Multi-specific antibody specifically binding to light and TSLP, and composition and use thereof

Abstract

The present invention relates to a multi-specific antibody or antigen-binding fragment specifically binding to LIGHT and TSLP, a composition comprising an antibody or antigen-binding fragment specifically binding to LIGHT and an antibody or antigen-binding fragment specifically binding to TSLP, and a pharmaceutical composition comprising the multi-specific antibody or composition, a preparation method for the pharmaceutical composition and a use thereof, including a method for using the pharmaceutical composition to prevent and treat inflammatory diseases, respiratory diseases, or autoimmune diseases.

Description

Multispecific antibodies specifically binding to LIGHT and TSLP, compositions, and applications thereof

[0001] Submit sequence listing as XML file

[0002] The contents of the following submitted XML file are incorporated herein by reference in their entirety: Sequence Listing in Computer Readable Form (CRF) (text name: LIGHT-TSLP.xml, recording date: 2025.03.14, size: 84KB). Technical Field

[0003] The present application relates to multispecific antibodies that specifically bind to LIGHT and TSLP, compositions comprising antibodies or antigen-binding fragments that specifically bind to LIGHT and antibodies or antigen-binding fragments that specifically bind to TSLP, and pharmaceutical compositions comprising the above-mentioned multispecific antibodies or compositions, preparation methods and uses thereof, including methods for preventing and treating inflammatory, respiratory or autoimmune diseases using the same. Background Art

[0004] Asthma is caused by a large number of cytokines secreted by various immune cells, such as T cells, B cells, mast cells, eosinophils, basophils, neutrophils and DC cells, as well as structural cells including epithelial cells and mesenchymal cells, which cause airway inflammation. Asthma can be categorized into the following types: H 2. Low / no T H 2) Classify.

[0005] Allergic asthma is widely believed to be caused by type 2 helper T lymphocytes (T H 2) driven immune inflammatory response (Moore, WC et al. J. Allergy Clin. Immunol. 119, 405–413 (2007)), which is associated with eosinophil classification and is associated with type 2 cytokines IL-4, IL-5, and IL-13 (Lambrecht et al., Immunity. 2019; 50(4): 975-991). H The 2-driven asthma subphenotype arises from a complex interplay between the innate and adaptive arms of the immune system, encompassing the full spectrum of disease severity and therefore closely associated with severe asthma (Wenzel, S. et al. Clin. Exp. Allergy 42, 650–658 (2012)).

[0006] Non-allergic or T H2. Low / absent responses are associated with a pauci-granulocytic and neutrophilic population, a group known as non-eosinophilic asthma (NEA), which accounts for 40% to 50% of all asthma patients (Stokes and Casale, Ann. Allergy Asthma Immunol. 2016 Aug; 117(2): 121-5). Cytokine pathways identified as promoting NEA include IL-6, IL-8, IL-17, IFNγ, TNFα, and G-CSF (Lambrecht et al. Immunity. 2019; 50(4): 975-991).

[0007] As a widespread respiratory disease, asthma results from a complex interaction between genetic factors and environmental factors such as aeroallergens and respiratory viruses. Particularly in the airway lumen, allergens can be taken up by dendritic cells (DCs), which process antigen molecules and present them to naive helper T cells (T cells). H 0). Then allergen-specific T H 2 cells are activated, which are responsible for the production of IL-4 and IL-13, promoting the synthesis of IgE antibodies controlled by B cells. H 2 cells release IL-5, which induces eosinophil maturation and survival. Regulatory T cells (Treg) are usually H 2. Cell-mediated responses produce immunosuppressive effects. When Treg cell function is defective, it can promote the above T H 2 cell activation and its downstream events. In addition to T H 2 Extracellular IL-9-releasing T cells H T cells can also be activated, leading to the growth and recruitment of mast cells, which release preformed and newly synthesized mediators after IgE-dependent degranulation. Other important T lymphocytes that contribute to the pathogenesis of asthma are T H 17 cells, which produce IL-17A and IL-17F, which in turn promote neutrophil recruitment and expansion. In addition, IL-12 promotes the release of IFN-γ, which can lead to T H1 cells are activated, especially in the case of persistent respiratory viral infection. Finally, the various mediators, cytokines (such as IL-4, IL-5, IL-13, IL-17, IFN-γ, etc.) and growth factors produced by these inflammatory cells may also affect the function and proliferation rate of airway structural cells, including epithelial cells, fibroblasts, smooth muscle cells and endothelial vascular cells (Pelaia, G., Vatrella, A., and Maselli, R. (2012) The potential of biologics for the treatment of asthma. Nat Rev Drug Discov 11, 958-972).

[0008] Full-length thymic stromal lymphopoietin (TSLP) was initially identified as a cytokine in the supernatant of mouse thymic stromal cells that can induce pre-B cell proliferation (Friend, et al., Exp Hematol. 22(3):321, 1994). Subsequently, human TSLP was identified (Quentmeier, et al., Leukemia. 2001 August; 15(8):1286-92, Reche, et al., J Immunol. 2001 July 1; 167(1):336-43). TSLP transmits signals through a heterodimeric receptor composed of the IL-7Rα subunit and TSLP-R. The main effector cells are DCs and IL2 cells. It can indirectly induce T cells by promoting DC maturation. H 0 cells to T H 2 and T H 17 differentiation, and then participate in T H Type 2 and non-T H Type 2 immune response. Existing studies have shown that TSLP is increased in the airways of asthmatic patients and is associated with T H 2 Cytokine and chemokine expression is associated with disease severity (Shikotra A et al. J Allergy Clin Immnol 2012; 129: 104-11e1-9).

[0009] LIGHT is a pleiotropic target, and its main effector cells are T H 1. T H 2. T H 17. Tc1 and myeloid cells. It not only acts as a T cell co-activation signal, but also promotes T H 0 cells to T H 1. T H 2. T H17 cells to participate in the adaptive immune response, and can also mediate the innate immune response by activating macrophages, eosinophils, neutrophils and other cells. In addition, LIGHT can also promote the differentiation of Tc1 cells and the formation of lymphocytes. Studies have shown that the increased level of LIGHT in the sputum of asthma patients is negatively correlated with lung function (for example, forced expiratory volume in 1 second (FEVi) and forced vital capacity (FVC)) (Romeo et al., J Allergy Clin Immunol. 2013; 131(2Suppl): AB203). The overall level of LIGHT is increased in patients with high neutrophil counts (Hastie et al., J Allergy Clin Immunol. 2010; 125(5): 1028-1036). Elevated LIGHT is associated with increased cell infiltration, T cell proliferation and T cell death in asthma patients. H 1 cytokine levels and decreased lung function (Romeo et al., J Allergy Clin Immunol. 2013; 131(2 Suppl): AB203).

[0010] Long-term control of asthma is typically achieved through the use of long-acting bronchodilators and inhaled corticosteroids as needed. For patients with particularly severe allergic asthma, biologics have been approved for treatment that target IgE (e.g., omalizumab) or the Th2-associated cytokine pathways of IL-4 and IL-5. However, several currently available biologics, such as anti-IL-4R antibodies (e.g., dupilumab) or anti-IL-5 / IL-5R antibodies (e.g., mepolizumab, benralizumab), are only indicated for patients with elevated eosinophils.

[0011] For patients with non-eosinophilic asthma, there are no approved drugs targeting NEA-related cytokine pathways (including IL-6, IL-8, IL-17, IFNγ, TNFα, and G-CSF), resulting in significant unmet medical needs for these patients. Commonly used asthma medications, such as inhaled corticosteroids (ICS), may increase neutrophil levels, exacerbating NEA. Existing drugs remain ineffective in controlling NEA in many patients (Esteban-Gorgojo et al., J Asthma Allergy. 2018;11:267-281). Tezepelumab (Amgen / AstraZeneca), an anti-TSLP monoclonal antibody, is currently the only drug with therapeutic efficacy in patients without elevated eosinophils, but it only reduces the annualized number of attacks by 39%, which is significantly lower than the 70% remission rate for patients with elevated eosinophils.

[0012] Is it possible to enable multi-target drugs to complement and / or enhance each other's effects on multiple pathways in the development of asthma to improve the effectiveness and / or safety of the drugs, thereby better meeting the treatment needs of people without elevated eosinophils, as well as people with elevated eosinophils or people with elevated eosinophils and neutrophils? This concept provides a new approach to the treatment of diseases or conditions related to inflammatory, respiratory or autoimmune diseases (e.g., asthma). This field still needs a new substance and treatment method that is more effective and covers a wider range of people.

[0013] The disclosures of all publications, patents, patent applications, and published patent applications mentioned in this application are hereby incorporated by reference in their entirety.

[0014] Application Overview

[0015] In one aspect, the present application provides multispecific antibodies (eg, bispecific antibodies) that specifically bind to LIGHT and specifically bind to TSLP, as well as pharmaceutical compositions comprising the multispecific antibodies (eg, bispecific antibodies).

[0016] In other embodiments, the present application also provides methods for preventing and / or treating diseases or conditions associated with inflammatory, respiratory or autoimmune diseases using the above-mentioned multispecific antibodies (e.g., bispecific antibodies) or pharmaceutical compositions thereof. In other embodiments, the present application also provides uses of the above-mentioned multispecific antibodies (e.g., bispecific antibodies) or pharmaceutical compositions thereof in the preparation of medicaments for preventing and / or treating diseases or conditions associated with inflammatory, respiratory or autoimmune diseases.

[0017] In some embodiments, the present application provides a multispecific antibody that binds to LIGHT and TSLP, comprising a first antigen-binding domain that specifically binds to LIGHT, and a second antigen-binding domain that specifically binds to TSLP, wherein the first antigen-binding domain that specifically binds to LIGHT comprises: a heavy chain variable region (V H ), the V H comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and a light chain variable region (V L ), the V Lcomprising: a light chain complementary determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 4, a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6; and the second antigen binding domain that specifically binds to TSLP comprising: (1) a heavy chain variable region (V H ), the V H comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 12, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16; and a light chain variable region (V L ), the V L comprising: a light chain complementary determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 18, a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20; or (2) a heavy chain variable region (V H ), the V H comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 13, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17; and a light chain variable region (V L ), the V L It comprises: a light chain complementary determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO:21, a LC-CDR2 comprising the amino acid sequence of SEQ ID NO:22, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO:23.

[0018] In some embodiments, the present application provides a multispecific antibody (e.g., a bispecific antibody) that binds to LIGHT and TSLP, comprising a first antigen-binding domain that specifically binds to LIGHT, and a second antigen-binding domain that specifically binds to TSLP, wherein the first antigen-binding domain that specifically binds to LIGHT comprises: V H , comprising the amino acid sequence shown in SEQ ID NO: 8 or a variant thereof, wherein the variant has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; and V L, comprising the amino acid sequence shown in SEQ ID NO: 11 or a variant thereof, wherein the variant has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 11; and wherein the second antigen-binding domain that specifically binds to TSLP comprises: (1) V H , comprising the amino acid sequence shown in SEQ ID NO: 24 or a variant thereof, wherein the variant has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 24; and V L , which comprises the amino acid sequence shown in SEQ ID NO: 26 or a variant thereof, wherein the variant has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 26; or (2) V H , comprising the amino acid sequence shown in SEQ ID NO: 25 or a variant thereof, wherein the variant has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 25; and V L , which comprises the amino acid sequence shown in SEQ ID NO: 27 or a variant thereof, wherein the variant has at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 27.

[0019] In some embodiments, the multispecific antibodies (e.g., bispecific antibodies) described herein comprise an Fc. In other embodiments, the Fc described herein is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD. In some embodiments, the Fc comprises an Fc variant. In some embodiments, the Fc is glycosylated. In some embodiments, the Fc is deglycosylated. In some embodiments, the Fc has reduced fucosylation or is afucosylated. In some embodiments, the Fc variant comprises a substitution at position 297. In some embodiments, the substitution at position 297 is 297Q. In some embodiments, the variable Fc region comprises substitutions at one or more of positions 234, 235, 239, 282, 289, 297, 312, 324, 330, 335, 337, 339, 356, 359, 361, 383, 384, 398, 400, 440, 422, and 442, numbered according to the EU index.

[0020] In some embodiments, the structure of the multispecific antibody (eg, bispecific antibody) is selected from DVD-Ig, Bs4Ab, Hetero H, CrossMab, IgG-(scFv)2, or scFv-Fab IgG.

[0021] In some embodiments, the multispecific antibody (e.g., bispecific antibody) has a DVD-Ig structure. In some embodiments, the multispecific antibody (e.g., bispecific antibody) comprises four polypeptide chains: wherein,

[0022] Both polypeptide chains contain V from N-terminus to C-terminus H 1-LV H 2-C H 1 structure, where V H 1 is the heavy chain variable region that specifically binds to LIGHT; V H 2 is the heavy chain variable region that specifically binds to TSLP; L is the connecting peptide; C H 1 is the heavy chain constant region C H 1 domain; wherein the polypeptide chain further comprises an Fc, the Fc comprising a C H 2 and C H 3 domains;

[0023] The other two polypeptide chains contain V L 1-LV L 2-C L structure, where V L 1 is the light chain variable region that specifically binds to LIGHT; V L 2 is the light chain variable region that specifically binds to TSLP; L is the connecting peptide; C L is the light chain constant region;

[0024] Among them, V H 1 and V L 1 constitutes an antigen binding domain (Fv) that can specifically bind to LIGHT, V H 2-C H 1 and V L 2-C L It consists of an antigen-binding domain (Fab) that can specifically bind to TSLP.

[0025] In other embodiments, the multispecific antibody (eg, bispecific antibody) comprises four polypeptide chains: wherein,

[0026] Both polypeptide chains contain V from N-terminus to C-terminus H 1-LV H 2-C H 1 structure, where V H 1 is the heavy chain variable region that specifically binds to TSLP; V H 2 is the heavy chain variable region that specifically binds to LIGHT; L is a connecting peptide; C H 1 is the heavy chain constant region C H 1 domain; wherein the polypeptide chain further comprises an Fc, the Fc comprising a C H 2 and CH 3 domains;

[0027] The other two polypeptide chains contain V L 1-LV L 2-C L 1 structure, where V L 1 is the light chain variable region that specifically binds to TSLP; V L 2 is the light chain variable region that specifically binds to LIGHT; L is a connecting peptide; C L is the light chain constant region;

[0028] Among them, V H 1 and V L 1 constitutes an antigen binding domain (Fv) that can specifically bind to TSLP, V H 2-C H 1 and V L 2-C L It comprises an antigen-binding domain (Fab) that can specifically bind to LIGHT.

[0029] In some embodiments, the multispecific antibodies (e.g., bispecific antibodies) described herein that specifically bind to LIGHT and TSLP comprise:

[0030] (a) the amino acid sequence of SEQ ID NO: 37 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 37; and / or the amino acid sequence of SEQ ID NO: 38 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 38; or

[0031] (b) the amino acid sequence of SEQ ID NO: 39 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 39; and / or the amino acid sequence of SEQ ID NO: 40 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 40; or

[0032] (c) the amino acid sequence of SEQ ID NO: 41 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 41; and / or the amino acid sequence of SEQ ID NO: 42 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 42; or

[0033] (d) the amino acid sequence of SEQ ID NO: 43 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 43; and / or the amino acid sequence of SEQ ID NO: 44 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 44.

[0034] In some embodiments, the multispecific antibodies (e.g., bispecific antibodies) described herein that specifically bind to LIGHT and TSLP comprise:

[0035] (a) the amino acid sequence of SEQ ID NO: 57 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 57; and / or the amino acid sequence of SEQ ID NO: 38 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 38; or

[0036] (b) the amino acid sequence of SEQ ID NO: 58 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 58; and / or the amino acid sequence of SEQ ID NO: 40 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 40; or

[0037] (c) the amino acid sequence of SEQ ID NO: 59 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 59; and / or the amino acid sequence of SEQ ID NO: 42 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 42; or

[0038] (d) the amino acid sequence of SEQ ID NO: 60 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 60; and / or the amino acid sequence of SEQ ID NO: 44 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 44.

[0039] In some embodiments, the multispecific antibody (e.g., bispecific antibody) has a Hetero H, CrossMab structure. In some embodiments, the multispecific antibody (e.g., bispecific antibody) comprises four polypeptide chains: wherein,

[0040] A polypeptide chain from N-terminus to C-terminus contains V H 1-C H 1 structure, where V H1 is the heavy chain variable region that specifically binds to LIGHT; C H 1 is the heavy chain constant region C H 1 domain, wherein the polypeptide chain further comprises an Fc, the Fc comprising a C H 2 and C H 3 domains; and

[0041] A polypeptide chain from N-terminus to C-terminus contains V L 1-C L , where V L 1 is the light chain variable region that specifically binds to LIGHT, C L is the light chain constant region; and

[0042] A polypeptide chain from N-terminus to C-terminus contains V H 2-C L , where V H 2 is the heavy chain variable region that specifically binds to TSLP, C L is a light chain constant region, wherein the polypeptide chain further comprises an Fc comprising a C H 2 and C H 3 domains; and

[0043] A polypeptide chain from N-terminus to C-terminus contains V L 2-C H 1, where V L 2 is the light chain variable region that specifically binds to TSLP, C H 1 is the heavy chain constant region C H 1 domain.

[0044] Among them, V H 1-C H 1 and V L 1-C L Composition of the antigen binding domain (Fab) that specifically binds to LIGHT, V H 2-C L and V L 2-C H 1 constitutes the antigen binding domain (Fab) that specifically binds to TSLP.

[0045] In some embodiments, the multispecific antibody (eg, bispecific antibody) comprises four polypeptide chains: wherein,

[0046] A polypeptide chain from N-terminus to C-terminus contains V H 1-C H 1 structure, where V H 1 is the heavy chain variable region that specifically binds to TSLP; C H 1 is the heavy chain constant region C H 1 domain, wherein the polypeptide chain further comprises an Fc, the Fc comprising a CH 2 and C H 3 domains; and

[0047] A polypeptide chain from N-terminus to C-terminus contains V L 1-C L , where V L 1 is the light chain variable region that specifically binds to TSLP, C L is the light chain constant region; and

[0048] A polypeptide chain from N-terminus to C-terminus contains V H 2-C L , where V H 2 is the heavy chain variable region that specifically binds to LIGHT, C L is a light chain constant region, wherein the polypeptide chain further comprises an Fc comprising a C H 2 and C H 3 domains; and

[0049] A polypeptide chain from N-terminus to C-terminus contains V L 2-C H 1, where V L 2 is the light chain variable region that specifically binds to LIGHT, C H 1 is the heavy chain constant region C H 1 domain.

[0050] Among them, V H 1-C H 1 and V L 1-C L Composition of the antigen binding domain (Fab) that specifically binds to TSLP, V H 2-C L and V L 2-C H 1 constitutes the antigen-binding domain (Fab) that specifically binds to LIGHT.

[0051] In some embodiments, the multispecific antibodies (e.g., bispecific antibodies) described herein that specifically bind to LIGHT and TSLP comprise:

[0052] (a) the amino acid sequence of SEQ ID NO: 47 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 47; and / or the amino acid sequence of SEQ ID NO: 48 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 48; and / or the amino acid sequence of SEQ ID NO: 45 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 45; and / or the amino acid sequence of SEQ ID NO: 46 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 46; or

[0053] (b) the amino acid sequence of SEQ ID NO:47 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:47; and / or the amino acid sequence of SEQ ID NO:48 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:48; and / or the amino acid sequence of SEQ ID NO:49 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:49; and / or the amino acid sequence of SEQ ID NO:50 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:50; or

[0054] (c) the amino acid sequence of SEQ ID NO:51 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:51; and / or the amino acid sequence of SEQ ID NO:52 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:52; and / or the amino acid sequence of SEQ ID NO:53 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:53; and / or the amino acid sequence of SEQ ID NO:54 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:54; or

[0055] (d) the amino acid sequence of SEQ ID NO:55 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:55; and / or the amino acid sequence of SEQ ID NO:56 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:56; and / or the amino acid sequence of SEQ ID NO:53 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:53; and / or the amino acid sequence of SEQ ID NO:54 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:54.

[0056] In some embodiments, the multispecific antibodies (e.g., bispecific antibodies) described herein that specifically bind to LIGHT and TSLP comprise:

[0057] (a) the amino acid sequence of SEQ ID NO:46 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:46; and / or the amino acid sequence of SEQ ID NO:48 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:48; and / or the amino acid sequence of SEQ ID NO:61 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:61; and / or the amino acid sequence of SEQ ID NO:62 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:62; or

[0058] (b) the amino acid sequence of SEQ ID NO:48 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:48; and / or the amino acid sequence of SEQ ID NO:50 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:50; and / or the amino acid sequence of SEQ ID NO:62 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:62; and / or the amino acid sequence of SEQ ID NO:63 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:63; or

[0059] (c) the amino acid sequence of SEQ ID NO:52 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:52; and / or the amino acid sequence of SEQ ID NO:54 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:54; and / or the amino acid sequence of SEQ ID NO:64 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:64; and / or the amino acid sequence of SEQ ID NO:65 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:65; or

[0060] (d) the amino acid sequence of SEQ ID NO:54 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:54; and / or the amino acid sequence of SEQ ID NO:56 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:56; and / or the amino acid sequence of SEQ ID NO:65 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:65; and / or the amino acid sequence of SEQ ID NO:66 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:66.

[0061] In some embodiments, a method for treating a desired individual disease or condition is provided, comprising administering to the individual an effective amount of any multispecific antibody (e.g., bispecific antibody) as described above or a pharmaceutical composition comprising the same. In some embodiments, the use of any multispecific antibody (e.g., bispecific antibody) as described above in the preparation of a pharmaceutical composition for treating a desired individual disease or condition is provided. In some embodiments, the use of any multispecific antibody (e.g., bispecific antibody) as described above or a pharmaceutical composition comprising the same in the preparation of a medicine for treating a disease or condition is provided. In some embodiments, the disease or condition includes a disease or condition associated with an inflammatory disease, a respiratory disease, or an autoimmune disease. In some embodiments, the disease or condition comprises asthma, lupus nephritis, IgA nephropathy, type 1 diabetes, inflammatory bowel disease (Crohn's disease, ulcerative colitis), eosinophilic esophagitis, adult respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis, COVID19, airway inflammation, lung disease, bronchiolitis, inflammatory disease, allergic reaction, lupus, atopic dermatitis, arthritis, herpes (e.g., dermatitis herpetiformis), chronic idiopathic urticaria, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, transplant rejection, allograft rejection, graft-versus-host disease (GVHD), psoriasis, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia.

[0062] In one aspect, the present application provides a pharmaceutical composition comprising: (i) an antibody or antigen-binding fragment that specifically binds to LIGHT, and (ii) an antibody or antigen-binding fragment that specifically binds to TSLP.

[0063] In one aspect, the present application provides a method for treating and / or preventing a disease or condition in an individual in need thereof, comprising administering to the individual an effective amount of (i) an antibody or antigen-binding fragment that specifically binds to LIGHT and (ii) an antibody or antigen-binding fragment that specifically binds to TSLP, or a pharmaceutical composition comprising the antibody or antigen-binding fragment that specifically binds to LIGHT and the antibody or antigen-binding fragment that specifically binds to TSLP.

[0064] In another aspect, the present application provides the use of an antibody or antigen-binding fragment that specifically binds to LIGHT and an antibody or antigen-binding fragment that specifically binds to TSLP in the preparation of a pharmaceutical composition for treating a disease or condition in a desired individual. In another aspect, the present application provides the use of an antibody or antigen-binding fragment that specifically binds to LIGHT and an antibody or antigen-binding fragment that specifically binds to TSLP, or a pharmaceutical composition comprising an antibody or antigen-binding fragment that specifically binds to LIGHT and an antibody or antigen-binding fragment that specifically binds to TSLP, in the preparation of a medicament for treating a disease or condition.

[0065] In some embodiments, in the pharmaceutical compositions, methods or uses described herein, the antibody or antigen-binding fragment that specifically binds to LIGHT comprises: a heavy chain variable region (V H ), the V H comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and a light chain variable region (V L ), the V L comprising: a light chain complementary determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 4, a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6; and the antibody or antigen-binding fragment that specifically binds to TSLP comprises: (1) a heavy chain variable region (V H ), the V H comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 12, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16; and a light chain variable region (V L ), the V L comprising: a light chain complementary determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 18, a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20; or (2) a heavy chain variable region (V H ), the V Hcomprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 13, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17; and a light chain variable region (V L ), the V L It comprises: a light chain complementary determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO:21, a LC-CDR2 comprising the amino acid sequence of SEQ ID NO:22, and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO:23.

[0066] In some embodiments, in the pharmaceutical compositions, methods or uses described herein, the antibody or antigen-binding fragment that specifically binds to LIGHT comprises: V H , comprising the amino acid sequence of SEQ ID NO: 8 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 8; and V L , comprising the amino acid sequence of SEQ ID NO: 11 or a variant thereof, wherein the variant has at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 11; and the antibody or antigen-binding fragment that specifically binds to TSLP, comprising: (1) V H , comprising the amino acid sequence of SEQ ID NO: 24 or a variant thereof, said variant having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 24; and V L , which comprises the amino acid sequence of SEQ ID NO: 26 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 26; or (2) V H , comprising the amino acid sequence of SEQ ID NO: 25 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 25; and V L , comprising the amino acid sequence of SEQ ID NO: 27 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 27.

[0067] In one aspect, the present application provides a method for treating and / or preventing a disease or condition in an individual in need thereof, comprising administering to the individual an effective amount of any antibody or antigen-binding fragment and / or multispecific antibody (e.g., bispecific antibody) and / or any pharmaceutical composition described herein.

[0068] In certain embodiments, according to any method described herein, the disease or illness include one or more symptoms caused by an inflammatory disease, a respiratory disease, or an autoimmune disease. In certain embodiments, the symptoms include asthma, lupus nephritis, IgA nephropathy, type 1 diabetes, inflammatory bowel disease (Crohn's disease, ulcerative colitis), eosinophilic esophagitis, adult respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis, COVID19, airway inflammation, lung disease, bronchiolitis, inflammatory disease, allergic reaction, lupus, atopic dermatitis, arthritis, herpes (e.g., dermatitis herpetiformis), chronic idiopathic urticaria, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, transplant rejection, allograft rejection, graft-versus-host disease (GVHD), psoriasis, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia.

[0069] In some embodiments, the present application provides an isolated nucleic acid molecule encoding any multispecific antibody (e.g., a bispecific antibody) as described above. In some embodiments, a vector is provided, comprising any nucleic acid molecule as described above. In some embodiments, a host cell is provided, comprising any multispecific antibody (e.g., a bispecific antibody), any nucleic acid molecule, or any vector as described above. In some embodiments, a method for preparing a multispecific antibody (e.g., a bispecific antibody) that specifically binds to LIGHT and TSLP is provided, comprising: a) culturing any host cell as described above under conditions that effectively express a multispecific antibody (e.g., a bispecific antibody) that specifically binds to LIGHT and TSLP; and b) obtaining the expressed multispecific antibody (e.g., a bispecific antibody) in the host cell.

[0070] Also provided are pharmaceutical compositions, kits, and articles of manufacture comprising any of the multispecific antibodies (eg, bispecific antibodies), nucleic acid molecules, vectors, or host cells described above. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1A shows an exemplary structural schematic diagram of a DVD-Ig (Dual-variable domain-Ig) multispecific antibody; Figure 1B shows an exemplary structural schematic diagram of a Bs4Ab multispecific antibody; Figure 1C shows an exemplary structural schematic diagram of a Hetero H, CrossMab multispecific antibody; Figure 1D shows an exemplary structural schematic diagram of an IgG-(scFv)2 multispecific antibody; and Figure 1E shows an exemplary structural schematic diagram of an scFv-Fab IgG multispecific antibody.

[0072] Figure 2 shows the inhibitory activity results of exemplary anti-LIGHT-TSLP bispecific antibodies hum_LT-m37-14-TSLP-0107-DVD-IgG1, hum_LT-m37-14-TSLP-0202-DVD-IgG1, hum_LT-m37-14-TSLP-0107-CrossMab-IgG1, and hum_LT-m37-14-TSLP-0202-CrossMab-IgG1 on the luciferase signal initiated by free LIGHT-activated HVEM.

[0073] Figure 3A shows the results of the inhibitory activity of the exemplary anti-LIGHT-TSLP bispecific antibody hum_LT-m37-14-TSLP-0202-CrossMab-IgG1 on the release of cytokines IL-5 and IL-13 from PBMCs. Figure 3B shows the results of the inhibitory activity of the exemplary anti-LIGHT-TSLP bispecific antibody hum_LT-m37-14-TSLP-0202-CrossMab-IgG1 on the release of cytokine IL-17A from PBMCs. Figure 3C shows the results of the inhibitory activity of the exemplary anti-LIGHT-TSLP bispecific antibodies TSLP-0202-hum_LT-m37-14-DVD-IgG1, hum_LT-m37-14-TSLP-0107-CrossMab-IgG1, and hum_LT-m37-14-TSLP-0202-CrossMab-IgG1 on the release of cytokine IL-13 from PBMCs.

