) inhibits or blocks the initiation of STAT-6 induced by TSLP and / or IL-13; (h) inhibits or blocks the secretion of cytokines (such as CCL26, CCL17, CCL22, IL-4, IL-13 or IL-5) induced by TSLP and / or IL-13. An isolated nucleic acid molecule or a set of nucleic acid molecules comprising a nucleotide sequence encoding the bispecific antibody of any one of claims 1-40. A vector comprising the isolated nucleic acid molecule or a set of nucleic acid molecules of claim 41; Preferably, the vector is a cloning vector or an expression vector. A host cell comprising the isolated nucleic acid molecule or a set of nucleic acid molecules of claim 41, or the vector of claim 42. A method of making the bispecific antibody of any one of claims 1-40, comprising culturing the host cell of claim 43 under conditions that allow expression of the bispecific antibody, and recovering the bispecific antibody from the cultured host cell culture. A conjugate comprising the bispecific antibody of any one of claims 1-40 and a conjugating moiety linked thereto; Preferably, the conjugating moiety is selected from a therapeutic agent. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1 to 40, or the isolated nucleic acid molecule or set of nucleic acid molecules of claim 41, or the vector of claim 42, or the host cell of claim 43, or the conjugate of claim 45, and a pharmaceutically acceptable carrier and / or excipient. The pharmaceutical composition of claim 46, wherein, The pharmaceutical composition further comprises an additional pharmaceutically active agent; Preferably, the additional pharmaceutically active agent is selected from the group consisting of: immunosuppressants, bronchodilators, other antagonists or antibodies of cytokines or cytokine receptors, antibiotics, radiation therapy, leukotriene antagonists, PDE4 inhibitors, antihistamines or antitussive drugs. The pharmaceutical composition of claim 47, wherein, The bispecific antibody and the additional pharmaceutically active agent are provided as separate components or as a mixed component. The pharmaceutical composition of any one of claims 46 to 48 for use in preventing and / or treating and / or adjuvant therapy of a disease associated with TSLP and / or IL-13 in a subject; Preferably, the disease is characterized by elevated and / or excessive TSLP and / or IL-13 expression and activity; Preferably, the disease is an allergic inflammatory or autoimmune disease; Preferably, the disease is selected from the group consisting of: atopic dermatitis (AD), asthma, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis, allergic conjunctivitis, allergic rhinitis (AR), Netherton syndrome, eosinophilic esophagitis (EOE), food allergy, allergic diarrhea, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis (ABPA), allergic fungal sinusitis, rheumatoid arthritis, systemic sclerosis, keloids, ulcerative colitis, chronic rhinosinusitis (CRS) and nasal polyps, chronic eosinophilic pneumonia, eosinophilic bronchitis, Churg-Strauss syndrome, hypereosinophilic syndrome, eosinophilic granulomatosis with polyangiitis, inflammatory bowel disease, urticaria, systemic mastocytosis, cutaneous mastocytosis, recurrent spontaneous angioedema, diabetes mellitus, myasthenia gravis, gastritis, pemphigus, primary biliary cirrhosis, multiple sclerosis, lupus, colitis, rheumatoid, psoriasis and thyroid disease. The pharmaceutical composition of any one of claims 46 to 49 for use in eliciting at least one of the following biological activities in a subject: (a) inhibiting or blocking the binding of TSLP to TSLPR (e.g., inhibiting or blocking the binding of TSLP to TSLPR / IL-7Rα); (b) inhibiting or blocking the binding of IL-13 to IL-13 receptor (e.g., inhibiting or blocking the binding of IL-13 to IL-13Rα-1 / IL-4Rα); (c) down-regulating or abrogating the activity of TSLP; (d) down-regulating or abrogating the activity of IL-13; (e) down-regulating or blocking the expression of OX40L; (f) inhibiting or blocking TSLP and / or IL-13 induced activation and / or proliferation of mast cells, DCs, NKT cells; (g) inhibiting or blocking TSLP and / or IL-13 induced STAT-6 priming; (h) inhibits or blocks TSLP and / or IL-13 induced secretion of cytokines such as CCL26, CCL17, CCL22, IL-4, IL-13 or IL-5. The use of the bispecific antibody of any one of claims 1 to 40, or the isolated nucleic acid molecule or set of nucleic acid molecules of claim 41, or the vector of claim 42, or the host cell of claim 43, or the conjugate of claim 45, or the pharmaceutical composition of any one of claims 46 to 50, for the manufacture of a medicament for the prevention and / or treatment and / or co-treatment of a disease associated with TSLP and / or IL-13 in a subject. Use of claim 51, wherein, said disease is characterized by elevated TSLP and / or IL-13 expression and / or excessive TSLP and / or IL-13 activity; Preferably, said disease is an allergic inflammatory or autoimmune disease; Preferably, said disease is selected from the group consisting of atopic dermatitis (AD), asthma, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis, allergic conjunctivitis, allergic rhinitis (AR), Netherton syndrome, eosinophilic