[0074] Figure 4 shows the pharmacodynamics of the bispecific anti-LIGHT-TSLP POC molecule in a chicken ovalbumin-induced mouse asthma model. Figure 4A shows the inhibitory activity of the bispecific anti-LIGHT-TSLP POC molecule on inflammatory cell infiltration in mouse bronchoalveolar lavage fluid. Figure 4B shows the inhibitory activity of the bispecific anti-LIGHT-TSLP POC molecule on albumin levels in mouse bronchoalveolar lavage fluid. Figure 4C shows the inhibitory activity of the bispecific anti-LIGHT-TSLP POC molecule on IgE levels in mouse serum. Figure 4D shows the results of HE staining of mouse lung tissue sections.

[0075] Figure 5 shows the pharmacodynamics of the bispecific anti-LIGHT-TSLP POC molecule in a mouse asthma model induced by chicken ovalbumin and LPS. Figure 5A shows the inhibitory activity of the bispecific anti-LIGHT-TSLP POC molecule on inflammatory cell infiltration in mouse bronchoalveolar lavage fluid. Figure 5B shows the inhibitory activity of the bispecific anti-LIGHT-TSLP POC molecule on albumin levels in mouse bronchoalveolar lavage fluid.

[0076] Detailed description of this application

[0077] In one aspect, the present application provides antibodies or antigen-binding fragments that specifically bind to LIGHT. In another aspect, the present application provides multispecific antibodies (eg, bispecific antibodies) that specifically bind to LIGHT and TSLP.

[0078] In another aspect, the present application further provides a pharmaceutical composition comprising an antibody or antigen-binding fragment that specifically binds to LIGHT and an antibody or antigen-binding fragment that specifically binds to TSLP. In another aspect, the present application further provides a pharmaceutical composition comprising a multispecific antibody (e.g., a bispecific antibody) that specifically binds to LIGHT and TSLP.

[0079] On the other hand, the present application also provides a method for treating a disease or condition in an individual in need thereof, comprising administering to the individual an effective amount of an antibody that specifically binds to LIGHT or a pharmaceutical composition comprising the same, a multispecific antibody (e.g., a bispecific antibody) that specifically binds to LIGHT and TSLP or a pharmaceutical composition comprising the same, or an antibody or antigen-binding fragment that specifically binds to LIGHT and an antibody or antigen-binding fragment that specifically binds to TSLP or a pharmaceutical composition comprising the same.

[0080] On the other hand, the present application provides uses of an antibody that specifically binds to LIGHT or a pharmaceutical composition comprising the same, a multispecific antibody (e.g., a bispecific antibody) that specifically binds to LIGHT and TSLP or a pharmaceutical composition comprising the same, or an antibody or antigen-binding fragment that specifically binds to LIGHT and an antibody or antigen-binding fragment that specifically binds to TSLP or a pharmaceutical composition comprising the same in the preparation of a medicament for preventing or treating inflammatory diseases, respiratory diseases, or autoimmune diseases.

[0081] Through a combination of scFv phage library screening, humanization, risk point modification, and appropriately designed biochemical and biological experiments, antibodies or antigen-binding fragments that specifically bind to LIGHT were identified. Through a combination of scFv phage library screening, affinity maturation, and appropriately designed biochemical and biological experiments, antibodies or antigen-binding fragments that specifically bind to TSLP were identified (see WO2022166739A1). At the same time, multispecific antibodies (e.g., bispecific antibodies) that specifically bind to LIGHT and TSLP were prepared. When used in the form of (i) a pharmaceutical composition, or (ii) a multispecific antibody (e.g., a bispecific antibody), or (iii) a combined application for the prevention and / or treatment of related diseases, a cumulative or synergistic effect can be achieved.

[0082] The present application also provides nucleic acids encoding antibodies or antigen-binding fragments that specifically bind to LIGHT, multispecific antibodies (e.g., bispecific antibodies) that specifically bind to LIGHT and TSLP, vectors and host cells containing the above nucleic acids, and methods for preparing the above multispecific antibodies.

[0083] definition

[0084] As described herein, " treatment (treatment) " or " treatment (treating) " is a method for obtaining beneficial or desired results, including clinical results. In view of the purpose of the application, the beneficial or desired clinical results, including but not limited to the following one or more: alleviate one or more symptoms caused by the disease, alleviate the degree of disease, stabilize the disease (for example, prevent or delay disease worsening), prevent or delay the spread of the disease (for example, transfer), prevent or delay disease recurrence, delay or slow down disease progression, improve the disease state, alleviate the disease (partial or complete), reduce the dosage of one or more other drugs required for the treatment of the disease, delay disease progression, improve or improve quality of life, gain weight, and / or prolong life. At the same time, " treatment " also includes the minimizing of disease pathology results (for example, for asthma, forced expiratory volume). The method of the present application takes into account any one or more aspects of these treatments.

[0085] The term "prevent" and similar words, such as "prevented," "preventing," "prevention," or "prophylactic," refer to a method of preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of a disease or condition, such as asthma. It also refers to delaying the occurrence or recurrence of a disease or condition, or delaying the occurrence or recurrence of the symptoms of a disease or condition. As used herein, "prevention" and similar words also include reducing the intensity, impact, symptoms, and / or burden of a disease or condition before it occurs or recurs. As used herein, "prevention" and similar words also include reducing the risk and susceptibility of a disease or condition, such as asthma, from occurring or recurring.

[0086] Antibodies or antigen-binding fragments As described in this application, the term "antibody" is broad and includes various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies and antigen-binding fragments thereof, as long as they exhibit the desired antigen-binding activity. A full-length antibody comprises two heavy chains and two light chains. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in the two chains typically include three highly variable loops, known as complementary determining regions (CDRs) (light chain (LC) CDRs include LC-CDR1, LC-CDR2, and LC-CDR3, and heavy chain (HC) CDRs include HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries of the antibodies or antigen-binding fragments disclosed herein can be defined or identified by the Kabat, Chothia, or Al-Lazikani conventions (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). The three CDR regions of the heavy or light chain are inserted between flanking segments called framework regions (FRs), which are more highly conserved than the CDR regions and form a scaffold that supports the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit various effector functions. Antibodies are classified based on the amino acid sequence of their heavy chain constant regions. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by having α, δ, ε, γ, and μ heavy chains, respectively. Several major antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0087] As used herein, the term "antigen-binding fragment" includes an antibody fragment, including, for example, a diabody, Fab, Fab', F(ab')2, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide-stabilized diabody (dsdiabody), a single-chain antibody (scFv), an scFv dimer (divalent diabody), a multispecific antibody composed of an antibody fragment comprising one or more CDRs, a single-domain antibody, a nanobody, a domain antibody, a divalent domain antibody, or any other antibody fragment that can bind to an antigen but does not comprise a complete antibody structure. Fab (fragment antigen-binding) as used herein is a fragment comprising the V domain of an antibody. L domain, V H domain, C L domain, and C H Antigen-binding fragments also include fusion proteins comprising the above-mentioned antibody fragments. Antigen-binding fragments are capable of binding to the same antigen as the parent antibody or parent antibody fragment (such as a parent scFv). In some embodiments, the antigen-binding fragment may include one or more CDRs from a specific human antibody, which are transplanted into the framework regions of one or more different human antibodies.

[0088] As described in the present application, the term "multispecific antibody" refers to an antibody molecule (e.g., bispecific antibody) that has binding specificity for at least two different antigens or epitopes in one molecule. Preferably, the multispecific antibody is a bispecific antibody. As described in the present application, the term "bispecific antibody" refers to an antibody molecule that has binding specificity for two different antigens or epitopes in one molecule. The production process of multispecific antibodies (e.g., bispecific antibodies) includes the design of complete molecules, the synthesis and cloning of the nucleotide sequence of each domain, the expression of mammalian cells, and the purification of the final product. The structure of exemplary multispecific antibodies (e.g., bispecific antibodies) includes structures known in the art, for example, DVD-Ig structure, Bs4Ab structure, Hetero H, CrossMab structure, IgG-(scFv) 2 structure or scFv-Fab IgG structure, etc. (e.g., see review document Labrijn AF, et al. Nat Rev Drug Discov. 2019Aug; 18(8): 585-608).

[0089] As described in this application, the term "antigen binding domain" refers to the part of an antigen binding molecule that specifically binds to an antigen. More specifically, the term "antigen binding domain" refers to a part of an antibody that includes a region that specifically binds to and is complementary to part or all of an antigen. If it is a large antigen, the antigen binding molecule may only bind to a specific part of the antigen, which is called an antigen epitope. For example, the antigen binding domain can be provided by one or more variable regions (also referred to as variable domains). Preferably, the antigen binding domain includes the variable region of the antibody light chain (V L ) and antibody heavy chain variable region (V H In one aspect, the antigen binding domain is capable of binding to its antigen and blocking or partially blocking the function of the antigen. Antigen binding domains that specifically bind to LIGHT include antibodies and antigen binding fragments further defined herein.

[0090] As used herein, the term "epitope" refers to a specific group of atoms or amino acids on an antigen to which an antibody or antibody portion binds. If two antibodies or antibody portions exhibit competitive binding to an antigen, they likely bind to the same epitope on the antigen.

[0091] As described herein, a first antibody "competes" for binding to a second antibody for a LIGHT target when the first antibody inhibits binding of the second antibody to the LIGHT target by at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) at equimolar concentrations, and vice versa. PCT Publication WO 03 / 48731 describes a high-throughput antibody "epitope binning" method based on cross-competition.

[0092] As used herein, the terms "specifically bind," "specifically recognize," or "specific for" refer to a measurable and reproducible interaction, such as the binding of a target to an antibody that can confirm the presence of the target in a heterogeneous population of molecules, including biomolecules. For example, an antibody's ability to specifically recognize a target (which may be an epitope) means that the antibody binds to the target with greater affinity, avidity, greater ease, and / or greater persistence than it binds to other targets. In some embodiments, an antibody that specifically recognizes an antigen reacts with one or more antigenic determinants of the antigen with an affinity that is at least 10 times greater than its binding affinity to other targets.

[0093] As used herein, an "isolated" antibody is an antibody that (1) is not related to naturally occurring proteins, (2) does not contain other proteins from the same source, (3) is expressed by cells of a different species, or (4) does not exist in nature.

[0094] As used herein, the term "isolated nucleic acid" refers to a nucleic acid of genomic, cDNA, or synthetic origin, or a combination thereof. Depending on its origin, the "isolated nucleic acid" (1) is unrelated to all or part of a polynucleotide found in nature, (2) is operably linked to a polynucleotide to which it is not naturally associated, or (3) does not occur in nature as part of a longer sequence.

[0095] As used herein, the term "CDR" or "complementarity determining region" refers to the non-contiguous antigen binding sites found within the variable regions of heavy and light chain polypeptides. In the literature Kabat et al., J. Biol. Chem. 252: 6609-6616 (1977); Kabat et al., USDept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196: 901-917 (1987); Al-Lazikani B. et al. al., J. Mol. Biol., 273: 927-948 (1997); MacCallum et al., J. Mol. Biol. 262: 732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Lefranc MPet These specific regions are described in, for example, Desmond et al., Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001), where these definitions include overlap or subsets of amino acid residues when compared to one another. However, any definition used to designate a CDR of an antibody, grafted antibody, or variant thereof is included within the scope of the term as defined and used herein. Table 1 lists the positions of the amino acid residues included in the CDRs defined by the various references cited above for comparison. Algorithms and binding interfaces for CDR prediction are known in the art, including, for example, Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Ehrenmann F. et al., Nucleic Acids Res., 38: D301-D307 (2010); and Adolf-Bryfogle J. et al., Nucleic Acids Res., 43: D432-D438 (2015). The contents of the references cited in this paragraph are incorporated herein by reference in their entirety for use in this application and in one or more claims that may be included in this application.

[0096] Table 1: CDR Definition 1 The amino acid residue numbers refer to the nomenclature of Kabat et al.2 The amino acid residue numbers refer to the nomenclature of Chothia et al. 3 The amino acid residue numbers refer to the nomenclature of MacCallum et al. 4 The amino acid residue numbers refer to the nomenclature of Lefranc et al. 5 The amino acid residue numbers refer to the nomenclature of Honegger and Plückthun.

[0097] The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to the corresponding sequence in an antibody from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they have the biological activity described in the present application (see US Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)).

[0098] "Fv" is the smallest antibody fragment that contains a complete antigen recognition and binding site. This fragment is a dimer formed by a heavy chain variable region and a light chain variable region tightly non-covalently linked. Six hypervariable loops (3 loops each in the light chain and heavy chain) are derived from the folding of these two domains. The hypervariable loops provide the antibody with amino acid residues for binding to the antigen and give the antibody specificity for binding to the antigen. However, even a single variable region (or half of an Fv fragment, which contains only 3 CDRs specific for the antigen) has the ability to recognize and bind to the antigen, although its affinity is lower than that of the complete binding site.

[0099] "Single-chain Fv", also abbreviated as "sFv" or "scFv", is a Fv that contains V molecules linked into a single polypeptide chain. H and V L In some embodiments, the scFv polypeptide further comprises a V H and V L The linker polypeptide between the domains allows the scFv to form an ideal structure for antigen binding. For an overview of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0100] The term "diabodies" refers to double-chain antibodies H and V L A small antibody fragment prepared by constructing an scFv fragment (see above) with a short linker (e.g., 5-10 residues) between the chains, so that the variable regions pair between the chains rather than within the chains, resulting in a bivalent fragment, that is, a fragment with two antigen-binding sites. A bispecific diabody is a heterodimer of two "crossover" scFv fragments, in which the V domains of the two antibodies are H and V L The domains are located on different polypeptide chains. Diabodies are fully described in EP 404,097; WO 93 / 11161; Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0101] The "humanized" form of a non-human (such as a rodent) antibody is a chimeric antibody that includes minimal sequences from a non-human antibody. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which the hypervariable region (HVR) residues of the recipient antibody are replaced by hypervariable region residues from a non-human species such as a mouse, rat, rabbit or non-human primate with ideal antibody specificity, affinity and performance (donor antibody). In some cases, residues in the human immunoglobulin framework region (FR) are replaced by corresponding non-human residues. In addition, a humanized antibody can include residues that are not present in either the recipient antibody or the donor antibody. These modifications can further improve the performance of the antibody. Typically, a humanized antibody will comprise essentially at least one, usually two, variable regions, in which all or substantially all of the hypervariable loops correspond to the hypervariable loops of a non-human immunoglobulin, and all or substantially all of the framework regions are human immunoglobulin sequences. The human antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc), typically a constant region of a human immunoglobulin. For details, see Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992).

[0102] The "percent (%) amino acid sequence homology" or "homology" or "identity" of the polypeptide and antibody sequences identified herein is defined as the percentage of identical amino acid residues between the candidate sequence and the polypeptide sequence being compared. Amino acid sequence homology or identity can be determined by various alignment methods within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to maximize alignment over the full length of the compared sequences. However, for the purposes of this application, amino acid sequence homology or identity values ​​are generated using the sequence alignment computer program MUSCLE (Edgar, RC, Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, RC, BMC Bioinformatics 5(1):113, 2004).

[0103] The term "Fc (fragment crystallizable)" or "Fc region" refers to a polypeptide comprising the constant region of an intact antibody, excluding the C H 1 domain, and in some cases, part of the hinge, whether in monomeric or multimeric form. The original immunoglobulin source of the natural Fc is preferably human, and can be any immunoglobulin, for example, IgG1, IgG2, IgG3 or IgG4. The natural Fc is composed of monomeric polypeptides, which can be linked into dimer or multimer forms by covalent (i.e., disulfide bonds) and non-covalent associations. The Fc region of an immunoglobulin generally comprises the C heavy chain constant region. H 2 domains and C H 3 domains, and optionally including C H 4 domains.

[0104] In some embodiments, each of the two Fc monomers in the Fc dimer comprises an amino acid substitution that promotes heterodimerization of the two monomers. In some embodiments, heterodimerization of the Fc monomers can be promoted by introducing different but compatible substitutions such as "knob-into-hole" residue pairs into the two Fc monomers. The "knob-into-hole" technology is also disclosed in U.S. Patent Publication No. 8,216,805. In some embodiments, one Fc monomer comprises a knob mutation T366W, and the other Fc monomer comprises a hole mutation T366S, L358A, and Y407V. In some embodiments, two Cys residues (S354C on the "knob" side and Y349C on the "hole" side) that form a stabilized disulfide bridge are introduced.

[0105] The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR described herein is an FcR that binds to an IgG antibody (a gamma receptor), including receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA (activating receptor) and FcγRIIB (inhibiting receptor), which have similar amino acid sequences and differ primarily in the cytoplasmic domain. The cytoplasmic domain of the activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM). The cytoplasmic domain of the inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) (see M.in Annu. Rev. Immunol. 15:203-234 (1997). The term also includes allotypes, such as the FcγRIIIA allotypes: FcγRIIIA-Phe158, FcγRIIIA-Val158, FcγRIIA-R131, and / or FcγRIIA-H131. FcRs are described in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991), Capel et al., Immunomethods 4:25-34 (1994), and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). The term FcR in this application encompasses other types of FcRs, including those identified in the future. The term FcR also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgGs to the newborn (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).

[0106] The term "FcRn" refers to the neonatal Fc receptor (FcRn). FcRn is structurally similar to the major histocompatibility complex (MHC) and consists of an α chain non-covalently bound to β2 microglobulin. The various functions of the neonatal Fc receptor FcRn are described in Ghetie and Ward (2000) Annu. Rev. Immunol. 18, 739-766. FcRn plays an important role in the passive transport of immunoglobulins (IgGs) from mother to newborn and in regulating serum IgG levels. As a salvage receptor, FcRn can bind and transport endocytosed IgGs in their intact form within and between cells, protecting them from the default degradation pathway.

[0107] The human IgG heavy chain constant region "C H The "I domain" typically extends from amino acid position 118 to amino acid position 215 (EU numbering system).

[0108] The "hinge region" is generally defined as extending from Glu 216 to Pro 230 of human IgG1 (Burton, Molec. Immunol. 22: 161-206 (1985)). By placing the first and last cysteine ​​residues that form inter-heavy chain disulfide bonds in the same positions as in IgG1, the hinge regions of other IgG subtypes can be aligned with the IgG1 sequence.

[0109] Human IgG Fc region "C H The "2 domain" usually extends from amino acid position 231 to amino acid position 340. H The 2 domain is unique in that it does not pair closely with another region. Instead, it is located between the two C H Two N-terminally linked branched sugar chains are inserted between the two domains. It is speculated that sugars may serve as a substitute for domain-to-domain pairing to help maintain C H 2 domains are stabilized. Burton, Molec Immunol. 22: 161-206 (1985).

[0110] “C H The 3" domain includes the C-terminal residue extending from the C H The 2 domains (from amino acid 341 to the C-terminus of the antibody sequence, usually amino acid residue 446 or 447 of IgG).

[0111] A "functional Fc fragment" possesses the "effector functions" of a native Fc region sequence. Exemplary "effector functions" include C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and downregulation of cell surface receptors (e.g., B cell receptor; BCR). Such effector functions generally require the Fc region to be bound to a binding domain (e.g., an antibody variable region) and can be assessed using a variety of experimental methods known in the art.

[0112] Antibodies with IgG Fc variants having "altered" FcR binding affinity or ADCC activity have enhanced or diminished FcR binding activity and / or ADCC activity compared to the parent polypeptide or a polypeptide comprising a native Fc sequence. Fc variants that exhibit "enhanced binding" to an FcR have a higher binding affinity (e.g., a lower apparent Kd or IC50 value) for at least one FcR compared to the parent polypeptide or a polypeptide comprising a native IgG Fc sequence. In some embodiments, the binding ability is enhanced by 3-fold, e.g., 5, 10, 25, 50, 60, 100, 150, 200, or even up to 500-fold, or the binding ability is increased by 25% to 1000%, compared to the parent polypeptide. Fc variants that exhibit "decreased binding" to an FcR have a lower affinity (e.g., a higher apparent Kd or IC50 value) for at least one FcR compared to the parent polypeptide. The binding ability is decreased by 40% or more compared to the parent polypeptide.

[0113] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" is a form of cytotoxicity that refers to the binding of secreted Ig to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages), enabling these cytotoxic effector cells to specifically bind to target cells bearing antigen and subsequently kill the target cells using cytotoxins. Antibodies "arm" the cytotoxic cells and are required for this killing. Of the major cell types that mediate ADCC, NK cells express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcRs on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess the ADCC activity of a target molecule, an in vitro ADCC assay can be performed, as described in U.S. Patent Nos. 5,500,362 or 5,821,337. Effector cells suitable for such experiments include peripheral blood mononuclear cells (PBMC) and natural killer cells (NK). Alternatively, or in addition, the ADCC activity of the target molecule can also be assessed in vivo, for example, as described in the animal model disclosed in Clynes et al. PNAS (USA) 95: 652-656 (1998).

[0114] Polypeptides comprising Fc variants exhibit "enhanced ADCC activity" or are capable of more effectively mediating ADCC in the presence of human effector cells, compared to polypeptides comprising wild-type IgG Fc polypeptides or parent polypeptides. When tested in substantially the same quantity as polypeptides comprising wild-type IgG Fc polypeptides (or parent polypeptides), such polypeptides comprising Fc variants are capable of more effectively mediating ADCC, both in vitro and in vivo. Such variants are generally identified using any in vitro ADCC assay known in the art, such as assays or methods for identifying ADCC activity, such as in animal models. In some embodiments, such variants mediate ADCC 5- to 100-fold more efficiently, such as 25- to 50-fold more efficiently, than wild-type Fc (or parent polypeptide).

[0115] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate structural subclass) that binds to the cognate antigen. To assess complement activation, a CDC assay, such as that described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), can be performed. Polypeptide variants with altered Fc region amino acid sequences and increased or decreased C1q binding capacity are described in U.S. Patent No. 6,194,551 B1 and WO 99 / 51642. The contents of these patent publications are expressly incorporated herein by reference. See also Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0116] Unless otherwise indicated, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. A nucleotide sequence encoding a protein or RNA may also include introns, for example, a nucleotide sequence encoding a protein may contain introns in some forms.

[0117] The term "operably linked" refers to a functional connection between a regulatory sequence and a heterologous nucleotide sequence, thereby enabling expression of the latter. For example, a first nucleotide sequence is operably linked to a second nucleotide sequence when the first nucleotide sequence is in a functional relationship with the second nucleotide sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, if necessary, can link two protein coding regions in the same reading frame.

[0118] "Homologous" refers to the sequence similarity or sequence homology between two polypeptides or two nucleic acid molecules. If the same base or amino acid monomer subunit is present at the same position in two compared sequences, for example, if two DNA molecules both contain adenine at the same position, then the two DNA molecules are homologous at that position. The percentage homology between two sequences refers to the ratio of the number of matching or homologous positions shared by the two sequences to the total number of positions multiplied by 100. For example, if 6 out of 10 positions in two sequences are matched or homologous, then the two sequences have 60% homology. For example, the DNA sequences ATTGCC and TATGGC have 50% homology. Generally speaking, when aligning two sequences, the comparison is performed with the goal of obtaining maximum homology.

[0119] An "effective amount" of an antibody (including a multispecific antibody) or composition disclosed herein is an amount sufficient to achieve a specific purpose. An "effective amount" can be determined empirically and by known methods related to the purpose.

[0120] The term "therapeutically effective amount" refers to an amount of the antibody (including multispecific antibodies) or composition described herein that is effective in treating a disease or symptom of an individual. That is, an amount sufficient to reduce or improve the severity and / or duration of a disease or one or more symptoms thereof; to prevent the development of a disease, cause symptoms to subside, prevent the recurrence, development, onset or progression of one or more symptoms associated with the disease, detect the disease, or enhance / improve the preventive or therapeutic effect of another therapy (e.g., a preventive or therapeutic agent). For example, in the case of asthma, "asthma-related parameters" are used as indicators for evaluating the effectiveness of asthma treatment. For example, "asthma-related parameters" include: (a) forced expiratory volume in 1 second (FEV1); (b) peak expiratory flow (PEF), including morning PEF (AM PEF) and evening PEF (PM PEF); (c) use of inhaled bronchodilators such as albuterol or levalbuterol; (d) five-item Asthma Control Questionnaire (ACQ5) score; (d) nocturnal awakenings; and (e) 22-item Sino-Nasal Outcome Test (SNOT-22) score. The therapeutically effective amount of the antibodies or compositions disclosed herein can increase one or more of FEV1, AM PEF, or PM PEF from baseline, and / or reduce one or more of daily albuterol / levalbuterol usage, ACQ5 score, average number of awakenings at night, or SNOT-22 score from baseline. As used herein, the term "baseline" refers to the value of the asthma-related parameter of the patient before or at the time of taking the pharmaceutical composition of the present invention. In some embodiments, an improvement in an asthma-related parameter means an increase in FEV1 of at least 0.10 L from baseline. In some embodiments, an improvement in an asthma-related parameter means an increase in AM PEF of at least 10.0 L / min from baseline. In some embodiments, an improvement in an asthma-related parameter means an increase in PM PEF of at least 1.0 L / min from baseline. In some embodiments, an improvement in an asthma-related parameter means a reduction in albuterol / levalbuterol usage of at least 1 puff(s) per day from baseline. In some embodiments, an improvement in an asthma-related parameter means a decrease in ACQ5 score of at least 0.5 points from baseline. In some embodiments, an improvement in an asthma-related parameter is a decrease in the number of nocturnal awakenings by at least 0.2 from baseline. In some embodiments, an improvement in an asthma-related parameter is a decrease in the SNOT-22 score by at least 5 points from baseline. In some embodiments, a therapeutically effective amount is an amount that adequately controls the disease. In some embodiments, a therapeutically effective amount is an amount that prolongs a patient's survival. In certain embodiments, a therapeutically effective amount is an amount that improves a patient's progression-free survival.

[0121] As used herein, "pharmaceutically acceptable" or "pharmacologically compatible" refers to a material that is free of biological activity or other undesirable properties, e.g., a material that can be added to a pharmaceutical composition administered to a patient without causing a significant adverse biological reaction, or that does not interact in a deleterious manner with any other component contained in the composition. Pharmaceutically acceptable carriers or excipients preferably meet the required standards for toxicology or manufacturing testing and / or are included in the inactive ingredient guide compiled by the U.S. Food and Drug Administration.

[0122] The embodiments of the present application described herein should be understood to include "consisting of" and / or "consisting essentially of" embodiments.

[0123] In this application, "about" is mentioned as a numerical value or parameter, including (and describing) variations of the value or parameter itself. For example, the description of "about X" includes the description of "X".

[0124] As used herein, reference to a value or parameter "not" generally indicates and describes "other than" that value or parameter. For example, "the method cannot be used to treat infection type X" means that the method is generally used to treat infection types other than infection type X. As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0125] Antibodies or antigen-binding domains that specifically bind to LIGHT or TSLP

[0126] The present application provides antibodies or antigen-binding domains that specifically bind to LIGHT or TSLP, including, but not limited to, humanized antibodies, chimeric antibodies, mouse antibodies, human antibodies, and antibody molecules comprising heavy and / or light chain CDRs as described herein. In one aspect, the antibody or antigen-binding domain is an isolated antibody or antigen-binding domain that binds to LIGHT or TSLP. Antibodies or antigen-binding domains that specifically bind to LIGHT or TSLP include, in whole or in part, full-length antibodies (such as full-length IgG1, IgG2, or IgG4) that specifically bind to LIGHT or TSLP, single-chain antibodies that specifically bind to LIGHT or TSLP, multispecific (such as bispecific) antibodies that bind to LIGHT and TSLP, immunoconjugates that specifically bind to LIGHT or TSLP, and the like. In some embodiments, the antibody or antigen-binding domain that specifically binds to LIGHT or TSLP is a Fab, Fab', F(ab)'2, Fab'-SH, single-chain antibody (scFv), Fv fragment, dAb, Fd, nanobody, or double-chain antibody (diabody). In some embodiments, an antibody or antigen-binding domain that specifically binds to LIGHT or TSLP refers to an antibody or antigen-binding domain that binds to LIGHT or TSLP with an affinity that is at least 10 times greater (including, for example, 10 ... 2 , 10 3 , 10 4 , 10 5 , 10 6 , or 10 7 In some embodiments, a non-target is an antigen that is not LIGHT or TSLP.

[0127] Binding affinity can be determined by methods known in the art, such as ELISA, fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation analysis (RIA). Kd values ​​can be determined by methods known in the art, such as surface plasmon resonance (SPR) technology or biolayer interferometry (BLI) technology.

[0128] Although this application generally discusses antibodies or antigen-binding domains that specifically bind to LIGHT or TSLP comprising human sequences (e.g., human heavy and light chain variable regions comprising human CDR sequences), non-human antibodies are also contemplated. In some embodiments, non-human antibodies comprise human CDR sequences and non-human framework region sequences for antibodies or antigen-binding domains that specifically bind to LIGHT or TSLP as described herein. In some embodiments, non-human framework region sequences include any sequence used to generate heavy and / or light chain variable regions using one or more human CDR sequences as described herein, including, for example, mammals such as mice, rats, rabbits, pigs, cattle (e.g., cows, bulls, buffaloes), deer, sheep, goats, chickens, cats, dogs, ferrets, primates (e.g., marmosets, macaques), etc. In some embodiments, non-human antibodies or antigen-binding domains that specifically bind to LIGHT or TSLP include antibodies or antigen-binding domains that specifically bind to LIGHT or TSLP generated by grafting one or more human CDR sequences described herein into non-human framework regions (e.g., mouse or chicken framework region sequences).

[0129] Antibodies or antigen-binding domains that specifically bind to LIGHT

[0130] In one aspect, the present application provides antibodies or antigen-binding domains that specifically bind to LIGHT.

[0131] In some embodiments, the antibody or antigen binding domain that specifically binds to LIGHT binds to LIGHT. In some embodiments, the antibody or antigen binding domain that specifically binds to LIGHT is specific for LIGHT and does not cross-react with species or does not cross-react with other types of non-LIGHT. In some embodiments, the antibody or antigen binding domain that specifically binds to LIGHT cross-reacts with other types of non-LIGHT.

[0132] In some embodiments, the antibody or antigen binding domain that specifically binds to LIGHT comprises: a heavy chain variable region (V H ), the V H comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising GYFIN (SEQ ID NO: 1); HC-CDR2 comprising RIYPYNVDTFYNQNFKG (SEQ ID NO: 2); and HC-CDR3 comprising GTHYYGSSGAMDY (SEQ ID NO: 3), or the V H A variant comprising up to about 5 amino acid substitutions in the HC-CDRs; and a light chain variable region (V L ), the V Lcomprising: a light chain complementary determining region (LC-CDR) 1 comprising KASQNVGTAVA (SEQ ID NO: 4); a LC-CDR2 comprising SASNRYT (SEQ ID NO: 5); and a LC-CDR3 comprising QQYSSYPYT (SEQ ID NO: 6), or said V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0133] In some embodiments, the antibody or antigen binding domain that specifically binds LIGHT comprises:

[0134] (i)V H , comprising the amino acid sequence of SEQ ID NO: 7 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 7; and V L , comprising the amino acid sequence of SEQ ID NO: 10 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 10;

[0135] (ii)V H , comprising the amino acid sequence of SEQ ID NO: 8 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 8; and V L , comprising the amino acid sequence of SEQ ID NO: 11 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 11;

[0136] (iii)V H , comprising the amino acid sequence of SEQ ID NO: 9 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 9; and V L , comprising the amino acid sequence of SEQ ID NO: 11 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 11; or,

[0137] (iv)V H , comprising the amino acid sequence of SEQ ID NO: 7 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 7; and V L , comprising the amino acid sequence of SEQ ID NO: 11 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 11.

[0138] Antibodies or antigen-binding domains that specifically bind to TSLP

[0139] In one aspect, the present application provides antibodies or antigen-binding domains that specifically bind to TSLP.

[0140] In some embodiments, the antibodies or antigen-binding domains that specifically bind to TSLP described herein specifically bind to an epitope in TSLP. In some embodiments, the antibodies or antigen-binding domains that specifically bind to TSLP are specific for TSLP and do not cross-react with species or other non-TSLPs. In some embodiments, the antibodies or antigen-binding domains that specifically bind to TSLP cross-react with other non-TSLPs.

[0141] In some embodiments, the antibody or antigen-binding domain that specifically binds to TSLP described herein comprises: (a) a heavy chain variable region (V H ), the V H comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising SGYGWS (SEQ ID NO: 12); HC-CDR2 comprising YISYYGSISYNPSLKS (SEQ ID NO: 14); and HC-CDR3 comprising TNLLYFDS (SEQ ID NO: 16), or the V H A variant comprising up to about 5 amino acid substitutions in the HC-CDRs; and a light chain variable region (V L ), the V L comprising: a light chain complementary determining region (LC-CDR) 1 comprising RASQSVSNNLA (SEQ ID NO: 18); a LC-CDR2 comprising DASSRAT (SEQ ID NO: 19); and a LC-CDR3 comprising QQYSDWPQYT (SEQ ID NO: 20), or the V L A variant comprising a substitution of up to about 5 amino acids in the LC-CDRs; or (b) a heavy chain variable region (V H ), the V H comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising SYGIN (SEQ ID NO: 13); HC-CDR2 comprising VIVPLVGVTIYAEKFQG (SEQ ID NO: 15); and HC-CDR3 comprising GQEYFYWYFDL (SEQ ID NO: 17), or the V H A variant comprising up to about 5 amino acid substitutions in the HC-CDRs; and a light chain variable region (V L ), the V Lcomprising: a light chain complementary determining region (LC-CDR) 1 comprising SGSSSDIGGYNRVS (SEQ ID NO: 21); a LC-CDR2 comprising DVSKRPS (SEQ ID NO: 22); and a LC-CDR3 comprising SSYAGTDTFIL (SEQ ID NO: 23), or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0142] In some embodiments, the antibody or antigen binding domain that specifically binds to TSLP comprises:

[0143] (i)V H , comprising the amino acid sequence of SEQ ID NO: 24 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 24; and V L , comprising the amino acid sequence of SEQ ID NO: 26 or a variant thereof, said variant having at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 26; or,

[0144] (ii)V H , comprising the amino acid sequence of SEQ ID NO: 25 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 25; and V L , comprising the amino acid sequence of SEQ ID NO: 27 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 27.

[0145] With respect to the aforementioned antibodies or antigen-binding domains that specifically bind to LIGHT or TSLP, in some embodiments, the amino acid substitutions described above are limited to the "exemplary substitutions" shown in Table 8 of this application. In some embodiments, the amino acid substitutions are limited to the "preferred substitutions" shown in Table 8 of this application.

[0146] In some embodiments, the present application provides antibodies or antigen-binding domains that can competitively bind to LIGHT or TSLP with any of the above-mentioned antibodies that specifically bind to LIGHT or TSLP. In some embodiments, the present application provides antibodies or antigen-binding domains that competitively bind to the same epitope as any of the above-mentioned antibodies or antigen-binding domains that specifically bind to LIGHT or TSLP.

[0147] In some embodiments, competition assays can be used to identify monoclonal antibodies or antigen-binding domains that compete with the antibodies or antigen-binding domains described herein for binding to LIGHT or TSLP. Competition assays can determine whether two antibodies bind to the same epitope by recognizing identical or spatially overlapping epitopes or by competitively inhibiting antigen binding by one antibody. In certain embodiments, such competing antibodies bind to the same epitope as the antibodies described herein. Some exemplary competition assays include, but are not limited to, conventional assays described in Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Detailed exemplary methods for resolving epitopes bound by antibodies are described in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ). In some embodiments, each antibody is said to bind to the same epitope if it blocks 50% or more of the binding of the other antibody. In some embodiments, the antibody that competes with the antibody described herein that specifically binds to LIGHT or TSLP is a chimeric antibody, a humanized antibody, or a fully human antibody.

[0148] In some embodiments, the antibody or antigen binding domain that specifically binds to TSLP can be selected from the antibody or antigen binding domain that specifically binds to TSLP described in patent application WO2022166739A1. The sequences of exemplary antibodies or antigen binding domains that specifically bind to LIGHT and TSLP are shown in Tables 2 and 3, wherein the CDR numbering is performed according to the Kabat definition. Those skilled in the art will recognize that there are a variety of known algorithms to predict the position of CDRs and define the light and heavy chain variable regions of antibodies. The CDRs comprising the antibodies as described in this application, the V H and / or V L Sequences, but based on prediction algorithms other than those exemplified in the table below, of antibodies or antigen binding domains are also within the scope of this application.

[0149] Multispecific antibodies that specifically bind to LIGHT and TSLP

[0150] In one aspect, the present application provides a multispecific antibody (preferably, a bispecific antibody) comprising a first antigen-binding domain that specifically binds to LIGHT, and a second antigen-binding domain that specifically binds to TSLP.

[0151] In some embodiments, the first antigen binding domain that specifically binds to LIGHT comprises: a heavy chain variable region (V H ), the V H The heavy chain complementarity determining region (HC-CDR) 1 comprises GYFIN (SEQ ID NO: 1); HC-CDR2 comprises RIYPYNVDTFYNQNFKG (SEQ ID NO: 2); and HC-CDR3 comprises GTHYYGSSGAMDY (SEQ ID NO: 3); and a light chain variable region (V L ), the V L It comprises: a light chain complementarity determining region (LC-CDR) 1 comprising KASQNVGTAVA (SEQ ID NO: 4); a LC-CDR2 comprising SASNRYT (SEQ ID NO: 5); and a LC-CDR3 comprising QQYSSYPYT (SEQ ID NO: 6).

[0152] In some embodiments, the second antigen binding domain that specifically binds to TSLP comprises: (a) a heavy chain variable region (V H ), the V H comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising SGYGWS (SEQ ID NO: 12); HC-CDR2 comprising YISYYGSISYNPSLKS (SEQ ID NO: 14); and HC-CDR3 comprising TNLLYFDS (SEQ ID NO: 16); and a light chain variable region (V L ), the V L comprising: a light chain complementarity determining region (LC-CDR) 1 comprising RASQSVSNNLA (SEQ ID NO: 18); a LC-CDR2 comprising DASSRAT (SEQ ID NO: 19); and a LC-CDR3 comprising QQYSDWPQYT (SEQ ID NO: 20); or (b) a heavy chain variable region (V H ), the V H The heavy chain complementarity determining region (HC-CDR) 1 comprises SYGIN (SEQ ID NO: 13); HC-CDR2 comprises VIVPLVGVTIYAEKFQG (SEQ ID NO: 15); and HC-CDR3 comprises GQEYFYWYFDL (SEQ ID NO: 17); and a light chain variable region (V L ), the V LIt comprises: a light chain complementarity determining region (LC-CDR) 1 comprising SGSSSDIGGYNRVS (SEQ ID NO: 21); a LC-CDR2 comprising DVSKRPS (SEQ ID NO: 22); and a LC-CDR3 comprising SSYAGTDTFIL (SEQ ID NO: 23).

[0153] In some embodiments, the first antigen binding domain that specifically binds LIGHT comprises:

[0154] (a)V H , comprising the amino acid sequence of SEQ ID NO: 7 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 7; and V L , which comprises the amino acid sequence of SEQ ID NO: 10 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 10; or,

[0155] (b)V H , comprising the amino acid sequence of SEQ ID NO: 8 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 8; and V L , which comprises the amino acid sequence of SEQ ID NO: 11 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 11; or,

[0156] (c)V H , comprising the amino acid sequence of SEQ ID NO: 9 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 9; and V L , which comprises the amino acid sequence of SEQ ID NO: 11 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 11; or,

[0157] (d)V H , comprising the amino acid sequence of SEQ ID NO: 7 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 7; and V L , which comprises the amino acid sequence shown in SEQ ID NO:11 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence shown in SEQ ID NO:11.

[0158] In some embodiments, the second antigen binding domain that specifically binds TSLP comprises:

[0159] (a)V H , comprising the amino acid sequence of SEQ ID NO: 24 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 24; and V L , which comprises the amino acid sequence of SEQ ID NO: 26 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 26; or,

[0160] (b)V H , comprising the amino acid sequence of SEQ ID NO: 25 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 25; and V L , which comprises the amino acid sequence shown in SEQ ID NO:27 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity with the amino acid sequence shown in SEQ ID NO:27.

[0161] In some embodiments, the present application provides a multispecific antibody (preferably, a bispecific antibody) comprising a first antigen-binding domain that specifically binds to LIGHT and a second antigen-binding domain that specifically binds to TSLP, wherein the first antigen-binding domain comprises: V H , the V Hcomprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6; and wherein the second antigen binding domain comprises: V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16; and V L , the V L It comprises: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20.

[0162] In some embodiments, the present application provides a multispecific antibody (preferably, a bispecific antibody) comprising a first antigen-binding domain that specifically binds to LIGHT and a second antigen-binding domain that specifically binds to TSLP, wherein the first antigen-binding domain comprises: V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6; and wherein the second antigen binding domain comprises: V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 13, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17; and V L , the V LIt comprises: LC-CDR1 comprising the amino acid sequence of SEQ ID NO:21, LC-CDR2 comprising the amino acid sequence of SEQ ID NO:22, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO:23.

[0163] In some embodiments, the present application provides a multispecific antibody (preferably, a bispecific antibody) comprising a first antigen-binding domain that specifically binds to LIGHT and a second antigen-binding domain that specifically binds to TSLP, wherein the first antigen-binding domain comprises: V H , comprising the amino acid sequence shown in SEQ ID NO: 8 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; and V L , comprising the amino acid sequence of SEQ ID NO: 11 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 11; and wherein the second antigen binding domain comprises: V H , comprising the amino acid sequence of SEQ ID NO: 24 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 24; and V L , which comprises the amino acid sequence shown in SEQ ID NO: 26 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 26.

[0164] In some embodiments, the present application provides a multispecific antibody (preferably, a bispecific antibody) comprising a first antigen-binding domain that specifically binds to LIGHT and a second antigen-binding domain that specifically binds to TSLP, wherein the first antigen-binding domain comprises: V H , comprising the amino acid sequence shown in SEQ ID NO: 8 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; and V L , comprising the amino acid sequence of SEQ ID NO: 11 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 11; and wherein the second antigen binding domain comprises: V H , comprising the amino acid sequence of SEQ ID NO: 25 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 25; and V L, which comprises the amino acid sequence shown in SEQ ID NO: 27 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 27.

[0165] The structure of multispecific antibodies

[0166] As described above, the present application provides a multispecific antibody (preferably, a bispecific antibody) comprising a first antigen-binding domain that specifically binds to LIGHT and a second antigen-binding domain that specifically binds to TSLP. In some preferred embodiments, the structure of the multispecific antibody (preferably, a bispecific antibody) is selected from DVD-Ig, Hetero H, CrossMab, Bs4Ab, IgG-(scFv)2, or scFv-Fab IgG.

[0167] Multispecific antibodies with DVD-Ig structures

[0168] The dual variable domain immunoglobulin molecule DVD-Ig (Dual-variable domain-Ig) multispecific antibody structure (preferably, bispecific antibody) (see, for example, Wu C, et al. Molecular construction and optimization of anti-human IL-1alpha / beta dual variable domain immunoglobulin (DVD-Ig) molecules. MAbs. 2009 Jul-Aug; 1 (4): 339-47) is a V domain of a normal full-length IgG antibody. L and V H The N-terminus of each antibody is connected to the V L and V H domain, through two antibody V H With V L The interaction forms an antigen binding domain that can simultaneously bind to corresponding antigens, thereby achieving multispecificity (preferably, bispecificity). A schematic diagram of the typical structure of this multispecific antibody is shown in Figure 1A.

[0169] In some embodiments, according to any multispecific antibody described herein (preferably, a bispecific antibody), it has a DVD-IgG structure, which is a homodimer and a tetravalent multispecific antibody composed of two identical monomers, wherein each monomer is composed of two polypeptide chains (hereinafter referred to as heavy chain and light chain), including two antigen binding domains, one of which is Fv and the other is Fab, which bind to different antigens respectively, and the two binding domains are connected in series by a connecting peptide (L). In some preferred embodiments, the DVD-Ig structure further includes two Fc regions, the Fc region comprising C H 2 and C H 3 domains.

[0170] In a preferred embodiment of the present application, the multispecific antibody (preferably, a bispecific antibody) has a DVD-Ig structure, wherein two Fab antigen-binding domains specifically bind to a first antigen (e.g., LIGHT), and the other two Fv antigen-binding domains specifically bind to a second antigen (e.g., TSLP). In another preferred embodiment, wherein two Fv antigen-binding domains specifically bind to a first antigen (e.g., LIGHT), and the other two Fab antigen-binding domains specifically bind to a second antigen (e.g., TSLP).

[0171] In some embodiments, the heavy chain of the multispecific antibody (preferably, a bispecific antibody) comprises from N-terminus to C-terminus: V H 1-LV H 2-C H 1 structure. In some embodiments, the light chain of the multispecific antibody comprises, from N-terminus to C-terminus: V L 1-LV L 2-C L structure.

[0172] In some preferred embodiments, the heavy chain further comprises an Fc comprising a C H 2 and C H Therefore, in these embodiments, the heavy chain of the multispecific antibody (preferably, bispecific antibody) comprises from N-terminus to C-terminus: V H 1-LV H 2-C H 1-C H 2-C H 3 structure. In some embodiments, the light chain of the multispecific antibody comprises from N-terminus to C-terminus: V L 1-LV L 2-C L structure.

[0173] Among them, V H 1 and V L 1 are the heavy chain variable region and light chain variable region that specifically bind to an antigen; V H 2 and V L 2 are the heavy chain variable region and light chain variable region that specifically bind to another antigen; L is the connecting peptide; C H 1 is the heavy chain constant region C H 1 domain; C L Is the light chain constant region. H 1 and V L 1 constitutes an antigen-binding domain (Fv) of a multispecific antibody (preferably, a bispecific antibody); V H 2-C H 1 and V L 2-C L The other antigen-binding domain (Fab) constitutes a multispecific antibody (preferably, a bispecific antibody).

[0174] In some embodiments, the leucine (L) at position 234 and the leucine (L) at position 235 of the hinge region of the heavy chain are replaced with alanine (A), forming a combination mutation LALA. This combination of mutations can weaken the binding of the antibody Fc to the FcR receptors CD64, CD32A, CD16, and human complement component C1q, thereby weakening antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The numbering is according to the EU index as in Kabat.

[0175] The present application relates to a multispecific antibody that can bind to LIGHT and TSLP, which has the DVD-Ig structure as described above.

[0176] In some preferred embodiments of the present application, the V H 1 and V L 1 are the heavy chain variable region and light chain variable region that specifically bind to LIGHT, V H 1 and V L 1 constitutes an antigen binding domain (Fv) that specifically binds to LIGHT; V H 2 and V L 2 are the heavy chain variable region and light chain variable region that specifically bind to TSLP, V H 2-C H 1 and V L 2-C L It consists of an antigen-binding domain (Fab) that specifically binds to TSLP.

[0177] In other preferred embodiments of the present application, the V H 1 and V L1 are the heavy chain variable region and light chain variable region that specifically bind to TSLP, V H 1 and V L 1 constitutes the antigen binding domain (Fv) that specifically binds to TSLP; V H 2 and V L 2 are the heavy chain variable region and light chain variable region that specifically bind to LIGHT, V H 2-C H 1 and V L 2-C L It constitutes an antigen-binding domain (Fab) that specifically binds to LIGHT.

[0178] In some embodiments, the multispecific antibody can bind to LIGHT and TSLP simultaneously.

[0179] In some embodiments, the multispecific antibody (preferably, a bispecific antibody) according to the present application comprises the amino acid sequence of SEQ ID NO: 37 or a variant thereof, which has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 37; and / or the amino acid sequence of SEQ ID NO: 38 or a variant thereof, which has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 38.

[0180] In some embodiments, the multispecific antibody (preferably, a bispecific antibody) according to the present application comprises the amino acid sequence of SEQ ID NO: 39 or a variant thereof, which has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 39; and / or the amino acid sequence of SEQ ID NO: 40 or a variant thereof, which has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 40.

[0181] In some embodiments, the multispecific antibody (preferably, a bispecific antibody) according to the present application comprises the amino acid sequence of SEQ ID NO: 41 or a variant thereof, which has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 41; and / or the amino acid sequence of SEQ ID NO: 42 or a variant thereof, which has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 42.

[0182] In some embodiments, the multispecific antibody (preferably, a bispecific antibody) according to the present application comprises the amino acid sequence of SEQ ID NO: 43 or a variant thereof, which has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 43; and / or the amino acid sequence of SEQ ID NO: 44 or a variant thereof, which has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 44.

[0183] In some embodiments, the multispecific antibody (preferably, a bispecific antibody) described herein comprises the amino acid sequence of SEQ ID NO: 57 or a variant thereof, which has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 57; and / or the amino acid sequence of SEQ ID NO: 38 or a variant thereof, which has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 38.

[0184] In some embodiments, the multispecific antibody (preferably, a bispecific antibody) described herein comprises the amino acid sequence of SEQ ID NO: 58 or a variant thereof, which has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 58; and / or the amino acid sequence of SEQ ID NO: 40 or a variant thereof, which has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 40.

[0185] In some embodiments, the multispecific antibody (preferably, a bispecific antibody) according to the present application comprises the amino acid sequence of SEQ ID NO: 59 or a variant thereof, which has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 59; and / or the amino acid sequence of SEQ ID NO: 42 or a variant thereof, which has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 42.

[0186] In some embodiments, the multispecific antibody (preferably, a bispecific antibody) according to the present application comprises the amino acid sequence of SEQ ID NO: 60 or a variant thereof, which has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 60; and / or the amino acid sequence of SEQ ID NO: 44 or a variant thereof, which has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 44.

[0187] Hetero H, a bispecific antibody with a CrossMab structure

[0188] Hetero H, a multispecific antibody (preferably, a bispecific antibody) structure of CrossMab structure (see, for example, Klein C, et al. The use of CrossMAb technology for the generation of bi- and multispecific antibodies. MAbs. 2016 Aug-Sep; 8(6): 1010-20), is a heterodimer based on the exchange of antibody domains within one Fab arm of a bispecific IgG antibody, which can be the exchange of the entire Fab domain (CrossMab Fab) or the exchange of only the variable region of the Fab domain (CrossMab V H -V L ) or exchange of the constant regions only (CrossMab C H 1-C L ), thereby ensuring the correct pairing between the antibody light and heavy chains. In addition, to promote the mutual binding of heterodimers, the Fc region (which contains C H 2 and C H 3 domains) to design knobs-in-holes (KIH) structures, in which one of the C H The amino acid residues in the 3 domain are replaced with amino acid residues with larger side chain volume to form a "knob", and the C in another Fc H The amino acid residues in the 3 domains are replaced with amino acid residues with smaller side chain volumes to form a "hole." Simultaneously, two Cys residue mutations (S354C on the "knob" side and Y349C on the "hole" side) that can form stabilizing disulfide bridges are introduced. The typical structure of this bispecific antibody is shown in Figure 1C.

[0189] In other preferred embodiments, the multispecific antibody (preferably, a bispecific antibody) as described above further comprises two Fcs. In some embodiments, the KIH structure can promote the mutual binding of heterodimers.

[0190] In some embodiments, the Fc is derived from wild-type human IgG1 Fc. In other embodiments, the C H The 3 domain includes, but is not limited to, the following amino acid substitutions: S354C, T366W, Y349C, T366S, L368A, and Y407V, wherein the numbering is according to the EU index as in Kabat.

[0191] In some embodiments, according to any multispecific antibody described herein (preferably, a bispecific antibody), it has a Hetero H, CrossMab structure. This structure is a bivalent bispecific antibody composed of two monomers. The first monomer is composed of two polypeptide chains (hereinafter referred to as the first heavy chain and the first light chain) and contains an antigen binding domain (Fab); the second monomer is also composed of two polypeptide chains (hereinafter referred to as the second heavy chain and the second light chain) and contains another antigen binding domain (Fab). In some embodiments, in the Fab of the first monomer and / or the second monomer, the light chain constant region (C L ) and heavy chain constant region C H 1 domain (C H 1) positions can be interchanged; or the heavy chain variable region (V H ) and light chain variable region (V L ) positions can be interchangeable; or the light chain constant region (C L ) and heavy chain constant region C H 1 domain (C H 1) and the heavy chain variable region (V H ) and light chain variable region (V L ) can be replaced with each other at the same time to ensure the correct pairing between the light and heavy chains. In some embodiments, wherein the multispecific antibody (preferably, a bispecific antibody) further comprises two Fc, which comprises C H 2 and C H 3 domains.

[0192] In a preferred embodiment of the present application, the multispecific antibody (preferably, a bispecific antibody) has a Hetero H, CrossMab structure, wherein one antigen binding domain specifically binds to a first antigen (e.g., LIGHT), and the other antigen binding domain specifically binds to a second antigen (e.g., TSLP).

[0193] In some embodiments, the first heavy chain of the multispecific antibody (preferably, a bispecific antibody) comprises, from N-terminus to C-terminus: V H 1-C H 1 structure; the first light chain from N-terminus to C-terminus includes: V L 1-C L structure.

[0194] In some embodiments, the first heavy chain further comprises an Fc comprising a C H 2 and C H Therefore, in some embodiments, the first heavy chain of the multispecific antibody (preferably, a bispecific antibody) comprises from N-terminus to C-terminus: V H 1-C H 1-C H 2-CH 3 structure; the first light chain from N-terminus to C-terminus includes: V L 1-C L structure.

[0195] Among them, V H 1 and V L 1 are the heavy chain variable region and light chain variable region that specifically bind to the first antigen, C H 1 is the heavy chain constant region C H 1 domain, C L Is the light chain constant region. H 1-C H 1 and V L 1-C L Composition of the antigen binding domain (Fab) that binds the first antigen.

[0196] In some embodiments, the second heavy chain of the multispecific antibody (preferably, a bispecific antibody) comprises, from N-terminus to C-terminus: V H 2-C L Structure; In some embodiments, the second light chain of the multispecific antibody (preferably, a bispecific antibody) comprises from N-terminus to C-terminus: V L 2-C H 1 structure.

[0197] In some embodiments, the second heavy chain further comprises an Fc comprising a C H 2 and C H Therefore, in some embodiments, the second heavy chain of the multispecific antibody (preferably, bispecific antibody) comprises from N-terminus to C-terminus: V H 2-C L -C H 2-C H 3 structure. In some embodiments, the second light chain of the multispecific antibody (preferably, bispecific antibody) comprises from N-terminus to C-terminus: V L 2-C H 1 structure.

[0198] Among them, V H 2 and V L 2 are the heavy chain variable region and light chain variable region that specifically bind to the second antigen, C H 1 is the heavy chain constant region C H 1 domain, C L Is the light chain constant region. H 2-C L and V L 2-C H 1 constitutes the antigen-binding domain (Fab) that binds a second antigen.

[0199] In some embodiments, the C in the first monomer or the second monomer of the multispecific antibody (preferably, a bispecific antibody) L and C H 1 are replaced with each other. In some embodiments, V H 1 and V L 1 can be replaced with each other. In other embodiments, V H 2 and V L The positions of 2 can be interchanged.

[0200] In some embodiments, the C of the first heavy chain H The amino acid residues in the 3 domain are replaced by amino acid residues with larger side chain volume to form a "knob", and the C of the second heavy chain H In some other embodiments, the amino acid residues in the C domain of the second heavy chain are replaced with amino acid residues with smaller side chain volume to form a "hole". H The amino acid residues in the 3 domain are replaced by amino acid residues with larger side chain volume to form a "knob" and the C of the first heavy chain H The amino acid residues in the C3 domain are replaced with amino acid residues with smaller side chain volume to form a "hole". In some embodiments, the C H The 3 domain includes, but is not limited to, the following amino acid substitutions: S354C, T366W, Y349C, T366S, L368A, and Y407V, wherein the numbering is according to the EU index as in Kabat.

[0201] The present application relates to a multispecific antibody (preferably, a bispecific antibody) that can bind to LIGHT and TSLP, which has the Hetero H, CrossMab structure as described above.

[0202] In some preferred embodiments of the present application, V H 1 and V L 1 are the heavy chain variable region and light chain variable region that specifically bind to TSLP, V H 2 and V L 2 are the heavy chain variable region and light chain variable region that specifically bind to LIGHT, V H 1-C H 1 and V L 1-C L Composition of the antigen binding domain (Fab) that specifically binds to TSLP, V H 2-C L and V L 2-C H 1 constitutes the antigen-binding domain (Fab) that specifically binds to LIGHT.

[0203] In some other preferred embodiments of the present application, VH 1 and V L 1 are the heavy chain variable region and light chain variable region that specifically bind to LIGHT, V H 2 and V L 2 are the heavy chain variable region and light chain variable region that specifically bind to TSLP, V H 1-C H 1 and V L 1-C L Composition of the antigen binding domain (Fab) that specifically binds to LIGHT, V H 2-C L and V L 2-C H 1 constitutes an antigen binding domain (Fab) that specifically binds to TSLP. In some embodiments of the present application, the multispecific antibody (preferably, a bispecific antibody) can bind to LIGHT and TSLP simultaneously.

[0204] In some embodiments, the multispecific antibody (preferably, a bispecific antibody) according to the present application comprises the amino acid sequence of SEQ ID NO: 46 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 46; and / or the amino acid sequence of SEQ ID NO: 48 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 48. And / or the amino acid sequence of SEQ ID NO: 45 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 45. and / or the amino acid sequence of SEQ ID NO: 47 or a variant thereof, which variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47.