esophagitis (EOE), food allergy, allergic diarrhea, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis (ABPA), allergic fungal sinusitis, rheumatoid arthritis, systemic sclerosis, keloids, ulcerative colitis, chronic rhinosinusitis (CRS) and nasal polyps, chronic eosinophilic pneumonia, eosinophilic bronchitis, Churg-Strauss syndrome, hypereosinophilic syndrome, eosinophilic granulomatosis with polyangiitis, inflammatory bowel disease, urticaria, systemic mastocytosis, cutaneous mastocytosis, recurrent spontaneous angioedema, diabetes mellitus, myasthenia gravis, gastritis, pemphigus, primary biliary cirrhosis, multiple sclerosis, lupus, colitis, rheumatoid, psoriasis and thyroid disease. Use according to claim 52, wherein, said bispecific antibody, isolated nucleic acid molecule or set of nucleic acid molecules, vector, host cell, conjugate, or pharmaceutical composition is administered in combination, e.g. simultaneously, separately or sequentially, with another pharmaceutically active agent; Preferably, said another pharmaceutically active agent is selected from the group consisting of: immunosuppressants, bronchodilators, antagonists or antibodies of other cytokines or cytokine receptors, antibiotics, radiotherapy, leukotriene antagonists, PDE4 inhibitors, antihistamines or anti-tussive drugs. A method for the prevention and / or treatment and / or co-treatment of a disease associated with TSLP and / or IL-13 in a subject, said method comprising administering to a subject in need thereof an effective amount of the bispecific antibody of any one of claims 1 to 40, or the isolated nucleic acid molecule or set of nucleic acid molecules of claim 41, or the vector of claim 42, or the host cell of claim 43, or the conjugate of claim 45, or the pharmaceutical composition of any one of claims 46 to 50. The method of claim 54, wherein, said disease is characterized by elevated TSLP and / or IL-13 expression and / or excessive TSLP and / or IL-13 activity; Preferably, said disease is an allergic inflammatory or autoimmune disease; Preferably, said disease is selected from the group consisting of atopic dermatitis (AD), asthma, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis, allergic conjunctivitis, allergic rhinitis (AR), Netherton syndrome, eosinophilic esophagitis (EOE), food allergy, allergic diarrhea, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis (ABPA), allergic fungal sinusitis, rheumatoid arthritis, systemic sclerosis, keloids, ulcerative colitis, chronic rhinosinusitis (CRS) and nasal polyps, chronic eosinophilic pneumonia, eosinophilic bronchitis, Churg-Strauss syndrome, hypereosinophilic syndrome, eosinophilic granulomatosis with polyangiitis, inflammatory bowel disease, urticaria, systemic mastocytosis, cutaneous mastocytosis, recurrent spontaneous angioedema, diabetes mellitus, myasthenia gravis, gastritis, pemphigus, primary biliary cirrhosis, multiple sclerosis, lupus, colitis, rheumatoid, psoriasis and thyroid disease. said bispecific antibody, isolated nucleic acid molecule or set of nucleic acid molecules, vector, host cell, conjugate, or pharmaceutical composition is administered in combination, e.g. simultaneously, separately or sequentially, with another pharmaceutically active agent; Preferably, said another pharmaceutically active agent is selected from the group consisting of: immunosuppressants, bronchodilators, antagonists or antibodies of other cytokines or cytokine receptors, antibiotics, radiotherapy, leukotriene antagonists, PDE4 inhibitors, antihistamines or anti-tussive drugs. Preferably, the disease is an allergic inflammatory or autoimmune disease; Preferably, the disease is selected from the group consisting of atopic dermatitis (AD), asthma, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis, allergic conjunctivitis, allergic rhinitis (AR), Netherton syndrome, eosinophilic esophagitis (EOE), food allergy, allergic diarrhea, eosinophilic gastroenteritis, allergic bronchopulmonary aspergillosis (ABPA), allergic fungal sinusitis, rheumatoid arthritis, systemic sclerosis, keloids, ulcerative colitis, chronic rhinosinusitis (CRS) and nasal polyps, chronic eosinophilic pneumonia, eosinophilic bronchitis, Churg-Strauss syndrome, hypereosinophilic syndrome, eosinophilic granulomatosis with polyangiitis, inflammatory bowel disease, urticaria, systemic mastocytosis, cutaneous mastocytosis, recurrent spontaneous idiopathic angioedema, diabetes mellitus, myasthenia gravis, gastritis, pemphigus, primary biliary cirrhosis, multiple sclerosis, lupus, colitis, rheumatoid, psoriasis and thyroid disease. The method of claim 54 or 55, wherein, The method further comprises administering to the subject an additional pharmaceutically active agent, for example, simultaneously, separately or sequentially; Preferably, the additional pharmaceutically active agent is selected from the group consisting of: immunosuppressants, bronchodilators, other antagonists or antibodies of cytokines or cytokine receptors, antibiotics, radiotherapy, leukotriene antagonists, PDE4 inhibitors, antihistamines or anti-tussive drugs.

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