[0205] In some embodiments, the multispecific antibody (preferably, a bispecific antibody) according to the present application comprises the amino acid sequence of SEQ ID NO: 47 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 47; and / or the amino acid sequence of SEQ ID NO: 48 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 48; and / or the amino acid sequence of SEQ ID NO: 49 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 49; and / or the amino acid sequence of SEQ ID NO: 50 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 51. ID NO: 50 has at least about 80% (eg, at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0206] In some embodiments, the multispecific antibody (preferably, a bispecific antibody) according to the present application comprises the amino acid sequence of SEQ ID NO: 51 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 51; and / or the amino acid sequence of SEQ ID NO: 52 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 52; and / or the amino acid sequence of SEQ ID NO: 53 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 53; and / or the amino acid sequence of SEQ ID NO: 54 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 54; ID NO: 54 has at least about 80% (eg, at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0207] In some embodiments, the multispecific antibody (preferably, a bispecific antibody) according to the present application comprises the amino acid sequence of SEQ ID NO: 55 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 55; and / or the amino acid sequence of SEQ ID NO: 56 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 56; and / or the amino acid sequence of SEQ ID NO: 53 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 53; and / or the amino acid sequence of SEQ ID NO: 54 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: ID NO: 54 has at least about 80% (eg, at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0208] In some embodiments, the multispecific antibody (preferably, a bispecific antibody) according to the present application comprises the amino acid sequence of SEQ ID NO: 46 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 46; and / or the amino acid sequence of SEQ ID NO: 48 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 48; and / or the amino acid sequence of SEQ ID NO: 61 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 61; and / or the amino acid sequence of SEQ ID NO: 62 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: ID NO: 62 has at least about 80% (eg, at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0209] In some embodiments, the multispecific antibody (preferably, a bispecific antibody) according to the present application comprises the amino acid sequence of SEQ ID NO: 48 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 48; and / or the amino acid sequence of SEQ ID NO: 50 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 50; and / or the amino acid sequence of SEQ ID NO: 62 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 62; and / or the amino acid sequence of SEQ ID NO: 63 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: ID NO: 63 has at least about 80% (eg, at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0210] In some embodiments, the multispecific antibody (preferably, a bispecific antibody) according to the present application comprises the amino acid sequence of SEQ ID NO: 52 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 52; and / or the amino acid sequence of SEQ ID NO: 54 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 54; and / or the amino acid sequence of SEQ ID NO: 64 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 64; and / or the amino acid sequence of SEQ ID NO: 65 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 65; ID NO: 65 has at least about 80% (eg, at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0211] In some embodiments, the multispecific antibody (preferably, a bispecific antibody) according to the present application comprises the amino acid sequence of SEQ ID NO: 54 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 54; and / or the amino acid sequence of SEQ ID NO: 56 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 56; and / or the amino acid sequence of SEQ ID NO: 65 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 65; and / or the amino acid sequence of SEQ ID NO: 66 or a variant thereof, wherein the variant has at least about 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: ID NO: 66 has at least about 80% (eg, at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) sequence identity.

[0212] Bs4Ab structured bispecific antibody

[0213] The multispecific antibody of the Bs4Ab structure is a tetravalent antibody that includes a full-length IgG structure and achieves multispecificity (preferably, bispecificity) by inserting another binding unit scFv into its hinge region. The multispecific antibodies (e.g., bispecific antibodies) of the Bs4Ab structure are also described in the literature Bezabeh B, et al. Insertion of scFv into the hinge domain of full-length IgG1 monoclonal antibody results in tetravalent bispecific molecule with robust properties. MAbs. 2017 Feb / Mar; 9(2): 240-256. A schematic diagram of the typical structure of the bispecific antibody is shown in Figure 1B.

[0214] In some embodiments, the multispecific antibody (preferably, a bispecific antibody) described herein has a Bs4Ab structure comprising two identical monomers, each monomer comprising two polypeptide chains, namely a heavy chain and a light chain. Each monomer comprises two antigen-binding domains, one of which is a Fab and the other is a scFv. In some embodiments, the scFv is connected to the Fab via a first connecting peptide (L1) and to the Fc via a second connecting peptide (L2).

[0215] In some embodiments, the multispecific antibody (preferably, a bispecific antibody) further comprises an Fc comprising a C H 2 and C H 3 domains.

[0216] In some embodiments, the heavy chain of a multispecific antibody (preferably a bispecific antibody) comprises, from N-terminus to C-terminus: V H 1-C H 1-L1-V H 2-L3-V L 2 structure. In other embodiments, the heavy chain of the multispecific antibody (preferably, bispecific antibody) comprises from N-terminus to C-terminus: V H 1-C H 1-L1-V L 2-L3-V H 2 structure.

[0217] In some embodiments, the heavy chain further comprises an Fc comprising a C H 2 and C H Therefore, in some embodiments, the heavy chain of a multispecific antibody (preferably a bispecific antibody) comprises from N-terminus to C-terminus: V H 1-C H 1-L1-V H 2-L3-V L 2-L2-C H 2-C H 3 structure. In other embodiments, the heavy chain of the multispecific antibody (preferably, bispecific antibody) comprises from N-terminus to C-terminus: V H 1-C H 1-L1-V L 2-L3-V H 2-L2-C H 2-C H 3 structure.

[0218] In some embodiments, the light chain of a multispecific antibody (preferably, a bispecific antibody) comprises, from N-terminus to C-terminus: V L 1-C Lstructure.

[0219] Among them, V H 1 and V L 1 are the heavy chain variable region and light chain variable region that specifically bind to one of the antigens, V H 2 and V L 2 are the heavy chain variable region and light chain variable region that specifically bind to another antigen, C H 1 is the heavy chain constant region C H 1 domain, C L is the light chain constant region, L1, L2 and L3 are connecting peptides. H 1-C H 1 and V L 1-C L One of the antigen binding domains (Fab) of a multispecific antibody (preferably a bispecific antibody), V H 2-L3-V L 2 or V L 2-L3-V H 2 constitutes the other antigen-binding domain (scFv) of a multispecific antibody (preferably, a bispecific antibody).

[0220] In some embodiments, V H 1 and V L 1 are the heavy chain variable region and light chain variable region that specifically bind to TSLP, V H 1-C H 1 and V L 1-C L Composition of the antigen binding domain (Fab) that specifically binds to TSLP; V H 2 and V L 2 are the heavy chain variable region and light chain variable region that specifically bind to LIGHT, V H 2-L3-V L 2 or V L 2-L3-V H 2 constitutes an antigen binding domain (scFv) that specifically binds to LIGHT.

[0221] In other embodiments, V H 1 and V L 1 are the heavy chain variable region and light chain variable region that specifically bind to LIGHT, V H 1-C H 1 and V L 1-C L Composition of the antigen binding domain (Fab) that specifically binds to LIGHT; V H 2 and V L 2 are the heavy chain variable region and light chain variable region that specifically bind to TSLP, V H2-L3-V L 2 or V L 2-L3-V H 2 constitutes an antigen binding domain (scFv) that specifically binds to TSLP.

[0222] In some embodiments of the present application, one of the antigen binding domains (Fab or scFv) specifically binds to TSLP, and the other antigen binding domain (scFv or Fab) specifically binds to LIGHT. In some embodiments, the multispecific antibody (preferably, a bispecific antibody) can bind to LIGHT and TSLP simultaneously.

[0223] In some embodiments, the antigen binding domain scFv that specifically binds to LIGHT and TSLP includes genetically engineered cysteine ​​mutations by H and V L Two cysteine ​​mutations were introduced into the interface to obtain a disulfide bond-stabilized multispecific antibody (preferably, a bispecific antibody).

[0224] In some embodiments, the multispecific antibody (preferably, a bispecific antibody) comprises C H 1, which comprises the amino acid sequence shown in any one of SEQ ID NOs:35-36.

[0225] In some embodiments, the multispecific antibody (preferably, a bispecific antibody) comprises C H 2-C H 3, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 32-34. In some embodiments, the multispecific antibody (preferably, a bispecific antibody) comprises C H 2-C H 3, which comprises one or more substitutions at positions 22, 24, and 26 relative to the amino acid sequence shown in any one of SEQ ID NOs: 32-34. In some embodiments, the multispecific antibody (preferably, a bispecific antibody) comprises C H 2-C H 3, which comprises one or more substitutions of M22Y, S24T, T26E relative to the amino acid sequence of any one of SEQ ID NOs: 32-34. In some embodiments, the multispecific antibody (preferably, a bispecific antibody) comprises C H 2-C H 3, which comprises one or more substitutions at positions 198 and 204 relative to the amino acid sequence shown in any one of SEQ ID NOs: 32-34. In some embodiments, the multispecific antibody (preferably, a bispecific antibody) comprises CH 2-C H 3, which comprises one or more substitutions of M198L, N204S relative to the amino acid sequence shown in any one of SEQ ID NOs: 32-34.

[0226] Multispecific antibodies with IgG-(scFv)2 structure

[0227] IgG-(scFv)2 multispecific antibodies (e.g., bispecific antibodies) are antibodies in which scFv fragments that bind to other antigens are attached to the Fc termini of the two heavy chains of an IgG antibody to achieve multispecificity (preferably, bispecificity). Multispecific antibodies with an IgG-(scFv)2 structure are also described in the literature Coloma MJ, Morrison SL. Design and production of novel tetravalent bispecific antibodies. Nat Biotechnol. 1997 Feb; 15(2): 159-63. A schematic diagram of a typical bispecific antibody is shown in Figure 1D.

[0228] In some embodiments, the multispecific antibodies (preferably, bispecific antibodies) described herein have an IgG-(scFv)2 structure, which is composed of two identical monomers, each monomer containing two polypeptide chains, namely a heavy chain and a light chain; each monomer contains two antigen-binding domains, one of which is a Fab and the other is a scFv.

[0229] In some embodiments, the multispecific antibody (preferably, a bispecific antibody) further comprises an Fc comprising a C H 2 and C H The scFv is connected to the carboxyl terminus of Fc via a linker peptide (L).

[0230] In some embodiments, the heavy chain of a multispecific antibody (preferably a bispecific antibody) comprises, from N-terminus to C-terminus: V H 1-C H 1 structure. In some embodiments, the light chain of the multispecific antibody (preferably, a bispecific antibody) comprises from N-terminus to C-terminus: V L 1-C L .

[0231] In some embodiments, the heavy chain further comprises an Fc comprising a C H 2 and C H 3 domains.

[0232] In some embodiments, the heavy chain of a multispecific antibody (preferably a bispecific antibody) comprises, from N-terminus to C-terminus: V H 1-C H 1-C H 2-C H 3-LV H 2-L3-V L 2 structure. In other embodiments, the heavy chain of the multispecific antibody (preferably, bispecific antibody) comprises from N-terminus to C-terminus: V H 1-C H 1-C H 2-C H 3-LV L 2-L3-V H 2 Structure. In some embodiments, the light chain of a multispecific antibody (preferably, a bispecific antibody) comprises, from N-terminus to C-terminus: V L 1-C L structure.

[0233] Among them, V H 1 and V L 1 are the heavy chain variable region and light chain variable region that specifically bind to one of the antigens, V H 2 and V L 2 are the heavy chain variable region and light chain variable region that specifically bind to another antigen, C H 1 is the heavy chain constant region C H 1 domain, C L V is the light chain constant region, L and L3 are connecting peptides. H 1-C H 1 and V L 1-C L One of the antigen binding domains (Fab) of a multispecific antibody (preferably a bispecific antibody), V H 2-L3-V L 2 or V L 2-L3-V H 2. Another antigen-binding domain (scFv) constituting a multispecific antibody (preferably, a bispecific antibody)

[0234] In some embodiments of the present application, one of the antigen binding domains (Fab or scFv) specifically binds to TSLP, and the other antigen binding domain (scFv or Fab) specifically binds to LIGHT. In some embodiments, the multispecific antibody (preferably, a bispecific antibody) can bind to LIGHT and TSLP simultaneously.

[0235] In some embodiments, V H 1 and V L1 are the heavy chain variable region and light chain variable region that specifically bind to TSLP, V H 1-C H 1 and V L 1-C L Composition of the antigen binding domain (Fab) that specifically binds to TSLP; V H 2 and V L 2 are the heavy chain variable region and light chain variable region that specifically bind to LIGHT, V H 2-L3-V L 2 or V L 2-L3-V H 2 constitutes an antigen binding domain (scFv) that specifically binds to LIGHT.

[0236] In other embodiments, V H 1 and V L 1 are the heavy chain variable region and light chain variable region that specifically bind to LIGHT, V H 1-C H 1 and V L 1-C L Composition of the antigen binding domain (Fab) that specifically binds to LIGHT; V H 2 and V L 2 are the heavy chain variable region and light chain variable region that specifically bind to TSLP, V H 2-L3-V L 2 or V L 2-L3-V H 2 constitutes an antigen binding domain (scFv) that specifically binds to TSLP.

[0237] In some embodiments, the antigen binding domain scFv that specifically binds to LIGHT or TSLP includes genetically engineered cysteine ​​mutations by H and V L Two cysteine ​​mutations were introduced into the interface to obtain a disulfide bond-stabilized multispecific antibody (preferably, a bispecific antibody).

[0238] In some embodiments, the multispecific antibody (preferably, a bispecific antibody) comprises C H 1, which comprises the amino acid sequence shown in any one of SEQ ID NOs:35-36.

[0239] In some embodiments, the multispecific antibody (preferably, a bispecific antibody) comprises C H 2-C H 3, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 32-34. In some embodiments, the multispecific antibody (preferably, a bispecific antibody) comprises CH 2-C H 3, which comprises one or more substitutions at positions 22, 24, and 26 relative to the amino acid sequence shown in any one of SEQ ID NOs: 32-34. In some embodiments, the multispecific antibody (preferably, a bispecific antibody) comprises C H 2-C H 3, which comprises one or more substitutions of M22Y, S24T, T26E relative to the amino acid sequence of any one of SEQ ID NOs: 32-34. In some embodiments, the multispecific antibody (preferably, a bispecific antibody) comprises C H 2-C H 3, which comprises one or more substitutions at positions 198 and 204 relative to the amino acid sequence shown in any one of SEQ ID NOs: 32-34. In some embodiments, the multispecific antibody (preferably, a bispecific antibody) comprises C H 2-C H 3, which comprises one or more substitutions of M198L, N204S relative to the amino acid sequence shown in any one of SEQ ID NOs: 32-34.

[0240] Multispecific antibodies with scFv-Fab IgG structures

[0241] scFv-Fab IgG structure multispecific antibodies are heterodimeric antibodies, IgG antibody structures, in which one Fab arm is replaced with an scFv structure that binds to a different antigen, thereby achieving multispecificity (preferably, bispecificity). In a preferred embodiment of the present application, the typical structure of this bispecific antibody is shown in Figure 1E.

[0242] In some embodiments, the multispecific antibodies (preferably, bispecific antibodies) described herein have an scFv-Fab IgG structure in the form of a heterodimer. The heterodimeric antibody comprises a first monomer and a second monomer, wherein the first monomer is composed of two polypeptide chains (hereinafter referred to as the first heavy chain and the light chain) and comprises an antigen-binding domain (Fab) capable of binding to one antigen; and the second monomer is composed of one polypeptide chain (hereinafter referred to as the second heavy chain) and comprises an antigen-binding domain (scFv) capable of binding to the other antigen.

[0243] In some embodiments, the multispecific antibody (preferably, a bispecific antibody) further comprises two Fcs comprising C H 2 and C H The 3 domains further contain amino acid substitutions in the two Fc domains, which can promote the mutual binding of heterodimers.

[0244] In some embodiments, the first heavy chain of any multispecific antibody (preferably, a bispecific antibody) described herein comprises, from N-terminus to C-terminus: V H 1-C H 1 structure. The light chain contains V L 1-C L structure.

[0245] In some embodiments, the first heavy chain further comprises an Fc comprising a C H 2 and C H 3 domains.

[0246] In some embodiments, the first heavy chain comprises, from N-terminus to C-terminus: V H 1-C H 1-C H 2-C H 3 structures, the light chain contains V L 1-C L structure.

[0247] Among them, V H 1 and V L 1 are the heavy chain variable region and light chain variable region that specifically bind to one of the antigens, C H 1 is the heavy chain constant region C H 1 domain, C L V is the light chain constant region. H 1-C H 1 and V L 1-C L It constitutes one of the antigen binding domains (Fab).

[0248] In some embodiments, the second heavy chain of any multispecific antibody (preferably, a bispecific antibody) described herein comprises, from N-terminus to C-terminus: V H 2-L3-V L 2 structure. In some other embodiments, the second heavy chain of any multispecific antibody (preferably, bispecific antibody) described herein comprises, from N-terminus to C-terminus: V L 2-L3-V H 2 structure.

[0249] In some embodiments, the second heavy chain further comprises an Fc comprising a C H 2 and C H 3 domains.

[0250] In some embodiments, the second heavy chain comprises, from N-terminus to C-terminus: V H 2-L3-V L 2-C H 2-C H3 structure. In some other embodiments, the second heavy chain of any multispecific antibody (preferably, bispecific antibody) described herein comprises, from N-terminus to C-terminus: V L 2-L3-V H 2-C H 2-C H 3 structure.

[0251] Where V H 2 and V L 2 are the heavy chain variable region and light chain variable region that specifically bind to another antigen, and L3 is a connecting peptide. H 2-L3-V L 2 or V L 2-L3-V H 2 constitutes another antigen-binding domain (scFv).

[0252] In some embodiments, V H 1 and V L 1 are the heavy chain variable region and light chain variable region that specifically bind to TSLP, V H 1-C H 1 and V L 1-C L Composition of the antigen binding domain (Fab) that specifically binds to TSLP. H 2 and V L 2 are the heavy chain variable region and light chain variable region that specifically bind to LIGHT, V H 2-L3-V L 2 or V L 2-L3-V H 2 constitutes an antigen binding domain (scFv) that specifically binds to LIGHT.

[0253] In some embodiments, V H 1 and V L 1 are the heavy chain variable region and light chain variable region that specifically bind to LIGHT, V H 1-C H 1 and V L 1-C L Composition of the antigen binding domain (Fab) that specifically binds to LIGHT. H 2 and V L 2 are the heavy chain variable region and light chain variable region that specifically bind to TSLP, V H 2-L3-V L 2 or V L 2-L3-V H 2 constitutes an antigen binding domain (scFv) that specifically binds to TSLP.

[0254] In some embodiments, one of the antigen binding domains (Fab or scFv) specifically binds to LIGHT, and the other antigen binding domain (scFv or Fab) specifically binds to TSLP. In some embodiments, the multispecific antibody (preferably, a bispecific antibody) can simultaneously bind to LIGHT and TSLP.

[0255] In some embodiments, the antigen binding domain scFv that specifically binds to LIGHT or TSLP includes genetically engineered cysteine ​​mutations by H and V L Two cysteine ​​mutations were introduced into the interface to obtain a disulfide bond-stabilized multispecific antibody (preferably, a bispecific antibody).

[0256] In some embodiments, the Fc is derived from human wild-type IgG1. In another embodiment, the Fc in one monomer comprises, but is not limited to, the following amino acid substitutions E357Q, and S364K relative to human wild-type IgG1; the Fc in another monomer comprises, but is not limited to, the following amino acid substitutions Q295E, L368D, K370S, N384D, Q418E, and N421D relative to human wild-type IgG1, wherein the numbering is according to the EU index as in Kabat. In some embodiments, the V in an exemplary linked scFv is H 1 and V L The connecting peptide of 1 (eg, L3) comprises the sequence GKPGSGKPGSGKPGSGKPGS (SEQ ID NO: 78).

[0257] Connector peptide

[0258] Connecting peptides (or, may be referred to as "linkers") can be used to connect the domains and / or structural regions of the heavy chains of multispecific antibodies (preferably, bispecific antibodies) into a continuous molecule. The multispecific antibodies (preferably, bispecific antibodies) may include additional linkers, such as flexible linkers that connect the variable heavy chain and light chain of scFv. In some embodiments, the multispecific antibodies (preferably, bispecific antibodies) may include additional linkers, such as flexible linkers that connect the variable heavy chain and light chain of scFv and other linkers for connecting other binding units to the core structure of the multispecific antibody (preferably, bispecific antibody).

[0259] In one embodiment, the present invention relates to a polypeptide chain comprising at least 4 residues. The position of a connecting peptide (or joint) is typically, hydrophilic, and the joints themselves rarely or do not form a secondary structure (joint site or flexible joint site). After the molecule is assembled, at least 4 amino acid whose joints can be used to connect domains and / or districts close to each other. Longer joints can also be used. In certain embodiments, the joint can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 100, 125, 150, 175 or 200 residues. When using multiple joints to connect the various parts of the molecule, the joint can be identical or different (for example, identical or different length and / or amino acid sequence).

[0260] In certain aspects, the connecting peptide comprises or consists of a glycine-serine linker. As used herein, the term "glycine-serine linker" refers to a peptide consisting of glycine and serine residues. Exemplary glycine-serine linkers include those of the general formula (Gly4Ser) n An amino acid sequence of , wherein n is a positive integer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10). A preferred glycine-serine linker is (Gly4Ser)2, i.e., GGGGSGGGGS (SEQ ID NO: 67). A preferred glycine-serine linker is (Gly4Ser)4, i.e., GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 68). A preferred glycine-serine linker is (Gly4Ser)3, i.e., GGGGSGGGGSGGGGS (SEQ ID NO: 69). In other aspects, two or more glycine-serine linkers are tandem in a connecting peptide. In certain aspects, the connecting peptide comprises at least a portion of the hinge region (e.g., derived from an IgG1, IgG2, IgG3 or IgG4 molecule) and a series of glycine-serine residues (e.g., a glycine-serine linker, such as (G4S) n .

[0261] In certain embodiments, L1 and / or L2 include a hinge portion and a linker portion, such as a linker portion comprising a glycine-serine linker. In other aspects, L1 and / or L2 include only a hinge portion or only a linker portion, such as a glycine-serine linker. In certain aspects, L1 and L2 include a glycine-serine linker. In certain aspects, the glycine-serine linker portions of L1 and L2 are the same length, while in other aspects, the glycine-serine linker portions of L1 and L2 are different in length. When a multispecific antibody (preferably a bispecific antibody) comprises an scFv, the heavy chain and light chain of the scFv can be connected by a flexible linker. In certain embodiments, such a flexible linker generally does not include a hinge portion, but is a glycine-serine linker or other flexible linker. The length and amino acid sequence of the flexible linker that interconnects the scFv domains can be selected and optimized.

[0262] In some embodiments, the connecting peptide (e.g., L1 and / or L2) comprises a glycine-serine or all-glycine linker and a portion or a modified portion of the hinge region. In certain aspects, the connecting peptide (L1) connecting one antigen-binding domain (e.g., Fab or scFv) and the other antigen-binding domain (e.g., scFv or Fab) in a multispecific antibody (preferably a bispecific antibody) comprises the amino acid sequence EPKSDKTGGGGSGGGGS (SEQ ID NO: 72) or EPKSCGKTGGGGSGGGGS (SEQ ID NO: 73) or EPKSCGGGGSGGGGS (SEQ ID NO: 74). In certain aspects, the connecting peptide (L2) connecting the antigen-binding domain scFv and Fc in the multispecific antibody (preferably, a bispecific antibody) comprises the amino acid sequence GGGGSGGGGSEPKSDKTHTCPPCP (SEQ ID NO: 75) or GGGGSGGGGSCPPCP (SEQ ID NO: 76) or GGGGSGGGGSDKTHTCPPCP (SEQ ID NO: 77). In some embodiments, the connecting peptide (L2) connecting the antigen-binding domain and the carboxyl-terminal C-terminal end of the Fc region in the multispecific antibody (preferably, a bispecific antibody) comprises the amino acid sequence GGGGSGGGGSEPKSDKTHTCPPCP (SEQ ID NO: 75) or GGGGSGGGGSCPPCP (SEQ ID NO: 76) or GGGGSGGGGSDKTHTCPPCP (SEQ ID NO: 77). H The connecting peptide of 3 (eg, L) comprises the amino acid sequence GGGGSGGGGTGGGGS (SEQ ID NO: 79).

[0263] In some embodiments, the multispecific antibody (preferably, a bispecific antibody) may optionally comprise additional connecting peptides in addition to the connecting peptides connecting one antigen binding domain to another antigen binding domain or connecting one of the antigen binding domains to Fc (e.g., L1 and L2). The length and sequence of these additional connecting peptides are independently selected. For example, the multispecific antibody (preferably, a bispecific antibody) may further comprise a flexible connecting peptide (L3) that connects the variable heavy chain and light chain (V1) in the antigen binding domain scFv. HSCFV and V LSCFV ). This flexible connecting peptide may include a glycine-serine linker. Typically, this linker does not include a hinge portion. In some embodiments, the flexible connecting peptide (L3) connecting the variable heavy chain and light chain of the scFv comprises the amino acid sequence ASTKGP (SEQ ID NO: 70) or TVAAP (SEQ ID NO: 71). In some embodiments, the flexible connecting peptide (L3) connecting the variable heavy chain and light chain of the scFv comprises the sequence GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 68).

[0264] Exemplary antibody sequences are shown in Tables 2 to 6, where CDR numbering is based on the EU index as in Kabat. Those skilled in the art will recognize that there are a variety of known algorithms for predicting the positions of CDRs and defining antibody light and heavy chain variable regions. The CDRs, VDRs, and VDRs of antibodies or antigen-binding domains that specifically bind to LIGHT or TSLP as described herein, as well as multispecific antibodies (preferably bispecific antibodies) that specifically bind to LIGHT and TSLP, are described herein. H and / or V L Sequences, but based on prediction algorithms other than those exemplified in the table below, are also within the scope of this application.

[0265] The antibody or antigen-binding domain that specifically binds to TSLP can be selected from the anti-TSLP antibodies described in patent application WO2022166739A1, which is incorporated into the present application by reference.

[0266] Table 2-1: CDR sequences of exemplary antibodies or antigen-binding domains that specifically bind to LIGHT

[0267] Table 2-2: CDR sequences of exemplary antibodies or antigen-binding domains that specifically bind to TSLP

[0268] Table 3-1: V of exemplary antibodies or antigen-binding domains that specifically bind to LIGHT H / V L sequence

[0269] Table 3-2: Vs of exemplary antibodies or antigen-binding domains that specifically bind to TSLP H / V L sequence

[0270] Table 4: Exemplary antibody constant region sequences

[0271] Table 5-1: DVD-IgG structure Sequences of partial heavy chains (excluding Fc domain) and light chains of exemplary bispecific antibodies that specifically bind to LIGHT and TSLP

[0272] Table 5-2: Sequences of partial heavy chains (excluding Fc domain) and light chains of Hetero H, CrossMab structures of exemplary bispecific antibodies that specifically bind to LIGHT and TSLP

[0273] Table 6-1: DVD-IgG structures Full-length sequences of heavy and light chains of exemplary bispecific antibodies that specifically bind to LIGHT and TSLP

[0274] Table 6-2: Hetero H, CrossMab structure. Full-length heavy and light chain sequences of exemplary bispecific antibodies that specifically bind to LIGHT and TSLP.

[0275] Table 7: Exemplary connecting peptide (or linker) sequences

[0276] Antibody composition

[0277] In one aspect, the present application provides a pharmaceutical composition comprising: (i) an antibody or antigen-binding fragment that specifically binds to LIGHT and (ii) an antibody or antigen-binding fragment that specifically binds to TSLP.

[0278] In some embodiments, a pharmaceutical composition is provided, comprising: (i) an antibody or antigen-binding fragment that specifically binds to LIGHT and (ii) an antibody or antigen-binding fragment that specifically binds to TSLP, wherein the antibody or antigen-binding fragment that specifically binds to LIGHT comprises: a heavy chain variable region (V H ), the V HThe heavy chain complementarity determining region (HC-CDR) 1 comprises GYFIN (SEQ ID NO: 1); HC-CDR2 comprises RIYPYNVDTFYNQNFKG (SEQ ID NO: 2); and HC-CDR3 comprises GTHYYGSSGAMDY (SEQ ID NO: 3); and a light chain variable region (V L ), the V L It comprises: a light chain complementarity determining region (LC-CDR) 1 comprising KASQNVGTAVA (SEQ ID NO: 4); a LC-CDR2 comprising SASNRYT (SEQ ID NO: 5); and a LC-CDR3 comprising QQYSSYPYT (SEQ ID NO: 6).

[0279] In some embodiments, a pharmaceutical composition is provided, comprising: (i) an antibody or antigen-binding fragment that specifically binds to LIGHT and (ii) an antibody or antigen-binding fragment that specifically binds to TSLP, wherein the antibody or antigen-binding fragment that specifically binds to TSLP comprises: (a) a heavy chain variable region (V H ), the V H comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising SGYGWS (SEQ ID NO: 12); HC-CDR2 comprising YISYYGSISYNPSLKS (SEQ ID NO: 14); and HC-CDR3 comprising TNLLYFDS (SEQ ID NO: 16); and a light chain variable region (V L ), the V L comprising: a light chain complementarity determining region (LC-CDR) 1 comprising RASQSVSNNLA (SEQ ID NO: 18); a LC-CDR2 comprising DASSRAT (SEQ ID NO: 19); and a LC-CDR3 comprising QQYSDWPQYT (SEQ ID NO: 20); or (b) a heavy chain variable region (V H ), the V H The heavy chain complementarity determining region (HC-CDR) 1 comprises SYGIN (SEQ ID NO: 13); HC-CDR2 comprises VIVPLVGVTIYAEKFQG (SEQ ID NO: 15); and HC-CDR3 comprises GQEYFYWYFDL (SEQ ID NO: 17); and a light chain variable region (V L ), the V LIt comprises: a light chain complementarity determining region (LC-CDR) 1 comprising SGSSSDIGGYNRVS (SEQ ID NO: 21); a LC-CDR2 comprising DVSKRPS (SEQ ID NO: 22); and a LC-CDR3 comprising SSYAGTDTFIL (SEQ ID NO: 23).

[0280] In some embodiments, the antibody or antigen-binding fragment that specifically binds to LIGHT in the pharmaceutical composition comprises:

[0281] (a)V H , comprising the amino acid sequence of SEQ ID NO: 7 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 7; and V L , comprising the amino acid sequence of SEQ ID NO: 10 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 10;

[0282] (b)V H , comprising the amino acid sequence of SEQ ID NO: 8 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 8; and V L , comprising the amino acid sequence of SEQ ID NO: 11 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 11;

[0283] (c)V H , comprising the amino acid sequence of SEQ ID NO: 9 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 9; and V L , comprising the amino acid sequence of SEQ ID NO: 11 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 11; or

[0284] (d)V H , comprising the amino acid sequence of SEQ ID NO: 7 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 7; and V L , comprising the amino acid sequence of SEQ ID NO: 11 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 11.

[0285] In some embodiments, the antibody or antigen-binding fragment that specifically binds to TSLP in the pharmaceutical composition comprises:

[0286] (a)V H , comprising the amino acid sequence of SEQ ID NO: 24 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 24; and V L , comprising the amino acid sequence of SEQ ID NO: 26 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 26; or

[0287] (b)V H , comprising the amino acid sequence of SEQ ID NO: 25 or a variant thereof, said variant having at least about 80% sequence identity to the amino acid sequence of SEQ ID NO: 25; and V L , comprising the amino acid sequence of SEQ ID NO: 27 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 27.

[0288] In some embodiments, a pharmaceutical composition is provided comprising: an antibody or antigen-binding fragment that specifically binds to LIGHT and an antibody or antigen-binding fragment that specifically binds to TSLP, wherein the antibody or antigen-binding fragment that specifically binds to LIGHT comprises: V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6, or the V L wherein the antibody or antigen-binding fragment that specifically binds to TSLP comprises: V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 12, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the VL comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 18, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0289] In some embodiments, a pharmaceutical composition is provided comprising: an antibody or antigen-binding fragment that specifically binds to LIGHT and an antibody or antigen-binding fragment that specifically binds to TSLP, wherein the antibody or antigen-binding fragment that specifically binds to LIGHT comprises: V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6, or the V L wherein the antibody or antigen-binding fragment that specifically binds to TSLP comprises: V H , the V H comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 13, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17, or the V H A variant comprising up to about 5 amino acid substitutions in its HC-CDRs; and V L , the V L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 21, LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 23, or the V L A variant comprising up to about 5 amino acid substitutions in its LC-CDRs.

[0290] In some embodiments, a pharmaceutical composition is provided comprising: an antibody or antigen-binding fragment that specifically binds to LIGHT and an antibody or antigen-binding fragment that specifically binds to TSLP, wherein the antibody or antigen-binding fragment that specifically binds to LIGHT comprises: V H , comprising the amino acid sequence shown in SEQ ID NO: 8 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; and V L , comprising the amino acid sequence of SEQ ID NO: 11 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 11; and wherein the antibody or antigen-binding fragment that specifically binds to TSLP comprises: V H , comprising the amino acid sequence of SEQ ID NO: 24 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 24; and V L , which comprises the amino acid sequence shown in SEQ ID NO: 26 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 26.

[0291] In some embodiments, a pharmaceutical composition is provided comprising: an antibody or antigen-binding fragment that specifically binds to LIGHT and an antibody or antigen-binding fragment that specifically binds to TSLP, wherein the antibody or antigen-binding fragment that specifically binds to LIGHT comprises: V H , comprising the amino acid sequence shown in SEQ ID NO: 8 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; and V L , comprising the amino acid sequence of SEQ ID NO: 11 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 11; and wherein the antibody or antigen-binding fragment that specifically binds to TSLP comprises: V H , comprising the amino acid sequence of SEQ ID NO: 25 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence of SEQ ID NO: 25; and V L , which comprises the amino acid sequence shown in SEQ ID NO: 27 or a variant thereof, wherein the variant has at least about 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 27.

[0292] Binding affinity

[0293] Binding affinity can be expressed as Kd, Koff, Kon or Ka. As used in this application, the term "Koff" refers to the rate constant at which the antigen binding domain dissociates from the antigen binding domain / antigen complex, as measured by a kinetic selection device. The term "Kon" refers to the association rate constant of the antibody binding to the antigen to form the antigen binding domain / antigen complex. The dissociation constant "Kd" used in this application refers to the dissociation constant when a specific antibody-antigen interaction occurs, and refers to the antigen concentration required when the antigen occupies half of all antibody binding domains in the antibody molecule solution and reaches equilibrium, which is equal to Koff / Kon. The determination of Kd assumes that all binding molecules are in solution. In cases where the antigen binding domain is attached to the cell wall, for example in a yeast expression system, the corresponding dissociation rate constant is expressed using EC 50 It is expressed as , which is a good approximation of Kd. The affinity binding constant, Ka, is the reciprocal of the dissociation constant, Kd.

[0294] The equilibrium dissociation constant (Kd) can be used as an indicator of the affinity of the antigen-binding domain for the antigen. For example, simple analysis can be performed using antibodies labeled with various markers using the Scatchard method and a Biacore instrument (manufactured by Amersham Biosciences) to analyze the interaction between biomolecules by surface plasmon resonance according to the user manual or the accompanying kit. The Kd values ​​obtained using these methods are expressed in units of M. Antibodies that specifically bind to a target may have a Kd of ≤10 -7 M, ≤10 -8 M, for example ≤10 -9 M, ≤10 -10 M, ≤10 -11 M, ≤10 -12 M or ≤10 -13 Kd value of M.

[0295] The binding specificity of an antibody can be determined experimentally by methods known in the art, including, but not limited to, Western blots, ELISA, RIA, ECL, IRMA, EIA, BIAcore assays, and peptide scanning.

[0296] In some embodiments, the antibody or antigen-binding fragment that specifically binds to LIGHT specifically binds to the LIGHT target with a Kd value of 10 -7 M to 10 -13 M (e.g. 10 -7 M to 10 -13 M, 10 -8 M to 10 -13 M, 10 -9 M to 10 -13 M or 10 -10 M to 10-12 M). Therefore, in some embodiments, the Kd value of the binding between the antibody or antigen-binding fragment that specifically binds to LIGHT and LIGHT is 10 -7 M to 10 -13 M, 1×10 -7 M to 5×10 -13 M, 10 -7 M to 10 -12 M, 10 -7 M to 10 -11 M, 10 - 7 M to 10 -10 M, 10 -7 M to 10 -9 M, 10 -8 M to 10 -13 M, 1×10 -8 M to 5×10 -13 M, 10 -8 M to 10 -12 M, 10 - 8 M to 10 -11 M, 10 -8 M to 10 -10 M, 10 -8 M to 10 -9 M, 5×10 -9 M to 1×10 -13 M, 5×10 -9 M to 1×10 -12 M, 5×10 -9 M to 1×10 -11 M, 5×10 -9 M to 1×10 -10 M, 10 -9 M to 10 -13 M, 10 -9 M to 10 -12 M, 10 -9 M to 10 -11 M, 10 -9 M to 10 -10 M, 5×10 -10 M to 1×10 -13 M, 5×10 -10 M to 1×10 -12 M, 5×10 -10 M to 1×10 -11 M, 10 -10 M to 10 -13 M, 1×10 -10 M to 5×10 -13 M, 1×10 -10 M to 1×10-12 M, 1×10 -10 M to 5×10 -12 M, 1×10 -10 M to 1×10 -11 M, 10 -11 M to 10 -13 M, 1×10 -11 M to 5×10 -13 M, 10 -11 M to 10 -12 M, 10 -12 M to 10 -13 In some embodiments, the Kd value of the binding between the antibody or antigen-binding fragment that specifically binds to LIGHT and LIGHT is 10 -7 M to 10 -13 M.

[0297] In some embodiments, the Kd value for binding between an antibody or antigen-binding fragment that specifically binds to LIGHT and a non-target is higher than the Kd value for binding between the antibody or antigen-binding fragment that specifically binds to LIGHT and a target, and in some embodiments cited in this application, the binding affinity of the antibody or antigen-binding fragment that specifically binds to LIGHT and a target (e.g., LIGHT) is higher than the binding affinity of the antibody or antigen-binding fragment that specifically binds to LIGHT and a non-target. In some embodiments, non-target refers to non-LIGHT. In some embodiments, the Kd value for binding between an antibody or antigen-binding fragment that specifically binds to LIGHT and a non-LIGHT target is at least 10 times the Kd for binding between the antibody or antigen-binding fragment that specifically binds to LIGHT and the target LIGHT, for example, 10 - 10 2 times, 10 2 -10 3 times, 10 3 -10 4 times, 10 4 -10 5 times, 10 5 -10 6 times, 10 6 -10 7 times, 10 7 -10 8 times, 10 8 -10 9 times, 10 9 -10 10 times, 10 10 -10 11 times, 10 11 -10 12 times.

[0298] In some embodiments, any of the multispecific antibodies described herein comprises a first antigen binding domain that specifically binds to LIGHT, and a second antigen binding domain that specifically binds to TSLP. The first antigen binding domain specifically binds to the LIGHT target with a Kd value of 10 -7 M to 10 -13 M (e.g. 10 -7 M to 10 -13 M, 10 -8 M to 10 -13 M, 10 -9 M to 10 -13 M or 10 -10 M to 10 -12 M). Therefore, in some embodiments, the Kd value of the binding between the first antigen binding domain and LIGHT is 10 -7 M to 10 -13 M, 1×10 -7 M to 5×10 -13 M, 10 -7 M to 10 -12 M, 10 -7 M to 10 -11 M, 10 -7 M to 10 -10 M, 10 -7 M to 10 -9 M, 10 -8 M to 10 -13 M, 1×10 -8 M to 5×10 -13 M, 10 -8 M to 10 -12 M, 10 -8 M to 10 -11 M, 10 -8 M to 10 -10 M, 10 -8 M to 10 -9 M, 5×10 -9 M to 1×10 -13 M, 5×10 -9 M to 1×10 -12 M, 5×10 -9 M to 1×10 -11 M, 5×10 - 9 M to 1×10 -10 M, 10 -9 M to 10 -13 M, 10 -9 M to 10 -12 M, 10 -9 M to 10 -11 M, 10 -9 M to 10-10 M, 5×10 -10 M to 1×10 -13 M, 5×10 -10 M to 1×10 -12 M, 5×10 -10 M to 1×10 -11 M, 10 -10 M to 10 -13 M, 1×10 - 10 M to 5×10 -13 M, 1×10 -10 M to 1×10 -12 M, 1×10 -10 M to 5×10 -12 M, 1×10 -10 M to 1×10 -11 M, 10 -11 M to 10 -13 M, 1×10 -11 M to 5×10 -13 M, 10 -11 M to 10 -12 M, 10 -12 M to 10 -13 In some embodiments, the Kd value of the binding between the first antigen binding domain and LIGHT is 10 -7 M to 10 -13 The second antigen binding domain specifically binds to the TSLP target with a Kd value of 10 -7 M to 10 -13 M (e.g. 10 -7 M to 10 -13 M, 10 -8 M to 10 -13 M, 10 -9 M to 10 - 13 M or 10 -10 M to 10 -12 M). Thus, in some embodiments, the Kd value for binding between the second antigen binding domain and TSLP is 10 -7 M to 10 -13 M, 1×10 -7 M to 5×10 -13 M, 10 -7 M to 10 -12 M, 10 -7 M to 10 -11 M, 10 -7 M to 10 -10 M, 10 -7 M to 10 -9 M, 10 -8 M to 10-13 M, 1×10 -8 M to 5×10 -13 M, 10 -8 M to 10 -12 M, 10 -8 M to 10 -11 M, 10 -8 M to 10 -10 M, 10 -8 M to 10 -9 M, 5×10 -9 M to 1×10 -13 M, 5×10 -9 M to 1×10 -12 M, 5×10 -9 M to 1×10 -11 M, 5×10 -9 M to 1×10 -10 M, 10 -9 M to 10 -13 M, 10 -9 M to 10 -12 M, 10 -9 M to 10 -11 M, 10 -9 M to 10 -10 M, 5×10 -10 M to 1×10 -13 M, 5×10 -10 M to 1×10 -12 M, 5×10 -10 M to 1×10 -11 M, 10 -10 M to 10 -13 M, 1×10 -10 M to 5×10 -13 M, 1×10 -10 M to 1×10 -12 M, 1×10 -10 M to 5×10 -12 M, 1×10 -10 M to 1×10 -11 M, 10 -11 M to 10 -13 M, 1×10 -11 M to 5×10 -13 M, 10 -11 M to 10 -12 M, 10 -12 M to 10 -13 In some embodiments, the Kd value of the binding between the second antigen binding domain and TSLP is 10 -7 M to 10 -13 M.

[0299] In some embodiments, any of the multispecific antibodies described in the present application comprises a first antigen binding domain that specifically binds to LIGHT, and a second antigen binding domain that specifically binds to TSLP. The Kd value of the binding between the first antigen binding domain and the non-target is higher than the Kd value of the first antigen binding domain and the target, and in some embodiments cited in the present application, the binding affinity of the first antigen binding domain to the target (e.g., LIGHT) is higher than the binding affinity of the first antigen binding domain to the non-target. In some embodiments, the non-target refers to non-LIGHT. In some embodiments, the Kd value of the binding of the first antigen binding domain to the non-LIGHT target is at least 10 times the Kd of the binding between the first antigen binding domain and the target LIGHT, for example 10 - 10 2 times, 10 2 -10 3 times, 10 3 -10 4 times, 10 4 -10 5 times, 10 5 -10 6 times, 10 6 -10 7 times, 10 7 -10 8 times, 10 8 -10 9 times, 10 9 -10 10 times, 10 10 -10 11 times, 10 11 -10 12 times. Wherein the Kd value of the binding between the second antigen binding domain and the non-target is higher than the Kd value of the second antigen binding domain and the target, and in some embodiments cited in this application, the binding affinity of the second antigen binding domain to the target (e.g., TSLP) is higher than the binding affinity of the second antigen binding domain to the non-target. In some embodiments, the non-target refers to non-TSLP. In some embodiments, the Kd value of the binding between the second antigen binding domain and the non-TSLP target is at least 10 times the Kd of the binding between the second antigen binding domain and TSLP, for example 10 - 10 2 times, 10 2 -10 3 times, 10 3 -10 4 times, 10 4 -10 5 times, 10 5 -10 6 times, 10 6 -10 7 times, 10 7-10 8 times, 10 8 -10 9 times, 10 9 -10 10 times, 10 10 -10 11 times, 10 11 -10 12 times.

[0300] Preparation of nucleic acids, vectors and antibodies

[0301] Nucleic acids and vectors

[0302] Nucleic acid molecules encoding antibodies or antigen-binding fragments that specifically bind LIGHT, as well as multispecific antibodies that specifically bind LIGHT and TSLP, are also contemplated.

[0303] In some embodiments, the present application provides a nucleic acid (or a group of nucleic acids) encoding an antibody or antigen-binding fragment that specifically binds to LIGHT, or a nucleic acid encoding a multispecific antibody that specifically binds to LIGHT and TSLP, including any of the antibodies or antigen-binding fragments that specifically bind to LIGHT or the multispecific antibodies that specifically bind to LIGHT and TSLP described herein. In some embodiments, the nucleic acid (or a group of nucleic acids) encoding the antibody, antigen-binding fragment, or multispecific antibody described herein may further include a nucleic acid sequence encoding a polypeptide tag (e.g., a protein purification tag, a His-tag, an HA tag).

[0304] At the same time, the present application also provides antibodies or antigen-binding fragments that specifically bind to LIGHT, and multispecific antibodies that specifically bind to LIGHT and TSLP; or nucleic acid molecules encoding the antibodies or antigen-binding fragments; or isolated host cells containing vectors carrying the nucleic acid molecules.

[0305] The present application also includes variants of these nucleic acid sequences. For example, variants include nucleotide sequences that hybridize to a nucleic acid sequence encoding an antibody, antigen-binding fragment, or multispecific antibody of the present application under at least moderately stringent hybridization conditions.

[0306] The present application also provides a vector into which the nucleic acid sequence of the present application can be inserted.

[0307] In brief, a natural or synthetic nucleic acid encoding an antibody, antigen-binding fragment, or multispecific antibody is inserted into a suitable expression vector such that the nucleic acid is operably linked to 5' and 3' regulatory elements, such as a promoter (e.g., a lymphocyte-specific promoter) and a 3' untranslated region (UTR), to express the antibody, antigen-binding fragment, or multispecific antibody. The vector is suitable for replication and integration in eukaryotic host cells. Typical cloning and expression vectors contain transcriptional and translational terminators, initiation sequences, and promoters that regulate the expression of the target nucleic acid sequence.

[0308] The nucleic acids described herein can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Nucleic acid delivery methods are known in the art. For example, see US Pat. Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety. In some embodiments, the present application also provides gene therapy vectors.

[0309] Nucleic acids can be cloned into many types of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0310] In addition, the expression vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described in, for example, Green and Sambrook (2013, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and other virology or molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and slow viruses. Typically, suitable vectors include a replication origin that works in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers (see, for example, WO 01 / 96584; WO 01 / 29058; and US Pat. No. 6,326,193).

[0311] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Techniques known in the art can be applied to insert the selected gene into a vector and package it in retroviral particles. The recombinant virus is then isolated and delivered to the cells of the subject in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow for long-term stable integration of transgenes and their propagation in daughter cells. Lentiviral vectors have additional advantages over tumor-derived retroviruses, such as mouse leukemia viruses, because they can transduce non-dividing cells, such as hepatocytes. At the same time, they also have the additional advantage of low immunogenicity.

[0312] Other promoter elements, such as enhancers, regulate the frequency of transcription initiation. These are typically located 30-110 bp upstream of the start site, although many promoters have recently been found to contain functional elements downstream of the start site. The spacing between promoter elements is often flexible, so that promoter function is maintained even when elements are swapped or moved relative to one another. In the thymidine kinase (Tk) promoter, activity begins to decline only when the spacing between promoter elements increases to 50 bp.

[0313] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a very strong constitutive promoter sequence that can drive high-level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is the elongation factor 1α (EF-1α) promoter. However, other constitutive promoters may also be used, including but not limited to, simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus long terminal repeat (HIV-LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, including but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. In addition, this application should not be limited to the use of only constitutive promoters. Inducible promoters are also considered in this application. The use of an inducible promoter provides a molecular switch that can activate expression of an operably linked polynucleotide sequence when such expression is desired, and deactivate expression when such expression is not desired. Inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0314] In some embodiments, expression of the antibody, antigen-binding fragment, or multispecific antibody is inducible. In some embodiments, the nucleic acid sequence encoding the antibody, antigen-binding fragment, or multispecific antibody is operably linked to an inducible promoter, including any of the inducible promoters described herein.

[0315] Inducible promoter

[0316] The use of an inducible promoter provides a molecular switch that turns on the expression of an operably linked polynucleotide sequence when expression is desired, and turns off expression when expression is not desired. Exemplary inducible promoters suitable for use in eukaryotic cells include, but are not limited to, hormone-regulated elements (e.g., see Mader, S. and White, JH (1993) Proc. Natl. Acad. Sci. USA 90:5603-5607), synthetic ligand-regulated elements (see Spencer, DM et al (1993) Science 262:1019-1024), and ionizing radiation-regulated elements (see Manome, Y. et al (1993) Biochemistry 32:10607-10613; Datta, R. et al (1992) Proc. Natl. Acad. Sci. USA 89:1014-10153). Other exemplary inducible promoters suitable for use in in vivo or in vitro mammalian systems are described in Gingrich et al. (1998) Annual Rev. Neurosci 21:377-405. In some embodiments, the inducible promoter system used to express the antibody, antigen-binding fragment, or bispecific antibody is the Tet system. In some embodiments, the inducible promoter system used to express the antibody, antigen-binding fragment, or bispecific antibody is the E. coli lac repression system.

[0317] An exemplary inducible promoter system used in this application is the Tet system. This system is based on the Tet system described by Gossen et al. (1993). In an exemplary embodiment, the target polynucleotide is controlled by a promoter comprising one or more Tet operator (TetO) sites. In the inactive state, the Tet repressor (TetR) binds to the TetO site and inhibits transcription of the promoter. In the active state, for example, in the presence of an inducer such as tetracycline (Tc), anhydrotetracycline, doxycycline (Dox) or its active analogs, the inducer releases TetR from TetO, thereby causing transcription to occur. Doxycycline is a member of the tetracycline antibiotic family, and its chemical name is 1-dimethylamino-2,4a,5,7-pentahydroxy-11-methyl-4,6-dioxy-1,4a,11,11a,12,12a-hexahydrotetraene-3-carboxamide.

[0318] In one embodiment, TetR is codon-optimized for expression in mammalian cells, such as mouse or human cells. Due to the degeneracy of the genetic code, most amino acids are encoded by more than one codon, so that the sequence of a given nucleic acid has a large number of variants without any change in the amino acid sequence it encodes. However, many organisms differ in codon usage, also known as "codon preference" (i.e., the preference for using a specific codon for a given amino acid). Codon preference is generally associated with the presence of a dominant tRNA species for a particular codon, which in turn improves the efficiency of mRNA translation. Therefore, coding sequences derived from specific species (e.g., prokaryotes) can be customized by codon optimization to improve their expression in different species (e.g., eukaryotes).

[0319] Other specific variants of the Tet system include the following "Tet-Off" and "Tet-On" systems. In the Tet-off system, transcription is inactivated in the presence of Tc or Dox. In this system, a tetracycline-regulated transcription activator protein (tTA), composed of a fusion of TetR and the strong transcriptional activation domain of herpes simplex virus VP16, regulates the expression of the target nucleic acid under the transcriptional control of a tetracycline-responsive promoter element (TRE). The TRE element consists of a TetO sequence fused in series with a promoter (usually a minimal promoter sequence derived from the immediate early promoter of human cytomegalovirus). In the absence of Tc or Dox, tTA binds to TRE and activates transcription of the target gene. In the presence of Tc or Dox, tTA cannot bind to TRE and the target gene cannot be expressed.

[0320] In contrast, in the Tet-On system, transcription is activated in the presence of either Tc or Dox. The Tet-On system is based on the reverse tetracycline-regulated transcriptional activator rtTA. Like tTA, rtTA is a fusion protein consisting of the TetR repressor and the VP16 transactivation domain. However, a four-amino acid change in the DNA-binding region of TetR alters rtTA's binding properties, allowing it to only recognize the tetO sequence within the target transgene's TRE in the presence of Dox. Therefore, in the Tet-On system, rtTA can activate transcription of its TRE-regulated target gene only in the presence of Dox.

[0321] Another inducible promoter system is the lac repressor system of Escherichia coli (see Brown et al., Cell 49: 603-612 (1987)). The lac repressor system functions by regulating the transcription of a target polynucleotide operably linked to a promoter comprising the lac operator (lacO). The lac repressor (lacR) binds to LacO, thereby preventing transcription of the target polynucleotide. Expression of the target polynucleotide is induced by a suitable inducing agent, for example, isopropyl-β-D-thiogalactopyranoside (IPTG).

[0322] In order to evaluate the expression of a polypeptide or portion thereof, the expression vector to be introduced into the cell may also contain a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from a cell population transfected or infected by a viral vector. In other aspects, the selectable marker can be carried on a separate DNA fragment and used in co-transfection experiments. Either the selectable marker gene or the reporter gene can be flanked by suitable regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neo and similar genes.

[0323] Reporter genes can be used to identify potential transfected cells and evaluate the function of regulatory sequences. Generally, a reporter gene is a gene that is not present in or is not expressed by a recipient organism or tissue, and encodes a polypeptide whose expression is characterized by some properties that are easy to detect, such as enzymatic activity. After DNA is introduced into the recipient cells, the expression of the reporter gene is detected at the appropriate time. Suitable reporter genes can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein. Suitable expression systems are well known and can be prepared or obtained commercially by known techniques. Generally, a construct with a minimum 5' flanking region that can show the highest expression level of a reporter gene is identified as a promoter. This type of promoter region can be connected to a reporter gene and used to assess the ability of certain substances in regulating promoter-driven transcription.

[0324] In some embodiments, nucleic acids encoding any one of the antibodies or antigen-binding fragments or multispecific antibodies described herein are provided. In some embodiments, the nucleic acid includes one or more nucleic acid sequences encoding the heavy and light chains of the antibodies or antigen-binding fragments or multispecific antibodies. In some embodiments, each of the one or more nucleic acid sequences is contained in a separate vector. In some embodiments, at least some nucleic acid sequences are contained in the same vector. In some embodiments, all nucleic acid sequences are contained in the same vector. The vector can be selected from, for example, mammalian expression vectors and viral vectors (such as vectors derived from retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses).

[0325] Methods for introducing genes into cells and expressing them are known in the art. In the context of expression vectors, the vectors can be readily introduced into host cells, such as mammalian cells, bacteria, yeast, or insect cells, by any method known in the art. For example, expression vectors can be introduced into host cells by physical, chemical, or biological methods.

[0326] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, biolistic methods, microinjection, electroporation, and the like. Methods for preparing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Green and Sambrook (2013, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). In some embodiments, polynucleotides are introduced into host cells by calcium phosphate transfection.

[0327] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus type 1, adenoviruses, and adeno-associated viruses, among others. See, for example, US Pat. Nos. 5,350,674 and 5,585,362.

[0328] Chemical methods for introducing polynucleotides into host cells include colloidal dispersion systems, such as polymer complexes, nanocapsules, microspheres, magnetic beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery vehicle in vivo and in vitro is a liposome (e.g., an artificial membrane vesicle).

[0329] In another aspect, the nucleic acid can be bound to a lipid. The lipid-bound nucleic acid can be encapsulated into the aqueous interior of a liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linker molecule that binds to the liposome and the oligonucleotide, embedded in the liposome, form a complex with the liposome, dispersed in a solution containing lipids, mixed with lipids, bound to lipids, suspended in lipids, contained in or mixed with micelles, or otherwise bound to lipids. Lipid, lipid / DNA, or lipid / expression vector-related compositions are not limited to any particular structure in solution. For example, they may exist as a bilayer structure, as micelles, or in a "collapsed" structure. They may also simply be dispersed in solution, possibly forming aggregates of uneven size or shape. Lipids are fatty substances that can be naturally occurring or synthetic. For example, lipids include fat droplets that naturally occur in the cytoplasm, as well as a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0330] Regardless of the method used to introduce the exogenous nucleic acid into the host cell or otherwise expose the cell to the inhibitor of the present application, a variety of experiments can be performed to confirm the presence of the recombinant DNA sequence in the host cell. Such experiments include, for example, "molecular biology" experiments well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR, and PCR; "biochemical" experiments, such as detecting the presence or absence of a particular polypeptide, such as by immunological methods (ELISAs and Western blots) or identification by the experiments described in this application, all fall within the scope of this application.

[0331] Preparation of antibodies or antigen-binding fragments and multispecific antibodies

[0332] In some embodiments, the antibody or antigen-binding fragment (e.g., an antibody or antigen-binding fragment that specifically binds LIGHT) is a monoclonal antibody. In some embodiments, the antibody or antigen-binding fragment or multispecific antibody is derived from a monoclonal antibody. In some embodiments, the antibody, antigen-binding fragment or multispecific antibody comprises a V from a monoclonal antibody. H and V L In some embodiments, the antibody, antigen-binding fragment or multispecific antibody further comprises a C from a monoclonal antibody. H 1 and C Lregion, or variants thereof. Monoclonal antibodies can be prepared using methods known in the art, such as hybridoma methods, yeast display, phage display methods, or recombinant DNA methods. In addition, exemplary yeast display and phage display methods are described in this application and in the following examples. Multispecific antibodies can be prepared using methods known in the art, such as chemical coupling, hybridoma methods, and genetic engineering methods.

[0333] In the hybridoma method, hamsters, mice or other suitable host animals are usually immunized with an immunizing agent to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro. The immunizing agent may include a polypeptide or fusion protein of the target protein. Typically, if human cells are needed, peripheral blood lymphocytes (PBLs) are used, and if non-human mammalian derived cells are needed, spleen cells or lymph node cells are used. Lymphocytes are fused with immortalized cell lines using an appropriate fusion agent, such as polyethylene glycol, to form hybridoma cells. Immortalized cell lines are typically transformed mammalian cells, especially myeloma cells of rodent, bovine and human origin. Rat or mouse myeloma cell lines are typically used. Hybridoma cells can be cultured in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of unfused immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), hybridoma cell culture medium typically includes hypoxanthine, aminopterin, and thymidine (HAT medium), which prevents the growth of HGPRT-deficient cells.

[0334] In some embodiments, the immortalized cell line is effectively fused, ensures high-level stable expression of the antibody by the selected antibody-producing cells, and is sensitive to certain culture media, such as HAT medium. In some embodiments, the immortalized cell line is a mouse myeloma cell line, which can be obtained from, for example, the Salk Cell Collection in San Diego, California and the American Type Culture Collection in Manassas, Virginia. Human myeloma and mouse-human hybrid myeloma cell lines are also described for use in preparing human monoclonal antibodies.

[0335] The presence of monoclonal antibodies against the polypeptide in the culture medium of the hybridoma cells can then be determined. The binding specificity of the monoclonal antibodies produced by the hybridoma cells can be determined by immunoprecipitation or in vitro binding experiments, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques or analytical methods are known in the art. The binding affinity of the monoclonal antibodies can be determined by Scatchard analysis as described in, for example, Munson and Pollard, Anal. Biochem., 107:220 (1980).

[0336] After identifying the desired hybridoma cells, the target clones can be subcloned by limiting dilution and cultured by standard methods. Suitable culture media for this purpose include, for example, modified Eagle medium (DMEM) and RPMI-1640 culture medium. Alternatively, hybridoma cells can be grown in mammalian ascites.

[0337] The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0338] In some embodiments, according to any of the antibodies or antigen-binding fragments or multispecific antibodies described herein, the antibodies or antigen-binding fragments or multispecific antibodies comprise sequences selected from clones of an antibody library (e.g., a phage library displaying scFv or Fab fragments). The clones can be identified by methods for screening combinatorial libraries of antibody fragments having the desired activity. For example, various methods are known in the art for generating phage display libraries and screening these libraries to obtain antibodies with the desired binding properties. These methods are reviewed, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and in, for example, McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al. It is further described in Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004).

[0339] In certain phage display methods, the repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, and then screened for phage that can bind to the antigen, as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Phage typically display antibody fragments in the form of scFv fragments or Fab fragments. Library phage derived from immune sources provide high-affinity antibodies to the immunogen without the need to construct hybridoma cells. Alternatively, natural libraries (e.g., from humans) can be cloned to provide a single source of antibodies to a variety of non-self antigens and self antigens without any immunization, as described in Griffiths et al., EMBO J, 12:725-734 (1993). Finally, natural libraries can also be prepared by cloning non-rearranged V-gene fragments from stem cells and using PCR primers containing random sequences to encode CDR3 hypervariable regions and complete rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, US Pat. No. 5,750,373 and US Patent Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936 and 2009 / 0002360.

[0340] The antibody or antigen-binding fragment or multispecific antibody is prepared by screening the library for antigen-binding portions that can specifically bind to a target (e.g., LIGHT) through phage display. The library can be a human scFv phage display library with at least 1×10 9 (For example, at least 1×10 9 , 2.5×10 9 , 5×10 9 , 7.5×10 9 , 1×10 10 , 2.5×10 10 , 5×10 10 , 7.5×10 10 or 1×10 11) kinds of diverse unique human antibody fragments. In some embodiments, the library is a human natural library, constructed by DNA extracted from PMBCs and spleens of healthy subjects, comprising all human heavy and light chain subfamilies. In some embodiments, the library is a human natural library, constructed by DNA extracted from PMBCs isolated from patients with various diseases, such as patients with autoimmune diseases, cancer patients, and patients with infectious diseases. In some embodiments, the library is a semisynthetic human library, wherein the heavy chain CDR3 is completely random, and all amino acids (except cysteine) are present at any given position with the same probability. (See, for example, Hoet, RM et al., Nat. Biotechnol. 23 (3): 344-348, 2005). In some embodiments, the heavy chain CDR3 length of the semisynthetic human library is between 5 and 24 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24) amino acids. In some embodiments, the library is a fully synthetic phage display library. In some embodiments, the library is a non-human phage display library.

[0341] The phage clone that target antigen (for example, LIGHT) has high affinity can be screened by the iterative combination of phage and target antigen, described target antigen is combined with solid support (for example, the pearl for solution elutriation or the mammalian cell for cell elutriation), next remove unconjugated phage, and wash-out specifically binds phage.Subsequently, the phage clone of wash-out combination is also used for infecting suitable host cell, for example E.coli XL1-Blue, is expressed and purified.Can be by many rounds of elutriation (for example, 2,3,4,5,6 or more rounds), for example solution elutriation, cell elutriation or both are combined with the phage clone of enrichment specific binding target antigen.The phage clone of enrichment can detect by any method known in the art with the specific combination of target antigen, including for example ELISA and FACS.

[0342] Monoclonal antibodies can also be prepared by recombinant DNA methods, such as those described in US Patent No. 4,816,567. The DNA encoding the monoclonal antibodies described in this application can be easily isolated and sequenced by conventional methods (e.g., by oligonucleotide probes that can specifically bind to the genes encoding the light and heavy chains of mouse antibodies). The hybridoma cells described above or the antigen-specific phage clones of this application can be used as the source of such DNA. After isolation, the DNA can be placed in an expression vector, which is then transfected into a host cell, such as a simian COS cell, a Chinese hamster ovary carcinoma (CHO) cell, or a myeloma cell that does not produce immunoglobulins, to obtain monoclonal antibodies synthesized in recombinant host cells. The DNA can also be modified, for example, by replacing homologous non-human sequences with coding sequences for human heavy and light chain constant structures and / or framework regions (U.S. Patent No. 4,816,567; Morrison et al., supra), or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to an immunoglobulin coding sequence. Such non-immunoglobulin polypeptides can replace the constant region of the antibody in the present application, or can replace one antigen binding site in the variable region of the antibody in the present application to form a chimeric bivalent antibody. In some embodiments, additional variable regions directed against different epitopes or antigens can be included to generate chimeric multispecific antibodies.

[0343] The antibody can be a monovalent antibody. Methods for preparing monovalent antibodies are known in the art. For example, a method involves recombinant expression of immunoglobulin light chains and modified heavy chains. The heavy chain is typically truncated at any position in the Fc region to prevent cross-linking of the heavy chains. Alternatively, the relevant cysteine ​​residues are replaced with other amino acid residues or deleted to prevent cross-linking.

[0344] In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce antibody fragments, particularly Fab fragments, can be accomplished using any method known in the art.

[0345] Chemical coupling is the earliest technology used to prepare multispecific antibodies. In 1985, Brennan first used the chemical conjugation of two monoclonal antibody G1 fragments to prepare multispecific antibodies (Brennan M, et al. Preparation of bispecific antibodies by chemical recombination of monoclonal immunoglobulin G1 fragments [J]. Science, 1985, 229(4708):81-83). There are two main methods for chemical conjugation: one is to directly conjugate two monoclonal antibodies or their derivatives to form a multispecific antibody; the other is to first dissociate the two monoclonal antibodies into free light and heavy chains through various physical and chemical methods, and then recombine these light and heavy chains. The advantages of chemical conjugation are rapidity, ease of operation, and high recovery rate, but it can also easily damage the antigen-binding domain of the antibody, affecting antibody activity, and easily form multimers.

[0346] The use of hybridoma cell lines to prepare multispecific antibodies involves fusing two different hybridoma cell lines through cell fusion technology, and then identifying and isolating cells that can produce specific therapeutic antibodies (Kohler, G, et al. Continuous cultures of fused cells secreting antibodies of predefined specificity [J]. J Immunol., 2005, 174(5): 2453-2455). Because two hybridoma cells can produce two different light-heavy chains, and these light-heavy chains can be randomly combined, the multispecific antibodies prepared using this method have a high degree of randomness and low production efficiency.

[0347] Genetic engineering technology is currently also used to prepare a variety of multispecific antibodies (Roland E K. Antibody-cytokine fusion proteins [J]. Arch Biochem Biophys., 2012, 526 (2): 194-205). Using genetic engineering to edit recombinant antibodies can solve the problem of random combination by limiting the selectivity of the two pairs of light-heavy chain binding in various ways. KiH (Knob into hole) and CrossMab are two commonly used technologies currently used to improve the light-heavy chain pairing problem. KiH technology, that is, in C H 3 domains introduced asymmetric mutation structures (“knob” mutation refers to the mutation in C HIn the 3 domain, a large amino acid residue is substituted for a smaller residue, while a "hole" mutation refers to the use of a small amino acid residue to replace a larger residue). The Fc region of the engineered multispecific antibody is more inclined to heterodimerize rather than homodimerize due to the influence of steric hindrance (Ridgway JB, et al. "Knobs-into-holes" engineering of antibody CH3 domains for heavy chain heterodimerization [J]. Protein Eng. 1996, 9 (7): 617-621). In the glycosylated C H Introducing a Y349C mutation into the 3 domain can form disulfide bonds between glycosylated heavy chains and enhance the stability of KiH (Kuglstatter A, et al. Structural differences between glycosylated, disulfide-linked heterodimeric knob-into-hole Fc fragment and its homodimeric knob-knob and hole-hole side products[J]. Protein Eng Des Sel., 2017, 30(9): 649-656).

[0348] In addition to steric effects, the charge effects of amino acid residues have also been exploited to enhance heterodimerization between the two heavy chains of multispecific antibodies. Through structural modeling and molecular design, one chain is positively charged, while the paired chain is negatively charged. This promotes heavy chain heterodimer formation through the pattern of like charges repelling and unlike charges attracting. Mutations such as K409D and D399K, K409D / K392D and D399K / E356K, or E356K / E357K / D399K and K370E / K409D / K439E in both chains have all been shown to enhance heterodimerization to some extent (IGAWA T, et al. Methods for producing polypeptides by regulating polypeptide; association: US, 20100015133A1[P]. 2006). Combining the steric effects of KiH with charge effects is also a strategy for enhancing heterodimerization.

[0349] CrossMab technology is a new antibody pairing technology developed by Roche based on KiH technology. It is a technology that exchanges the domains of the light chain and heavy chain of one Fab in a multispecific antibody, while the other does not. The exchanged light chain will contain a fragment of the homologous heavy chain, making it unable to pair with the unexchanged heavy chain, thereby ensuring the correct combination between the light chain and the heavy chain (Schaefer W, et al. Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies [J]. Proc Natl Acad Sci USA, 2011, 108 (27): 11187-11192). The structure includes "CrossMab Fab", "CrossMab V H -V L ” or “CrossMab C H 1-C L " and other forms.

[0350] Antibody variable regions with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant regions. Preferably, the fusion is to an immunoglobulin heavy chain constant region, which includes at least part of the hinge, C H 2 and C H 3 domains. In some embodiments, the heavy chain constant region C contains the necessary sites for light chain binding H The 1 domain is present in at least one fusion. DNA encoding the immunoglobulin heavy chain fusion, and if desired, DNA encoding the immunoglobulin light chain, is inserted into separate expression vectors and co-transfected into a suitable host organism. In some embodiments, antibody variable regions directed against different epitopes or different antigens can be fused to immunoglobulin constant region sequences to produce chimeric multispecific antibodies.

[0351] Fully human and humanized antibodies

[0352] The antibody or antigen-binding fragment or multispecific antibody can be a humanized antibody or a fully human antibody. The humanized form of a non-human (such as mouse) antibody portion is a chimeric immunoglobulin, immunoglobulin chain or its fragment (such as Fv, Fab, Fab', F(ab')2, scFv or other antigen-binding subsequences of an antibody), which generally includes a minimum sequence derived from a non-human immunoglobulin. Humanized antibodies include human immunoglobulins, immunoglobulin chains or their fragments (receptor antibodies), wherein the residues of the receptor CDR are replaced by non-human (donor antibody) CDR residues with desired specificity, affinity and performance, such as mouse, rat or rabbit CDRs. In certain embodiments, human immunoglobulin Fv framework region residues are replaced by corresponding non-human residues. Humanized antibodies can also include amino acid residues that are neither part of the receptor antibody nor in the CDR or framework region sequences introduced. Typically, a humanized antibody comprises at least one, typically two variable regions, wherein all or substantially all of the CDR regions correspond to the CDR regions of non-human immunoglobulins, and all or substantially all of the framework regions are human immunoglobulin consensus sequences.

[0353] Typically, a humanized antibody contains one or more amino acid residues introduced from a non-human source. Those non-human amino acid residues are generally referred to as "imported" residues, typically from the "imported" variable region. According to some embodiments, humanization can be performed essentially according to the following method of Winter and colleagues (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)), by replacing the corresponding sequences of a human antibody with rodent CDRs or CDR sequences. Thus, this "humanized" antibody portion (US Patent No. 4,816,567), which is substantially less than a complete human antibody, has its variable regions replaced by corresponding sequences from a non-human source. In practice, the humanized antibody portion is a typical human antibody portion in which some CDR residues and possibly some framework region residues are substituted by residues from analogous sites in rodent antibodies.

[0354] Generating partially human antibodies is an alternative to humanization. For example, it is now possible to prepare transgenic animals (e.g., mice) that can produce a complete library of fully human antibodies upon immunization without producing endogenous immunoglobulins. For example, it has been reported that homozygous deletion of the antibody heavy chain joining region (JH) gene in chimeric and germline mutant mice completely inhibits endogenous antibody production. Transferring the human germline immunoglobulin gene array into such germline mutant mice can produce fully human antibodies upon antigen stimulation, see, for example, akobovits et al., PNAS USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immunol., 7:33 (1993); US Patent Nos. 5,545,806, 5,569,825, 5,591,669; 5,545,807; and WO 97 / 17852. Alternatively, fully human antibodies can be prepared by introducing human immunoglobulin loci into transgenic animals (e.g., mice in which endogenous immunoglobulin genes have been partially or completely silenced). Upon antigen stimulation, the production of fully human antibodies is found to be very similar to that produced in humans in all aspects, including gene rearrangement, assembly, and antibody libraries. This method is described in, for example, US Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016, and Marks et al., Bio / Technology, 10:779-783 (1992); Lonberg et al., Nature, 368:856-859 (1994); Morrison, Nature, 368:812-813 (1994); Fishwild et al., Nature Biotechnology, 14:845-851 (1996); Neuberger, Nature Biotechnology, 14:826 (1996); Lonberg and Described in Huszar, Intern. Rev. Immunol., 13:65-93 (1995).

[0355] Human antibodies or human antibody portions can also be produced by in vitro activated B cells (see US Patents 5,567,610 and 5,229,275) or by using various techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). The techniques of Cole et al. and Boerner et al. can also be used to prepare fully human monoclonal antibodies. See Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985) and Boerner et al., J. Immunol., 147 (1):86-95 (1991).

[0356] Variants of antibodies or antigen-binding fragments

[0357] In some embodiments, amino acid sequences of variants of the antibodies or antigen-binding fragments provided herein (e.g., antibodies that specifically bind to LIGHT, or multispecific antibodies that specifically bind to LIGHT and TSLP) are also contemplated. For example, it may be necessary to improve the binding affinity and / or other biological activities of the antibody or antigen-binding fragment. The amino acid sequence of the antigen-binding entity variant can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antigen-binding entity or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues in the amino acid sequence of the antigen-binding entity. The final construction can be completed by any combination of deletions, insertions, and substitutions of amino acid residues to give it the desired characteristics. For example, antigen binding.

[0358] In some embodiments, variants of antibodies or antigen-binding fragments having one or more amino acid substitutions are provided. The target sites of the substitution mutations include hypervariable regions (HVRs) and framework regions (FRs). Amino acid substitutions can be introduced into the target antibody to screen for products of desired activity, for example, improved affinity or activity. In some embodiments, the amino acid substitutions described herein are limited to the "exemplary substitutions" in Table 8 of the present application. In some embodiments, the amino acid substitutions are limited to the "preferred substitutions" in Table 8 of the present application.

[0359] Conservative substitutions are shown in Table 8 below.

[0360] Table 8: Conservative Substitutions

[0361] Amino acids are divided into different categories based on the properties of their side chains:

[0362] a. Hydrophobic amino acids: Norleucine, Met, Ala, Val, Leu, Isoleucine;

[0363] b. Neutral hydrophilic amino acids: cysteine ​​Cys, serine Ser, threonine Thr, asparagine Asn, glutamine Gln;

[0364] c. Acidic amino acids: Aspartic acid Asp, glutamic acid Glu;

[0365] d. Basic amino acids: histidine His, lysine Lys, arginine Arg;

[0366] e. Contains amino acids that affect chain direction: glycine Gly, proline Pro;

[0367] f. Aromatic amino acids: tryptophan Trp, tyrosine Tyr, phenylalanine Phe.

[0368] Non-conservative amino acid substitutions include substituting one class for another.

[0369] An exemplary substitution variant is an affinity-matured antibody, which can be conveniently produced using, for example, affinity maturation techniques based on phage display. In short, one or more CDR residues are mutated, the variant antibody portion is displayed on phage, and variants with specific biological activity (e.g., based on RBC cell lysis inhibition assay or binding affinity) are screened. Changes (e.g., substitutions) can be made in the HVRs region to obtain improved RBC lysis inhibition assay or antibody affinity. Changes can be made in the "hotspots" of the HVR, i.e., residues encoded by codons that undergo high-frequency mutations during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and / or at specific deterministic residues (SDRs), and the resulting variant V can be detected. H and V L Methods for constructing and reselecting affinity maturation from secondary libraries have been described in some literature, for example, Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)).

[0370] In some affinity maturation embodiments, diversity is introduced into the variable genes selected for affinity maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide directed mutagenesis). A secondary library is then created. The library is screened to identify antibody variants with the desired affinity. Another method for introducing diversity includes HVR-mediated methods, in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding are specifically identified, for example, using alanine scanning mutagenesis or modeling. Typically, CDR-H3 and CDR-L3 regions are particularly key targets.

[0371] In some embodiments, substitutions, insertions or deletions may occur within one or more HVRs, as long as such changes do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative changes that do not substantially reduce binding affinity (e.g., conservative substitutions provided herein) may be produced in HVRs. These changes may occur outside of HVR "hot spots" or SDRs regions. In some embodiments, the variant V provided above H and V L Sequences, each HVR is either unchanged or contains no more than 1, 2 or 3 amino acid substitutions.

[0372] A useful method for identifying amino acid residues or regions in antibodies that can be targeted for mutation is called "alanine scanning mutagenesis," as described in Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, one or a group of target residues (e.g., charged residues such as arginine, aspartic acid, histidine, lysine, and glutamic acid) are replaced with neutral or negatively charged amino acids (e.g., alanine or glutamic acid) to determine whether the antibody-antigen interaction is affected. Substitutions can be further introduced at the amino acid position to demonstrate that the position has functional sensitivity to the initial substitution. Alternatively or additionally, the contact sites between the antibody and the antigen are identified by the crystal structure of the antigen-antibody complex. These contact site residues and adjacent residues can be targeted or eliminated as substitution candidates. Variants are screened to determine whether they have the desired properties.

[0373] Insertions of amino acid sequences include fusions at the amino and / or carboxyl termini ranging in length from one residue to polypeptides comprising 100 or more residues, and also include insertions of one or more amino acid residues within a sequence. Examples of terminal insertions include antibodies having a methionyl residue at the N-terminus. Other insertion variants of antibody molecules include fusions of an enzyme (e.g., ADEPT) or a polypeptide that increases the serum half-life of the antibody molecule to the N- or C-terminus of the antibody molecule.

[0374] Fc variants

[0375] In some embodiments, one or more amino acid modifications are introduced into the Fc region of an antibody or antigen-binding fragment described herein (e.g., a full-length antibody that specifically binds to LIGHT, a multispecific antibody that specifically binds to LIGHT and TSLP, or a fusion protein comprising the antibody, antigen-binding fragment, or multispecific antibody), thereby generating an Fc variant. In some embodiments, the Fc variant has enhanced ADCC potency, typically associated with receptors that bind to Fc (FcRs). In some embodiments, the Fc variant has reduced ADCC potency. There are many examples of how changes or mutations in the Fc sequence affect its potency, for example, WO 00 / 42072 and Shields et al. J Biol. Chem. 9(2): 6591-6604 (2001) describe antibody variants with enhanced or reduced binding to FcRs. The contents of these publications are incorporated herein by reference.

[0376] Antibody-dependent cell-mediated cytotoxicity (ADCC) is the mechanism of action of therapeutic antibodies against tumor cells. ADCC is a cell-mediated immune defense in which effector cells of the immune system actively lyse target cells (e.g., infected cells) when antigens on the surface of the target cell membrane are bound by specific antigen-binding moieties (e.g., antibodies that specifically bind to LIGHT, multispecific antibodies that specifically bind to LIGHT and TSLP). Typically, the ADCC effect involves NK cells activated by antibodies. NK cells express the Fc receptor CD16. This receptor recognizes and binds to the Fc portion of antibody molecules bound to the surface of target cells. The most common Fc receptors on the surface of NK cells are CD16 or FcγRIII. Binding of the Fc receptor to the Fc region of the antibody leads to activation of NK cells, release of cytolytic granules, and subsequent apoptosis of the target cells.

[0377] In some embodiments, the present application also provides antibodies or antigen-binding fragments that specifically bind to LIGHT, or multispecific antibody variants that specifically bind to LIGHT and TSLP (e.g., full-length antibodies that specifically bind to LIGHT, or multispecific antibodies that specifically bind to LIGHT and TSLP) that contain an Fc region with one or more effector functions, which makes it an ideal candidate antibody for the application. In the case of antibodies or antigen-binding fragments that specifically bind to LIGHT, or multispecific antibodies that specifically bind to LIGHT and TSLP, the in vivo half-life is important, but certain effector functions (such as CDC and ADCC) are unnecessary or harmful. The reduction / elimination of CDC and / or ADCC activity can be confirmed by performing in vitro and / or in vivo cytotoxicity assays. For example, Fc receptor (FcR) binding experiments can be performed to confirm that the antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding ability. Among the main cells that mediate ADCC, NK cells express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 of Ravetch and Kinet Annu. Rev. Immunol. 9: 457-492 (1991), page 464. Non-limiting examples of in vitro assessment of ADCC activity of target molecules are described in US Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83: 7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82: 1499-1502 (1985); US Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166: 1351-1361 (1987)). Alternatively, non-radioactive detection methods can be used (see, e.g., ACTI TM Flow cytometry nonradioactive cytotoxicity assay (Cell Technology, Inc. Mountain View, Calif.) and CYTOTOX 96 TMNon-radioactive cytotoxicity assays (Promega, Madison, Wis.) can be used. Effector cells used in such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, ADCC activity of the target molecule can be tested in vivo, for example, in animal models as described in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody does not bind to C1q and thus lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life can be determined using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0378] Antibodies with reduced effector function comprising one or more substitutions at residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region ( US Pat. No. 6,737,056 ). These Fc variants include Fc variants with substitutions at two or more residues at positions 265, 269, 270, 297, and 327, including an Fc variant known as "DANA" in which residues 265 and 297 are substituted with alanine ( US Pat. No. 7,332,581 ).

[0379] Such antibody variants with increased or decreased binding to FcRs have been described (see, eg, US Pat. No. 6,737,056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0380] In some embodiments, alterations in the Fc region result in altered (ie, increased or decreased) opsonization, as described in Moore et al., MAbs. 2(2): 181-189 (2010).

[0381] In some embodiments, a variant of an antibody or antigen-binding fragment that specifically binds to LIGHT, or a multispecific antibody that specifically binds to LIGHT and TSLP (e.g., a full-length antibody that specifically binds to LIGHT, or a multispecific antibody that specifically binds to LIGHT and TSLP) is provided, comprising an Fc variant having one or more amino acid substitutions that can extend half-life and / or enhance binding to an Fc receptor (FcRn). Antibodies with extended half-life and improved FcRn binding are described in US2005 / 0014934A1 (Hinton et al.). These antibodies comprise one or more amino acid substitutions in the Fc region that enhance binding of the Fc region to FcRn. These Fc variants comprise one or more substitutions at residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434 in the Fc region, such as a substitution at residue 434 in the Fc region ( U.S. Pat. No. 7,371,826 ).

[0382] See also Duncan & Winter, Nature 322:738-40 (1988); US Pat. No. 5,648,260; US Pat. No. 5,624,821 and WO 94 / 29351 for additional examples of Fc variants.

[0383] Also contemplated are antibodies or antigen-binding fragments that specifically bind to LIGHT, or multispecific antibodies that specifically bind to LIGHT and TSLP (e.g., full-length antibodies that specifically bind to LIGHT, or multispecific antibodies that specifically bind to LIGHT and TSLP) comprising any one of the Fc variants described herein or a combination thereof.

[0384] Glycosylation variants

[0385] In some embodiments, the antibodies or antigen-binding fragments that specifically bind to LIGHT, or the multispecific antibodies that specifically bind to LIGHT and TSLP (e.g., full-length antibodies that specifically bind to LIGHT, or multispecific antibodies that specifically bind to LIGHT and TSLP) provided herein are altered to increase or decrease the degree of glycosylation of the antibodies or antigen-binding fragments that specifically bind to LIGHT, or the multispecific antibodies that specifically bind to LIGHT and TSLP. By altering the amino acid sequence of the antibodies or antigen-binding fragments that specifically bind to LIGHT, or the multispecific antibodies that specifically bind to LIGHT and TSLP, or polypeptide portions thereof, to add or remove one or more glycosylation sites, it is possible to conveniently add or delete glycosylation sites on the antibodies or antigen-binding fragments that specifically bind to LIGHT, or the multispecific antibodies that specifically bind to LIGHT and TSLP.

[0386] Wherein the antibody or antigen-binding fragment that specifically binds to LIGHT, or the multispecific antibody that specifically binds to LIGHT and TSLP comprises an Fc region, the sugars attached thereto can be modified. Natural antibodies produced by mammalian cells typically comprise branched biantennary oligosaccharides that are typically attached to the Fc region C through an N-link. H The oligosaccharide may be linked to Asn297 of the 2 domains of the LIGHT domain, see, for example, Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharide may comprise a variety of sugars, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as trehalose linked to the GlcNAc in the "stem" portion of the biantennary oligosaccharide structure. In some embodiments, the antibody or antigen-binding fragment that specifically binds to LIGHT, or the multispecific antibody that specifically binds to LIGHT and TSLP of the present application may be oligosaccharide-modified to produce an antibody or antigen-binding fragment that specifically binds to LIGHT, or a multispecific antibody variant that specifically binds to LIGHT and TSLP, having certain improved properties.

[0387] C with Fc region H The N-glycans linked to the two domains are heterogeneous. Antibodies or Fc fusion proteins produced in CHO cells are fucosylated by fucosyltransferase activity (see Shoji-Hosaka et al., J. Biochem. 2006, 140:777-83). Typically, a small fraction of naturally occurring non-fucosylated IgGs can be detected in human serum. N-glycosylation of the Fc region is important for its binding to FcγRs; however, non-fucosylated N-glycans enhance the binding ability of Fc to FcγRIIIa. Enhanced binding to FcγRIIIa results in an enhanced ADCC effect, which is advantageous in certain antibody therapeutic applications requiring cytotoxicity.

[0388] In some embodiments, when Fc-mediated cytotoxicity is not desired, enhanced effector function may be detrimental. In some embodiments, the Fc fragment or C H 2 domains are non-glycosylated. In some embodiments, by H The N-glycosylation site in domain 2 was mutated to prevent its glycosylation.

[0389] In some embodiments, an antibody or antigen-binding fragment that specifically binds to LIGHT, or a multispecific antibody that specifically binds to LIGHT and TSLP (e.g., a full-length antibody that specifically binds to LIGHT, or a multispecific antibody that specifically binds to LIGHT and TSLP) variant is provided, which comprises an Fc region, wherein the carbohydrate structure attached to the Fc region has reduced fucose or lacks fucose, which may enhance ADCC function. Specifically, the present application provides an antibody or antigen-binding fragment that specifically binds to LIGHT, or a multispecific antibody variant that specifically binds to LIGHT and TSLP, which has reduced fucose relative to the same antibody or antigen-binding fragment that specifically binds to LIGHT, or a multispecific antibody that specifically binds to LIGHT and TSLP produced by wild-type CHO cells. That is, they are characterized in that they have a lower amount of fucose than antibodies produced by native CHO cells (e.g., CHO cells that produce native glycosylated forms, CHO cells containing native FUT8 genes). In some embodiments, the N-linked glycans of the antibody or antigen-binding fragment that specifically binds LIGHT, or the multispecific antibody that specifically binds LIGHT and TSLP have less than 50%, 40%, 30%, 20%, 10%, or 5% fucose. For example, the fucose content of the antibody or antigen-binding fragment that specifically binds LIGHT, or the multispecific antibody that specifically binds LIGHT and TSLP may be 1%-80%, 1%-65%, 5%-65%, or 20%-40%. In some embodiments, the N-linked glycans of the antibody or antigen-binding fragment that specifically binds LIGHT, or the multispecific antibody that specifically binds LIGHT and TSLP do not contain fucose, i.e., the antibody or antigen-binding fragment that specifically binds LIGHT, or the multispecific antibody that specifically binds LIGHT and TSLP is completely free of fucose, lacks fucose, or is defucosylated. The fucose content is determined by calculating the average fucose content within the sugar chains attached to Asn297 relative to the total amount of all sugar structures (such as complex, hybrid, or mannose structures) attached to Asn297 as measured by MALDI-TOF mass spectrometry, as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at position 297 of the Fc region (EU Fc region residue numbering system). However, due to minor sequence variations in antibodies, Asn297 may also be located ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. These fucosylation variants may have enhanced ADCC function. See, for example, US Patent Publication Nos. US2003 / 0157108 (Presta, L.) and US2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.).Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US ​​2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al. J. Mol. Biol. 336:1239-1249(2004); Cell lines capable of producing defucosylated antibodies include Lec13 CHO cells lacking protein fucosylation function (Ripka et al. Arch. Biochem. Biophys. 249: 533-545 (1986); US Pat Appl No US2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., especially Example 11), and gene knockout cell lines, such as CHO cells in which the α-1,6-fucosyltransferase gene, FUT8, is knocked out (see Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4): 680-688 (2006); and WO2003 / 085107).

[0390] Variants of antibodies or antigen-binding fragments that specifically bind to LIGHT, or multispecific antibodies that specifically bind to LIGHT and TSLP (e.g., full-length antibodies that specifically bind to LIGHT, or multispecific antibodies that specifically bind to LIGHT and TSLP) further provide bisected oligosaccharides, for example, wherein a biantennary oligosaccharide attached to the Fc region of the antibody or antigen-binding fragment that specifically binds to LIGHT, or multispecific antibodies that specifically bind to LIGHT and TSLP is bisected by GlcNAc. Such variants of antibodies or antigen-binding fragments that specifically bind to LIGHT, or multispecific antibodies that specifically bind to LIGHT and TSLP (e.g., full-length antibodies that specifically bind to LIGHT, or multispecific antibodies that specifically bind to LIGHT and TSLP) may have reduced fucosylation and / or enhanced ADCC function. Examples of such antibody variants are described in WO 2003 / 011878 (Jean-Mairet et al.); US Pat. No. 6,602,684 (Umana et al.); US 2005 / 0123546 (Umana et al.), and Ferrara et al., Biotechnology and Bioengineering, 93(5):851-861 (2006). Also provided are variants of antibodies or antigen-binding fragments that specifically bind to LIGHT, or multispecific antibodies that specifically bind to LIGHT and TSLP (e.g., full-length antibodies that specifically bind to LIGHT, or multispecific antibodies that specifically bind to LIGHT and TSLP), which have at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibodies or antigen-binding fragments that specifically bind to LIGHT, or multispecific antibody variants that specifically bind to LIGHT and TSLP may have enhanced CDC function. Such variants are described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0391] In some embodiments, the antibody or antigen-binding fragment that specifically binds to LIGHT, or the multispecific antibody (e.g., a full-length antibody that specifically binds to LIGHT, or a multispecific antibody that specifically binds to LIGHT and TSLP) variant that specifically binds to LIGHT and TSLP comprises an Fc region that can bind to FcγRIII. In some embodiments, the antibody or antigen-binding fragment that specifically binds to LIGHT, or the multispecific antibody (e.g., a full-length antibody that specifically binds to LIGHT, or a multispecific antibody that specifically binds to LIGHT and TSLP) variant that specifically binds to LIGHT and TSLP that comprises an Fc region has ADCC activity in the presence of human effector cells (e.g., T cells), or has enhanced ADCC activity in the presence of human effector cells compared to other identical antibodies or antigen-binding fragments that specifically bind to LIGHT, or multispecific antibodies that specifically bind to LIGHT and TSLP (e.g., full-length antibodies that specifically bind to LIGHT, or multispecific antibodies that specifically bind to LIGHT and TSLP) that have a human wild-type IgG1 Fc region.

[0392] Cysteine ​​engineered variants

[0393] In some embodiments, it is desirable to prepare cysteine-engineered antibodies or antigen-binding fragments that specifically bind to LIGHT, or multispecific antibodies that specifically bind to LIGHT and TSLP (e.g., full-length antibodies that specifically bind to LIGHT, or multispecific antibodies that specifically bind to LIGHT and TSLP), in which one or more amino acid residues are substituted with cysteine ​​residues. In some embodiments, the substituted residues occur at accessible sites of the antibody or antigen-binding fragment that specifically binds to LIGHT, or the multispecific antibody that specifically binds to LIGHT and TSLP. By replacing those residues with cysteine, reactive sulfhydryl groups are located at accessible sites of the antibody or antigen-binding fragment that specifically binds to LIGHT, or the multispecific antibody that specifically binds to LIGHT and TSLP, which can be used to conjugate the antibody or antigen-binding fragment that specifically binds to LIGHT, or the multispecific antibody that specifically binds to LIGHT and TSLP to other moieties, such as drug moieties or linker-drug moieties, to prepare antibody or antigen-binding fragment that specifically binds to LIGHT, or multispecific antibody that specifically binds to LIGHT and TSLP immunoconjugates as further described herein. Cysteine-engineered antibodies or antigen-binding fragments that specifically bind to LIGHT, or multispecific antibodies that specifically bind to LIGHT and TSLP (e.g., full-length antibodies that specifically bind to LIGHT, or multispecific antibodies that specifically bind to LIGHT and TSLP) can be prepared as described in, for example, US Pat. No. 7,521,541.

[0394] derivative

[0395] In some embodiments, the multispecific antibodies that specifically bind to LIGHT and TSLP provided herein (e.g., multispecific antibodies that specifically bind to LIGHT and TSLP) can be further modified to include other non-protein moieties that are known in the art and readily available. Suitable moieties for derivatizing multispecific antibodies that specifically bind to LIGHT and TSLP include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), dextran or poly(n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde has advantages in manufacturing due to its stability in water. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers attached to the multispecific antibody that specifically binds LIGHT and TSLP can vary, and if more than one polymer is attached, they can be the same or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, whether the desired property or function of the multispecific antibody that specifically binds LIGHT and TSLP is to be improved, whether the multispecific antibody derivative that specifically binds LIGHT and TSLP is to be used for treatment of a particular condition, etc.

[0396] Pharmaceutical composition

[0397] The present application also provides compositions (e.g., pharmaceutical compositions, also referred to herein as formulations) comprising any multispecific antibody that specifically binds to LIGHT and TSLP, nucleic acids encoding the antibodies or antigen-binding fragments, vectors comprising nucleic acids encoding the antibodies or antigen-binding fragments, or host cells comprising the nucleic acids or vectors described herein. In some embodiments, a pharmaceutical composition is provided comprising any multispecific antibody that specifically binds to LIGHT and TSLP described herein and a pharmaceutically acceptable carrier.

[0398] Suitable multispecific antibody formulations that specifically bind to LIGHT and TSLP can be prepared by mixing a multispecific antibody that specifically binds to LIGHT and TSLP of the desired purity with an optional pharmaceutically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) and prepared in the form of a lyophilized formulation or a liquid formulation. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citric acid, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethylammonium chloride; benzalkonium chloride; benzethonium chloride; phenol; butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol, and m-cresol); and low molecular weight (less than 1 0 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (such as zinc-protein complexes); and / or nonionic surfactants such as TWEEN TM , PLURONICS TM or polyethylene glycol (PEG); exemplary formulations are described in WO98 / 56418 and expressly incorporated herein by reference. Lyophilized formulations suitable for subcutaneous administration are described in WO97 / 04801. Such lyophilized formulations can be reconstituted with a suitable diluent to a high protein concentration formulation, and the reconstituted formulation can be administered subcutaneously to the subject to be treated in the present application. Cationic liposomes or liposomes can be used to deliver the multispecific antibodies that specifically bind to LIGHT and TSLP in the present application to cells.

[0399] In addition to the multispecific antibodies that specifically bind to LIGHT and TSLP, the formulations described herein may also contain one or more other active substances necessary to treat a specific condition, preferably substances with complementary activities and no adverse reactions to each other. For example, in addition to the multispecific antibodies that specifically bind to LIGHT and TSLP, it may be necessary to further include other therapeutically active substances, such as antibiotics. These molecules are present in combination in amounts effective for the intended purpose. The effective amount of the other active substances depends on the amount of the multispecific antibodies that specifically bind to LIGHT and TSLP in the formulation, the disease or condition, the treatment, and other factors as described above. These drugs are generally used in the same dosages and routes of administration as described herein, or at 1% to 99% of the currently used dosage.

[0400] The multispecific antibodies that specifically bind to LIGHT and TSLP can also be encapsulated in microcapsules prepared, for example, by coacervation techniques and interfacial polymerization, such as hydroxymethylcellulose or gelatin-microcapsules and poly(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions. Sustained-release formulations can be prepared.

[0401] Sustained-release formulations of multispecific antibodies that specifically bind to LIGHT and TSLP can be prepared. Suitable examples of sustained-release formulations include solid hydrophobic polymer semipermeable matrices containing the antibody (or fragment thereof), which are in the form of shaped articles, such as films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactic acid (US Pat. No. 3,773,919), copolymers of L-glutamic acid and ethyl L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT TM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprorelin acetate) and poly-D(-)-3-hydroxybutyric acid. Although polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can enable release of molecules for more than 100 days, certain hydrogels can release proteins for shorter periods of time. When encapsulated antibodies remain in the body for a long time, they can denature or aggregate due to exposure to a humid environment at 37°C, potentially leading to loss of bioactivity or altered immunogenicity. Rational strategies can be designed to stabilize multispecific antibodies that specifically bind to LIGHT and TSLP based on the corresponding mechanisms. For example, if the aggregation mechanism is found to be through the formation of intermolecular SS bonds through thiodisulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing in acidic solutions, controlling the water content, using appropriate additives, and developing specific polymer matrix compositions.

[0402] In some embodiments, the multispecific antibody that specifically binds LIGHT and TSLP is formulated in a buffer containing citrate, sodium chloride, acetate, succinate, glycine, polysorbate 80 (Tween 80), or any combination thereof. In some embodiments, the multispecific antibody that specifically binds LIGHT and TSLP is formulated in a buffer with a pH between 4 and 9.

[0403] Preparations for in vivo administration must be sterile. This can be readily achieved, for example, by filtration through sterile filtration membranes.

[0404] Methods of preventing or treating disease

[0405] On the one hand, the present application provides a method for preventing or treating an inflammatory, respiratory or autoimmune disease in an individual, the method comprising administering to the individual an effective amount of any multispecific antibody (preferably, a bispecific antibody) that specifically binds to LIGHT and TSLP as described herein, or a composition comprising the same. In some embodiments, the present application also provides the use of the above-mentioned multispecific antibody (preferably, a bispecific antibody) or the above-mentioned composition in the preparation of a medicament for preventing or treating an inflammatory, respiratory or autoimmune disease.

[0406] On the other hand, the present application also provides a method for preventing or treating an inflammatory, respiratory, or autoimmune disease in an individual, the method comprising administering to the individual an effective amount of (i) an antibody or antigen-binding fragment that specifically binds to LIGHT and (ii) an antibody or antigen-binding fragment that specifically binds to TSLP as described herein; or administering to the individual a composition comprising the above-mentioned (i) antibody or antigen-binding fragment that specifically binds to LIGHT and (ii) an antibody or antigen-binding fragment that specifically binds to TSLP. In some embodiments, the present application also provides the use of the above-mentioned antibody or composition in the preparation of a medicament for preventing or treating an inflammatory, respiratory, or autoimmune disease.

[0407] Described in this application, the disease and / or illness associated with inflammatory, respiratory or autoimmune disease include but are not limited to: asthma, lupus nephritis, IgA nephropathy, type 1 diabetes, inflammatory bowel disease (Crohn's disease, ulcerative colitis), eosinophilic esophagitis, adult respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis, COVID19, airway inflammation, lung disease, bronchiolitis, inflammatory disease, allergic reaction, lupus, atopic dermatitis, arthritis, herpes (e.g., dermatitis herpetiformis), chronic idiopathic urticaria, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, transplant rejection, allograft rejection, graft versus host disease (GVHD), psoriasis, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia. In some embodiments, the method for preventing or treating inflammatory, respiratory or autoimmune disease reduces the mortality caused by inflammatory, respiratory or autoimmune disease.

[0408] Methods for preventing or treating inflammatory, respiratory or autoimmune diseases using multispecific antibodies that specifically bind to LIGHT and TSLP

[0409] In some embodiments, the present application provides a method for preventing or treating an inflammatory, respiratory or autoimmune disease in an individual, the method comprising administering to the individual an effective amount of a multispecific antibody (preferably a bispecific antibody) that specifically binds to LIGHT and TSLP as described herein, or a composition comprising the multispecific antibody (preferably a bispecific antibody), wherein the bispecific antibody comprises a first antigen-binding domain that specifically binds to LIGHT and a second antigen-binding domain that specifically binds to TSLP.

[0410] In some embodiments, the present application provides the use of the multispecific antibody (preferably, a bispecific antibody) that specifically binds to LIGHT and TSLP, or a composition comprising the multispecific antibody (preferably, a bispecific antibody) in the preparation of a medicament for preventing or treating inflammatory, respiratory or autoimmune diseases, wherein the multispecific antibody (preferably, a bispecific antibody) comprises a first antigen-binding domain that specifically binds to LIGHT and a second antigen-binding domain that specifically binds to TSLP.

[0411] In some embodiments, a method for preventing and / or treating an inflammatory, respiratory or autoimmune disease in an individual is provided, comprising administering to the individual an effective amount of a multispecific antibody (preferably a bispecific antibody) as described herein that specifically binds to LIGHT and TSLP, or a composition comprising the multispecific antibody (preferably a bispecific antibody), which is more effective than administering an equivalent dose of an antibody that specifically binds to LIGHT or an equivalent dose of an antibody that specifically binds to TSLP.

[0412] In some embodiments, the method comprises administering an effective amount of a multispecific antibody (preferably, a bispecific antibody) as described herein that specifically binds to LIGHT and TSLP, which reduces the incidence of asthma exacerbations by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more compared to administering an equivalent dose of an antibody that specifically binds to LIGHT or an equivalent dose of an antibody that specifically binds to TSLP.

[0413] In some embodiments, the method comprises administering an effective amount of a multispecific antibody (preferably, a bispecific antibody) that specifically binds to LIGHT and TSLP as described herein, which improves one or more asthma-related parameters by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold or more compared to administering an equivalent dose of an antibody that specifically binds to LIGHT or an equivalent dose of an antibody that specifically binds to TSLP.

[0414] In some embodiments, the method comprises administering an effective amount of a multispecific antibody (preferably, a bispecific antibody) that specifically binds to LIGHT and TSLP as described herein, which improves one or more symptoms or indicators of upper respiratory tract inflammation by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold more than administration of an equivalent dose of an antibody that specifically binds to LIGHT or an equivalent dose of an antibody that specifically binds to TSLP.

[0415] Composition comprising an antibody that specifically binds to LIGHT and an antibody that specifically binds to TSLP for use in preventing or treating inflammatory, respiratory or autoimmune diseases

[0416] In some embodiments, a method for preventing or treating an inflammatory, respiratory, or autoimmune disease in an individual is provided, the method comprising administering to the individual an effective amount of a pharmaceutical composition comprising: (i) an antibody or antigen-binding fragment that specifically binds to LIGHT as described herein and (ii) an antibody or antigen-binding fragment that specifically binds to TSLP as described herein.

[0417] In some embodiments, provided is the use of a composition comprising (i) an antibody or antigen-binding fragment that specifically binds to LIGHT as described herein, and (ii) an antibody or antigen-binding fragment that specifically binds to TSLP as described herein, for the preparation of a medicament for preventing or treating inflammatory, respiratory, or autoimmune diseases.

[0418] In some embodiments, a method for preventing or treating an inflammatory, respiratory, or autoimmune disease in an individual is provided, comprising administering to the individual: (i) an antibody or antigen-binding fragment that specifically binds to LIGHT as described herein and (ii) an antibody or antigen-binding fragment that specifically binds to TSLP as described herein. In some embodiments, the antibody or antigen-binding fragment that specifically binds to LIGHT and the antibody or antigen-binding fragment that specifically binds to TSLP are administered simultaneously. In some embodiments, the antibody or antigen-binding fragment that specifically binds to LIGHT and the antibody or antigen-binding fragment that specifically binds to TSLP are administered sequentially.

[0419] In some embodiments, there is provided a use of (i) an antibody or antigen-binding fragment that specifically binds to LIGHT as described herein and (ii) an antibody or antigen-binding fragment that specifically binds to TSLP as described herein in the preparation of a medicament for preventing or treating an inflammatory, respiratory, or autoimmune disease. In some embodiments, the antibody or antigen-binding fragment that specifically binds to LIGHT and the antibody or antigen-binding fragment that specifically binds to TSLP are administered simultaneously. In some embodiments, the antibody or antigen-binding fragment that specifically binds to LIGHT and the antibody or antigen-binding fragment that specifically binds to TSLP are administered sequentially.

[0420] In some embodiments, the method comprises administering a pharmaceutical composition comprising: an antibody or antigen-binding fragment that specifically binds LIGHT as described herein and an antibody or antigen-binding fragment that specifically binds TSLP as described herein, wherein the molar ratio of the antibody or antigen-binding fragment that specifically binds LIGHT to the antibody or antigen-binding fragment that specifically binds TSLP is about 5: 1, 4: 1, 3: 1, 2: 1, 1: 1, 1: 2, 1: 3, 1: 4, or 1: 5. In some embodiments, the molar ratio of the antibody or antigen-binding fragment that specifically binds LIGHT to the antibody or antigen-binding fragment that specifically binds TSLP is about 2: 1 or 1: 1.

[0421] In some embodiments, a method of preventing or treating an inflammatory, respiratory, or autoimmune disease in an individual is provided, comprising administering to the individual an effective amount of (i) an antibody or antigen-binding fragment that specifically binds to LIGHT as described herein and (ii) an antibody or antigen-binding fragment that specifically binds to TSLP as described herein, wherein the method is more effective than administering an equivalent dose of the antibody or antigen-binding fragment that specifically binds to LIGHT or an equivalent dose of the antibody or antigen-binding fragment that specifically binds to TSLP.

[0422] In some embodiments, the method comprises administering an effective amount of (i) an antibody or antigen-binding fragment that specifically binds LIGHT as described herein and (ii) an antibody or antigen-binding fragment that specifically binds TSLP as described herein, which reduces the incidence of asthma exacerbations by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold more than administration of an equivalent dose of the antibody or antigen-binding fragment that specifically binds LIGHT or an equivalent amount of the antibody or antigen-binding fragment that specifically binds TSLP.

[0423] In some embodiments, the method comprises administering an effective amount of (i) an antibody or antigen-binding fragment that specifically binds LIGHT as described herein and (ii) an antibody or antigen-binding fragment that specifically binds TSLP as described herein, which improves one or more asthma-related parameters by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold more than administration of an equivalent dose of the antibody or antigen-binding fragment that specifically binds LIGHT or an equivalent amount of the antibody or antigen-binding fragment that specifically binds TSLP.

[0424] In some embodiments, the method comprises administering an effective amount of (i) an antibody or antigen-binding fragment that specifically binds LIGHT as described herein and (ii) an antibody or antigen-binding fragment that specifically binds TSLP as described herein, which improves one or more symptoms or indicators of upper respiratory tract inflammation by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or 100-fold more than administration of an equivalent dose of the antibody or antigen-binding fragment that specifically binds LIGHT or an equivalent amount of the antibody or antigen-binding fragment that specifically binds TSLP.

[0425] Products and kits

[0426] In some embodiments of the present application, a product is provided, comprising a substance that can be used to prevent or treat an inflammatory, respiratory, or autoimmune disease (e.g., asthma) in an individual, or for delivering an antibody or antigen-binding fragment (an antibody that specifically binds LIGHT) or a multispecific antibody (e.g., a multispecific antibody that specifically binds LIGHT and TSLP), or a pharmaceutical composition comprising an antibody or antigen-binding fragment that specifically binds LIGHT and an antibody or antigen-binding fragment that specifically binds TSLP, to cells attached to pathogens expressing LIGHT or TSLP. The product can include a container and a label or package insert on or accompanying the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be made of a variety of materials, such as glass or plastic. Typically, the container contains a composition that is effective for treating the disease or condition described herein and has a sterile port (e.g., the container can be an intravenous infusion bag or a vial with a cap pierceable by a hypodermic needle). At least one active substance in the composition is the antibody, antigen-binding fragment, or multispecific antibody described herein. The label or package insert indicates the specific condition that the composition can be used to treat. The label or package insert further includes instructions for administering the multispecific antibody or pharmaceutical composition to a patient. Compositions and kits comprising the combination therapy described herein are contemplated.

[0427] Package insert refers to the instructions generally included in the commercial packaging of the therapeutic product, which includes indications, usage, dosage, administration, contraindications and / or warning information related to the use of these therapeutic products. In certain embodiments, the package insert indicates that the composition can be used to treat bacterial infections. In certain embodiments, the package insert indicates that the composition can be used to treat inflammatory, respiratory or autoimmune diseases (e.g., asthma). In addition, the product can also include a second container, which includes a pharmaceutically acceptable buffer, such as antibacterial water for injection (BWFI), phosphate buffered saline, Green's solution or glucose solution. Other materials required from a commercial and user perspective can also be included, including other buffers, diluents, filters, needles and syringes.

[0428] Also provided are kits that can be used for various purposes, such as for preventing or treating inflammatory, respiratory, or autoimmune diseases (e.g., asthma) in individuals, or for delivering antibodies or antigen-binding fragments that specifically bind to LIGHT, or multispecific antibodies that specifically bind to LIGHT and TSLP (e.g., full-length antibodies that specifically bind to LIGHT, or multispecific antibodies that specifically bind to LIGHT and TSLP) to cells attached to pathogens expressing LIGHT and TSLP, optionally in combination with a product. The kits of the present application include one or more containers containing an antibody or antigen-binding fragment that specifically binds to LIGHT, or a composition that specifically binds to LIGHT and TSLP (or a single dose form and / or product), and in some embodiments, further contain another agent (e.g., an agent described herein) and / or instructions for use consistent with any of the methods described herein. The kit may further include instructions for selecting an appropriate individual for treatment. The instructions for use included with the kits herein are typically written instructions on a label or package insert (e.g., a paper sheet included with the kit), although machine-readable instructions (e.g., instructions on a magnetic or optical storage disc) are also acceptable.

[0429] For example, in some embodiments, the kit comprises a composition comprising an antibody or antigen-binding fragment that specifically binds to LIGHT or a multispecific antibody that specifically binds to LIGHT and TSLP (e.g., a full-length antibody that specifically binds to LIGHT or a multispecific antibody that specifically binds to LIGHT and TSLP). In some embodiments, the kit comprises: a) a composition comprising any of the antibodies or antigen-binding fragments that specifically bind to LIGHT or the multispecific antibodies that specifically bind to LIGHT and TSLP described herein, and b) at least one other agent in an effective amount that can enhance the effect (e.g., therapeutic effect, detection effect) of the antibody or antigen-binding fragment that specifically binds to LIGHT or the multispecific antibody that specifically binds to LIGHT and TSLP described herein. In some embodiments, the kit comprises: a) a composition comprising any of the antibodies or antigen-binding fragments that specifically bind to LIGHT or the multispecific antibodies that specifically bind to LIGHT and TSLP described herein, and b) instructions for administering the antibody or antigen-binding fragment that specifically binds to LIGHT or the multispecific antibody that specifically binds to LIGHT and TSLP to an individual for treating an inflammatory, respiratory, or autoimmune disease (e.g., asthma) in the individual. In some embodiments, the kit includes: a) a composition comprising any of the antibodies or antigen-binding fragments that specifically bind to LIGHT or the multispecific antibodies that specifically bind to LIGHT and TSLP described herein, and b) at least one other agent in an effective amount that can enhance the efficacy (e.g., therapeutic efficacy, detection efficacy) of the antibody or antigen-binding fragment that specifically binds to LIGHT or the multispecific antibody that specifically binds to LIGHT and TSLP, and c) instructions for administering the antibody or antigen-binding fragment that specifically binds to LIGHT or the multispecific antibody that specifically binds to LIGHT and TSLP and the other agent to an individual for treating an inflammatory, respiratory, or autoimmune disease (e.g., asthma) in the individual. The antibody or antigen-binding fragment that specifically binds to LIGHT or the multispecific antibody that specifically binds to LIGHT and TSLP and the other agent can be present in separate containers or in the same container. For example, the kit can include one specific composition or two or more compositions, wherein one composition includes an antibody or antigen-binding fragment that specifically binds to LIGHT or the multispecific antibody that specifically binds to LIGHT and TSLP, and the other composition includes another agent.

[0430] In some embodiments, the kit comprises one (or a set of) nucleic acids encoding an antibody or antigen-binding fragment that specifically binds to LIGHT or a multispecific antibody that specifically binds to LIGHT and TSLP (e.g., a full-length antibody that specifically binds to LIGHT or a multispecific antibody that specifically binds to LIGHT and TSLP). In some embodiments, the kit comprises: a) one (or a set of) nucleic acids encoding an antibody or antigen-binding fragment that specifically binds to LIGHT or a multispecific antibody that specifically binds to LIGHT and TSLP, and b) a host cell that expresses the nucleic acid (or nucleic acids). In some embodiments, the kit comprises: a) one (or a group of) nucleic acids encoding an antibody or antigen-binding fragment that specifically binds LIGHT or a multispecific antibody that specifically binds LIGHT and TSLP, and b) instructions for: i) expressing the antibody or antigen-binding fragment that specifically binds LIGHT or the multispecific antibody that specifically binds LIGHT and TSLP in a host cell, ii) preparing a composition comprising the antibody or antigen-binding fragment that specifically binds LIGHT or the multispecific antibody that specifically binds LIGHT and TSLP, and iii) administering the composition comprising the antibody or antigen-binding fragment that specifically binds LIGHT or the multispecific antibody that specifically binds LIGHT and TSLP to an individual to prevent or treat an inflammatory, respiratory, or autoimmune disease (e.g., asthma) in the individual. In some embodiments, the kit comprises: a) a nucleic acid (or a set of nucleic acids) encoding an antibody or antigen-binding fragment that specifically binds LIGHT or a multispecific antibody that specifically binds LIGHT and TSLP, b) a host cell that expresses the nucleic acid (or a set of nucleic acids), and c) instructions for: i) expressing the antibody or antigen-binding fragment that specifically binds LIGHT or the multispecific antibody that specifically binds LIGHT and TSLP in the host cell, ii) preparing a composition comprising the antibody or antigen-binding fragment that specifically binds LIGHT or the multispecific antibody that specifically binds LIGHT and TSLP, and iii) administering the composition comprising the antibody or antigen-binding fragment that specifically binds LIGHT or the multispecific antibody that specifically binds LIGHT and TSLP to an individual to prevent or treat an inflammatory, respiratory, or autoimmune disease (e.g., asthma) in the individual.

[0431] The test kits described herein are packaged in a suitable form. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed polyester film or plastic bags), etc. The test kits may optionally provide other components, such as buffers and instructional information. Therefore, the application also provides articles, which include vials (e.g., sealed vials), bottles, jars, flexible packaging, etc.

[0432] Instructions for use of a composition comprising an antibody or antigen-binding fragment that specifically binds LIGHT, or a multispecific antibody that specifically binds LIGHT and TSLP, typically include information such as dosage, dosing cycle, and route of administration. The container can be a unit dose, a bulk package (e.g., a multidose package), or a subunit dose. For example, a kit is provided that contains a sufficient dose of an antibody or antigen-binding fragment that specifically binds LIGHT, or a multispecific antibody that specifically binds LIGHT and TSLP (e.g., a full-length antibody that specifically binds LIGHT, or a multispecific antibody that specifically binds LIGHT and TSLP) as described herein to provide long-term, effective treatment for an individual, e.g., one week, eight days, nine days, ten days, eleven days, twelve days, thirteen days, two weeks, three weeks, four weeks, six weeks, eight weeks, three months, four months, five months, seven months, eight months, nine months, or longer. The kit can also contain multiple unit doses of an antibody or antigen-binding fragment that specifically binds LIGHT or a multispecific antibody pharmaceutical composition that specifically binds LIGHT and TSLP and instructions for use, and be packaged in quantities sufficient for storage and use in pharmacies, e.g., hospital pharmacies and compounding pharmacies.

[0433] Those skilled in the art will recognize that several embodiments are possible within the scope and purpose of this application. The application will now be described in more detail with reference to the following non-limiting examples. The following examples further illustrate the application, but should not be construed as limiting its scope in any way. DETAILED DESCRIPTION

[0434] Example 1: Screening, preparation and characterization of full-length anti-LIGHT antibodies

[0435] 1.1. Screening and Preparation of Full-Length Anti-LIGHT Antibodies

[0436] Purified human LIGHT antigen His-huLIGHT was used to immunize 6-8 week old BALB / c mice with equal parts (v / v) of adjuvant. Sera from immunized mice were collected, and the total IgG titer in the sera of immunized mice was measured by ELISA. After several rounds of immunization, a phage display library was established using the spleens of the mice. A solid phase screening strategy was used to isolate phage that specifically binds to human LIGHT from the above phage display library. After screening, individual positive clones were further tested for binding to human LIGHT by ELISA. The positive scFvs after multiple rounds of screening were sequenced to obtain the sequence of the candidate lead antibody LT-m37. The lead antibody LT-m37 was then humanized, and chemically unstable amino acid residues in the CDR region were optimized to obtain a series of humanized molecules. Among them, hum_LT-m37-13, hum_LT-m37-14, hum_LT-m37-15, and hum_LT-m37-16 antibodies were selected for subsequent verification and further development. Their specific amino acid sequences are shown in Table 2-1 and Table 3-1.

[0437] 1.2. Detection of the activity of humanized anti-LIGHT antibodies to inhibit free LIGHT in activating the LTβR reporter gene

[0438] Light activates the NF-κB pathway after binding to the LTβR receptor. A luciferase reporter cell line (Hela-Rc32-NF-κB-Luc) expressing the LTβR receptor was constructed to detect the ability of anti-light antibodies to inhibit luciferase signaling initiated by light (including free light and cell surface light) activating the LTβR receptor. This serves as a measure of the anti-light antibody's inhibitory effect on light-activated LTβR-mediated signaling pathways.

[0439] First, a luciferase reporter cell line responsive to LTβR receptor agonism (Hela-Rc32-NF-κB-Luc) was constructed. Briefly, the cervical cancer cell line Hela-Rc32 expresses the LTβR receptor. Because LIGHT activates the NF-κB pathway upon binding to the LTβR, the Hela-Rc32 cell line was transfected with the plasmid pGL4.32[luc2P / NF-κB-RE / Hygro] (Promega, Cat#E849A). The luciferase gene in this plasmid is located downstream of the NF-κB response element, and luciferase is activated when LIGHT binds to the LTβR.

[0440] The construction method is as follows: According to the manufacturer's operating instructions, The plasmid vector pGL4.32[luc2P / NF-κB-RE / Hygro] (Promega, Cat#E849A) was transfected into Hela-Rc32 cells (ATCC, CRL-2972) using 3000 reagent (Invitrogen, Cat#L3000-001). The cells were cultured in complete medium containing antibiotics (DMEM, 10% FBS, 400 μg / ml Hygromycin B, 1% Penicillin-Streptomycin (PS)) for monoclonal cell screening. Monoclonal cells were then selected and cultured at 1×10 4 Cells were seeded into a white, clear-bottomed 96-well plate, and 50 ng of purified His-huLIGHT protein and 200 μl of complete culture medium were added. The cells were incubated at 37°C, 5% CO₂ for 24 hours. Then, 100 μl of luciferase substrate was added to each well. After incubation in the dark for 5 minutes, fluorescence (RLU) was measured. Monoclonal cells with RLU values ​​> 1000 or a net RLU increase (LIGHT stimulation: control) ≥ 4 were expanded and screened. After three rounds of repeated screening, the Hela-Rc32-NF-κB-Luc luciferase reporter cell line was obtained.

[0441] Then, the LTβR receptor agonist-responsive luciferase reporter gene cell line Hela-Rc32-NF-κB-Luc was cultured at 1×10 4 The cells were seeded into a white transparent bottom 96-well plate at a density of 100 μl / well and cultured overnight in 200 μl complete medium (DMEM, 10% FBS, 400 μg / ml Hygromycin B, 1% PS) at 37°C and 5% CO2. After removing the cell culture medium, 50 μl / well of His-huLIGHT antigen at a concentration of 400 ng / ml and 50 μl / well of the anti-LIGHT antibody to be tested (starting at a concentration of 200 μg / ml and then diluted 1:3) were added. F19 antibody (also known as CERC-002 or Quisovalimab, Avalo Therapeutics) was used as a positive control. After gentle shaking and mixing, the plate was cultured at 37°C and 5% CO2 for 6 hours. Then, ONE-Glo was used to detect the presence of 1% leukemia cells. TM Luciferase Assay System (Promega, Cat. No. E6120) detection method: add 50 μl ONE-Glo to the reporter gene cells equilibrated to room temperature. TMBuffer, shake in the dark for 10 minutes, and immediately detect fluorescence readings using a microplate reader (Luminescence 1000ms). GraphPad Prism 8.0 was used to generate a signal intensity-antibody concentration curve and calculate the IC of the anti-LIGHT antibody to inhibit free LIGHT-activated LTβR-NF-κB signaling. 50 value.

[0442] The results are shown in Table 9-1. Humanized anti-LIGHT antibodies hum_LT-m37-13, hum_LT-m37-14, hum_LT-m37-15, and hum_LT-m37-16 (human IgG4 format) can effectively inhibit free LIGHT from activating LTβR to initiate luciferase signaling. This inhibitory activity is basically equivalent to or even better than that of the positive control antibody F19.

[0443] Table 9-1: Humanized anti-LIGHT antibodies inhibit the activity of free LIGHT in activating the LTβR reporter gene

[0444] 1.3. Detection of the activity of humanized anti-LIGHT antibodies in inhibiting cell surface LIGHT activation of the LTβR reporter gene

[0445] First, a stable cell line HEK293-LIGHT expressing LIGHT was constructed. The construction method was as follows: according to the manufacturer's instructions, 2000 reagent (Invitrogen, catalog number 11668-027), the plasmid expressing LIGHT (from Shutaishen) was transfected into HEK293 cells, and the cells were cultured in complete medium containing antibiotics (DMEM, 10% FBS, 300 μg / ml Zeocin, 1% PS) for monoclonal screening and expansion.

[0446] Approximately 10 monoclonal cells were selected for cell surface LIGHT labeling. First, 20 μg / mL of the anti-LIGHT antibody F23-hFc (Cerecor) was added to the cells and incubated at 4°C for 1 hour. The cells were then washed three times with DPBS buffer. A goat anti-human Fc-FITC secondary antibody (Southern Biotech, 2063-02, 1:1000 dilution) was added and incubated at 4°C in the dark for 1 hour. The cells were then washed three times with DPBS. Fluorescence was then detected by flow cytometry, and LIGHT-positive cells were sorted and collected.

[0447] The selected LIGHT-positive cell lines were then identified by ELISA. Briefly, 4×10 4The above-mentioned LIGHT-positive cells were seeded into a 96-well plate at 100 cells / well, cultured overnight at 37°C and 5% CO2, washed with 0.1% PBST buffer, and then 100 μL of 1% BSA solution was added to each well. The plates were blocked at 37°C for 30 minutes, and then gradient concentrations of anti-LIGHT antibody F23-IgG4 (Cerecor) were added. The plates were incubated at 37°C for 1 hour. After washing, anti-human Fc-AP secondary antibody and color developer PNPP were added. The OD405nm value was read with a microplate reader, and the HEK293-LIGHT stably transfected cell line was finally obtained.

[0448] Then, the LTβR receptor agonist-responsive luciferase reporter gene cell line Hela-Rc32-NF-κB-Luc was cultured at 1×10 4 Cells were seeded into a 96-well plate with a white transparent bottom at a density of 100 cells / well and cultured overnight at 37°C and 5% CO2. After removing the cell culture medium, cells were added at a density of 2×10 5 50 μl / well of HEK293-LIGHT stable cell suspension (1:1000 μg / ml) and 50 μl / well of serially diluted anti-LIGHT antibody to be tested (starting at 1600 μg / ml, diluted 1:3), with F19 antibody as a positive control. After gentle vortexing, incubate at 37°C, 5% CO2 for 6 hours. TM Luciferase Assay System detection method, detection of fluorescence readings (Luminescence 1000ms), the specific method is as described in Example 1.2.

[0449] The results are shown in Table 9-2. The anti-LIGHT antibodies hum_LT-m37-13, hum_LT-m37-14, hum_LT-m37-15, and hum_LT-m37-16 (human IgG4 format) can all effectively inhibit cell surface LIGHT from activating LTβR to initiate luciferase signaling, and the inhibitory activity is superior to that of the positive control antibody F19.

[0450] Table 9-2: Humanized anti-LIGHT antibodies inhibit the activity of cell surface LIGHT in activating LTβR reporter genes

[0451] 1.4. Detection of humanized anti-LIGHT antibody inhibiting free LIGHT from activating the HVEM reporter gene

[0452] In addition to LTβR, LIGHT also binds to the HVEM receptor. When LIGHT binds to HVEM, it can also activate the NF-κB signaling pathway. By constructing a luciferase reporter cell line (293T-HVEM-NF-κB-Luc) that responds to HVEM receptor stimuli, LIGHT binding to HVEM activates luciferase. This allows the use of anti-LIGHT antibodies to detect the inhibitory effect of free LIGHT on HVEM receptor activation and luciferase signaling, serving as a measure of the ability of anti-LIGHT antibodies to inhibit LIGHT-activated HVEM-mediated signaling pathway activity.

[0453] First, a luciferase reporter gene cell line responsive to HVEM receptor agonism (293T-HVEM-NF-κB-Luc) was constructed. Briefly, according to the manufacturer's instructions, a lentiviral packaging system was used to construct the nucleic acid sequence encoding the HVEM-GFP-Blasticidin fusion protein into a lentiviral vector shuttle plasmid (from Shutaishen) and package the recombinant lentivirus. 293T cells were then seeded into 24-well plates, and 5×10 4 Cells were plated at 4% CO / well, and the supernatant was discarded. The recombinant lentivirus was added at different infection ratios (1, 2.5, and 5 MOI). DMEM medium supplemented with 20% FBS was added to 1.2 ml and incubated at 37°C, 5% CO2 for 72 hours. The culture medium was then replaced with 10 μg / ml blasticidin for selection. After 10-14 days, cells positive for GFP fluorescence (the top 20% with strong signals) were sorted and collected by flow cytometry. Subsequently, the luciferase-expressing plasmid pGL4.32[luc2P / NF-κB-RE / Hygro] (Promega, Cat#E849A) was transfected into the above-mentioned GFP-positive cells, and then the GFP-positive cells were sorted and collected by flow cytometry. After that, single clones were screened in complete culture medium containing antibiotics (DMEM, 300 μg / ml Hygromycin B, 10 μg / ml Blasticidin, 1% PS). After expansion culture, the HVEM receptor stimulant-responsive luciferase reporter gene cell line 293T-HVEM-NF-κB-Luc was obtained.

[0454] Then, the luciferase reporter gene cell line 293T-HVEM-NF-κB-Luc, which responds to HVEM receptor agonism, was cultured at 1×10 4The cells / well were seeded into a white transparent bottom 96-well plate at a density of 100 μl and cultured overnight in 200 μl complete medium (DMEM, 10% FBS, 300 μg / ml Hygromycin B, 10 μg / ml Blasticidin, 1% PS) at 37°C and 5% CO2. After removing the cell culture medium, 50 μl / well of His-huLIGHT antigen at a concentration of 400 ng / ml and 50 μl / well of gradient diluted anti-LIGHT antibody to be tested (starting at a concentration of 200 μg / ml and then diluted 1:3) were added, with F19 antibody as a positive control. After gentle shaking and mixing, the plate was cultured at 37°C and 5% CO2 for 6 hours. The ONE-Glo® was used as described above. TM Fluorescence values ​​were measured using the Luciferase Assay System. GraphPad Prism 8.0 was used to generate a signal intensity-antibody concentration curve and calculate the IC value of each anti-LIGHT antibody's inhibitory activity against free LIGHT-activated HVEM-NF-κB signaling. 50 value.

[0455] As shown in Table 9-3, the exemplary humanized anti-LIGHT antibodies hum_LT-m37-14, hum_LT-m37-15, and hum_LT-m37-16 (human IgG4 format) can effectively inhibit free LIGHT from activating HVEM to initiate luciferase signaling, and the inhibitory activity is basically equivalent to that of the positive control antibody F19.

[0456] Table 9-3: Humanized anti-LIGHT antibodies inhibit the activity of free LIGHT in activating the HVEM reporter gene

[0457] Example 2: Preparation and characterization of anti-LIGHT-TSLP bispecific antibodies

[0458] In the following sequence design: V H and V L Represent the heavy chain variable region and light chain variable region of the antibody respectively; C H Represents the constant region of the antibody heavy chain, including C H 1. C H 2 and C H 3 domains; scFv is composed of the V H and V L A single-chain antibody connected by a linker peptide; IgG1 Fc represents the Fc region of the IgG1 subclass antibody, which contains C H 2 and C H 3 domains; C L Represents the light chain constant region.

[0459] 2.1. Construction of Hetero H, CrossMab Bispecific Antibody

[0460] Sequence design: CrossMab technology (see, for example, Klein C, et al. The use of CrossMAb technology for the generation of bi- and multispecific antibodies. MAbs. 2016 Aug-Sep; 8(6): 1010-20) is based on the exchange of antibody domains within one Fab arm of a bispecific IgG antibody, which can be the exchange of the entire Fab domain (CrossMab Fab) or the exchange of only the variable region in the Fab (CrossMab V H -V L ) or exchange of the constant regions only (CrossMab C H 1-C L ), thereby ensuring the correct pairing between the antibody light and heavy chains.

[0461] The bispecific antibodies of this structure used in this embodiment all involve C H 1-C L The KIH was designed to replace the C of a monomer in the IgG1 Fc region, and two Cys residue mutations (S354C on the “knob” side and Y349C on the “hole” side) were introduced to form a stabilizing disulfide bridge. H The threonine (T) at position 366 in region 3 is replaced by tryptophan (W) to form a "knobs" structure, and the C of the other paired monomer is replaced by H In region 3, the threonine (T) at position 366 was replaced with serine (S), the leucine (L) at position 368 was replaced with alanine (A), and the tyrosine (Y) at position 407 was replaced with valine (V) to form a "hole" structure. In addition, the leucine (L) at position 234 and the leucine (L) at position 235 in the hinge region of the antibody were replaced with alanine (A) to form the combined mutation LALA. The numbering is according to the EU index as in Kabat.

[0462] A schematic diagram of the Hetero H, CrossMab bispecific antibody structure used in this example is shown in Figure 1C. The sequence composition of the LIGHT-TSLP bispecific antibody under this structure is shown in Table 10-1, and the specific amino acid sequences of its partial heavy chain, full-length heavy chain, and light chain are shown in Table 5-2 and Table 6-2. Among them, the anti-TSLP antigen-binding domain in the bispecific antibody is derived from the TSLP antibodies TSLP-0107 and TSLP-0202 (see WO2022166739A1), and its amino acid sequences are shown in Table 2-2 and Table 3-2.

[0463] Bispecific antibody construction process: According to the operating instructions, the heavy chain variable region and light chain variable region sequences of the anti-TSLP antibody and the heavy chain variable region and light chain variable region sequences of the anti-LIGHT antibody were constructed into the eukaryotic expression vector pTTa1 by seamless cloning, obtaining heavy chain expression vectors expressing two heavy chains respectively, and light chain expression vectors expressing two light chains respectively, for a total of 4 expression vectors.

[0464] Expression and purification of bispecific antibodies: According to the operating instructions, the four expression plasmids of the above-mentioned bispecific antibodies were co-transfected into HEK293F cells, and the transfected 293F cells were cultured at 37°C, 5% CO2, and 120rpm for 6 days. The cell culture fluid was collected separately. The above-mentioned antibodies were purified using protein A resin (MabCap At 4FF 5ml pre-packed column, product number SA023C15, Changzhou Tiandi Renhe Biotechnology Co., Ltd.). The specific operation is as follows: First, equilibrate the protein A column with 6 column volumes of PBS buffer (containing 0.15M NaCl, pH 7.4) at a flow rate of 150cm / h (flow rate 5ml / min). The culture supernatant (adjusted to pH 7.2-7.4) was passed through the column at a flow rate of 150cm / h (flow rate 5ml / min). After further equilibration of the column, elution was performed using 6 column volumes of 0.1 M glycine buffer (containing 0.15 M NaCl, pH 3.2). The eluate was collected and the pH was adjusted to neutral. The target protein solution was replaced with PBS buffer using an ultrafiltration concentrator. After ultrafiltration and concentration, the target protein concentration was determined using a BCA protein quantification kit.

[0465] Table 10-1: Hetero H. CrossMab structure. Composition of heavy and light chains of a bispecific antibody that specifically binds to LIGHT and TSLP antigens.

[0466] 2.2. Construction of bispecific antibodies with DVD-Ig structure

[0467] Sequence design: DVD-Ig (Dual-variable domain-Ig) bispecific antibody (see, for example, Wu C, et al. Molecular construction and optimization of anti-human IL-1alpha / beta dual variable domain immunoglobulin (DVD-Ig) molecules. MAbs. 2009 Jul-Aug; 1(4): 339-47), which is a structure in which the V domain of another antibody is attached to the N-terminus of the light chain and heavy chain of a normal IgG antibody. L and V H domain, through two antibody V H With V L The interaction forms an antigen-binding domain, which can simultaneously bind to the corresponding antigen to achieve bispecificity. In addition, the bispecific antibody of the DVD-Ig structure used in this embodiment also introduces the combined mutation LALA (L234A, L235A) in the Fc region, as detailed in Example 2.1.

[0468] A schematic diagram of the structure of the DVD-Ig bispecific antibody used in the Examples of this application is shown in Figure 1A. Table 10-2 shows the composition of the anti-LIGHT-TSLP bispecific antibody with a DVD-Ig structure used in the Examples, and the specific amino acid sequences of its partial heavy chain, full-length heavy chain, and light chain are shown in Tables 5-1 and 6-1, respectively.

[0469] Bispecific antibody construction process: According to the operating instructions, a heavy chain expression vector (1) expressing two heavy chains and a light chain expression vector (1) expressing two light chains were constructed by seamless cloning, for a total of 2 expression vectors.

[0470] Expression and purification of bispecific antibodies: The specific operation steps are as described in Example 2.1.

[0471] Table 10-2: DVD-Ig structure Composition of heavy and light chains of bispecific antibodies that specifically bind to LIGHT and TSLP antigens

[0472] 2.3. Binding Activity Detection of Anti-LIGHT-TSLP Bispecific Antibodies

[0473] Preparation and expression of TSLP and LIGHT antigens:

[0474] Human LIGHT antigens include His-human LIGHT fusion protein (His-huLIGHT), human Fc-human LIGHT fusion protein (hFc-huLIGHT), and mouse Fc-human LIGHT fusion protein (mFc-huLIGHT). The preparation process is as follows: cDNAs encoding His-huLIGHT, hFc-huLIGHT, and mFc-huLIGHT are respectively constructed into mammalian cell expression vectors through subcloning. The above expression vector plasmids are transfected into 293F cells, and the cell culture fluid is collected after cultivation. The recombinant protein with the His tag is purified using a nickel column (Ni), and the recombinant protein with the Fc tag is purified using a Protein A affinity chromatography column according to the manufacturer's operating instructions.

[0475] Human TSLP antigens include human TSLP-mFc and human TSLP-his. The preparation and expression processes thereof can be found in patent application WO2022166739A1.

[0476] TSLP Antigen Binding Activity Assay: ELISA was used to assess the binding activity of anti-LIGHT-TSLP bispecific antibodies to TSLP. Briefly, 0.2 μg / well of human TSLP-mFc was coated onto a 96-well plate overnight at 4°C. The plate was washed with PBST and blocked with 50 μL of 4% milk for 1 hour at 37°C. Subsequently, 50 μL / well of serially diluted anti-LIGHT-TSLP bispecific antibodies to be tested (starting at 600 nM, serially diluted in a 1:4 ratio) were added and incubated at 37°C for 1 hour. The plates were then washed six times with PBST. 100 μL of anti-human kappa-HRP secondary antibody (1:4000, SouthernBiotech, E1920-MJ11B) was added to each well and incubated at 37°C for 1 hour. The plates were then washed six times with PBST. Add 100 μL TMB to each well and incubate at 37°C for 10-20 minutes. Terminate the reaction with 2M H2SO4 and read the absorbance at 450 nm using a microplate reader. Generate binding curves using GraphPad Prism 8.0 and calculate the EC values ​​for each antibody. 50 value.

[0477] Activity detection of binding to LIGHT antigen: The binding activity of the anti-LIGHT-TSLP bispecific antibody to the LIGHT antigen was detected by ELISA. The specific steps were as described in the previous section "Activity detection of binding to TSLP antigen", where the coated antigen was 0.1 μg / well His-huLIGHT.

[0478] Simultaneous Binding Activity Assay for TSLP and LIGHT Antigens: ELISA was used to assess the simultaneous binding activity of the anti-LIGHT-TSLP bispecific antibody to both LIGHT and TSLP antigens. Briefly, His-huLIGHT (primary antigen) was coated onto a 96-well plate at 0.1 μg / well and incubated overnight at 4°C. After blocking with milk, 50 μL / well of a serially diluted anti-LIGHT-TSLP bispecific antibody to be tested (starting at 600 nM, serially diluted 1:4) was added and incubated at 37°C for 1 hour. After washing, 100 μL of TSLP-mFc (secondary antigen) at 0.2 μg / well was added to each well and incubated at 37°C for 1 hour. The plates were then washed six times with PBST. Then, 100 μL of anti-mouse IgG-HRP secondary antibody (1:4000, Sigma, A9044-2ML) was added to each well and incubated at 37°C for 1 hour. The plates were then washed six times with PBST. Add 100 μL TMB to each well and incubate at 37°C for 10-20 minutes. Terminate the reaction with 2M H2SO4 and read the absorbance at 450 nm using a microplate reader. Generate binding curves using GraphPad Prism 8.0 and calculate the EC values ​​for each antibody. 50 value.

[0479] The results are shown in Table 11. Anti-LIGHT-TSLP bispecific antibodies, including hum_LT-m37-14-TSLP-0107-DVD-IgG1, hum_LT-m37-14-TSLP-0202-DVD-IgG1, TSLP-0107-hum_LT-m37-14-DVD-IgG1, TSLP-0202-hum_LT-m37-14-DVD-IgG1, and Hetero.H The CrossMab structures—hum_LT-m37-14-TSLP-0107-CrossMab-IgG1, hum_LT-m37-14-TSLP-0202-CrossMab-IgG1, TSLP-0107-hum_LT-m37-14-CrossMab-IgG1, and TSLP-0202-hum_LT-m37-14-CrossMab-IgG1—all effectively bound to the LIGHT antigen, with binding activity comparable to that of the anti-LIGHT antibody hum_LT-m37-14. They also effectively bound to the TSLP antigen. Furthermore, results showed that these bispecific antibodies could simultaneously bind to both the LIGHT and TSLP antigens with high activity.

[0480] Table 11: LIGHT and TSLP antigen binding activity of anti-LIGHT-TSLP bispecific antibodies

[0481] (Note: NA stands for NO Assay)

[0482] 2.4. Detection of the activity of the anti-LIGHT-TSLP bispecific antibody to inhibit free LIGHT from activating the HVEM reporter gene

[0483] The experimental protocol described in Example 1.4 was used to detect the inhibitory activity of the anti-LIGHT-TSLP bispecific antibody on the HVEM-mediated signaling pathway activated by free LIGHT, wherein the anti-LIGHT antibody hum_LT-m37-14 was used as a control.

[0484] The results are shown in Figure 2 . Exemplary anti-LIGHT-TSLP bispecific antibodies, including hum_LT-m37-14-TSLP-0107-DVD-IgG1 and hum_LT-m37-14-TSLP-0202-DVD-IgG1 with DVD-Ig structures, and hum_LT-m37-14-TSLP-0107-CrossMab-IgG1 and hum_LT-m37-14-TSLP-0202-CrossMab-IgG1 with Hetero.H CrossMab structures, can effectively inhibit free LIGHT from activating HVEM to initiate luciferase signaling.

[0485] In addition, other molecules TSLP-0107-hum_LT-m37-14-DVD-IgG1, TSLP-0202-hum_LT-m37-14-DVD-IgG1, TSLP-0107-hum_LT-m37-14-CrossMab-IgG1, and TSLP-0202-hum_LT-m37-14-CrossMab-IgG1 were also able to effectively inhibit free LIGHT from activating HVEM to initiate luciferase signaling (data not shown).

[0486] 2.5. Detection of the activity of the anti-LIGHT-TSLP bispecific antibody to inhibit free LIGHT from activating the LTβR reporter gene

[0487] The experimental protocol described in Example 1.2 was used to detect the inhibitory activity of the anti-LIGHT-TSLP bispecific antibody on the free LIGHT-activated LTβR-mediated signaling pathway, wherein the starting concentration of the bispecific antibody to be tested was 666.67 nM.

[0488] As shown in Table 12, exemplary anti-LIGHT-TSLP bispecific antibodies, including hum_LT-m37-14-TSLP-0202-DVD-IgG1 with a DVD-Ig structure, and hum_LT-m37-14-TSLP-0107-CrossMab-IgG1, hum_LT-m37-14-TSLP-0202-CrossMab-IgG1, TSLP-0107-hum_LT-m37-14-CrossMab-IgG1, and TSLP-0202-hum_LT-m37-14-CrossMab-IgG1 with a Hetero.H CrossMab structure, were able to effectively inhibit the luciferase signal initiated by free LIGHT-activated LTβR.

[0489] Table 12: Anti-LIGHT-TSLP bispecific antibodies inhibit the activity of free LIGHT in activating the LTβR reporter gene

[0490] 2.6. Detection of the activity of anti-LIGHT-TSLP bispecific antibody in inhibiting cell surface LIGHT activation of LTβR reporter gene

[0491] The experimental protocol described in Example 1.3 was used to detect the inhibitory activity of the anti-LIGHT-TSLP bispecific antibody on the cell surface LIGHT-activated LTβR-mediated signaling pathway, wherein the starting concentration of the bispecific antibody to be tested was 53333.33 nM.

[0492] As shown in Table 13, the exemplary anti-LIGHT-TSLP bispecific antibodies, includi...

Claims

1. A multispecific antibody comprising a first antigen-binding domain that specifically binds to LIGHT and a second antigen-binding domain that specifically binds to TSLP, wherein: The first antigen binding domain that specifically binds to LIGHT comprises: Heavy chain variable region (V H ), the V H comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 1; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and a light chain variable region (V L ), the V L comprising: a light chain complementarity determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 4; a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6; as well as, The second antigen binding domain that specifically binds to TSLP comprises: (1) Heavy chain variable region (V H ), the V H comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 12; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 14; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16; and a light chain variable region (V L ), the V L comprising: a light chain complementarity determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 18; a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19; and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20; or (2) Heavy chain variable region (V H ), the V H comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 13; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 15; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17; and a light chain variable region (V L ), the V L It comprises: a light chain complementary determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO:21; a LC-CDR2 comprising the amino acid sequence of SEQ ID NO:22; and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO:

23.

2. The multispecific antibody according to claim 1, wherein: The first antigen binding domain that specifically binds to LIGHT comprises: V H , comprising the amino acid sequence of SEQ ID NO: 8 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 8; and V L , comprising the amino acid sequence of SEQ ID NO: 11 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 11; as well as, The second antigen binding domain that specifically binds to TSLP comprises: (1)V H , comprising the amino acid sequence of SEQ ID NO: 24 or a variant thereof, said variant having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 24; and V L , comprising the amino acid sequence of SEQ ID NO: 26 or a variant thereof, said variant having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 26; or (2)V H , comprising the amino acid sequence of SEQ ID NO: 25 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 25; and V L , comprising the amino acid sequence of SEQ ID NO: 27 or a variant thereof, wherein the variant has at least 80% sequence identity with the amino acid sequence of SEQ ID NO:

27.

3. The multispecific antibody according to any one of claims 1 to 2, wherein the structure thereof is selected from DVD-Ig, Hetero H, CrossMab, Bs4Ab, IgG-(scFv)2 or scFv-Fab IgG.

4. The multispecific antibody according to any one of claims 1 to 3, which has a structure of DVD-Ig and comprises four polypeptide chains: in, The two polypeptide chains contain V from N-terminus to C-terminus H 1-LV H 2-C H 1 structure, where V H 1 is the heavy chain variable region that specifically binds to LIGHT; V H 2 is the heavy chain variable region that specifically binds to TSLP; L is the connecting peptide; C H 1 is the heavy chain constant region C H 1 domain; wherein the polypeptide chain further comprises an Fc, the Fc comprising a C H 2 and C H 3 domains; and The other two polypeptide chains contain V L 1-LV L 2-C L structure, where V L 1 is the light chain variable region that specifically binds to LIGHT; V L 2 is the light chain variable region that specifically binds to TSLP; L is the connecting peptide; C L is the light chain constant region.

5. The multispecific antibody according to any one of claims 1 to 3, which has a structure of DVD-Ig and comprises four polypeptide chains: in, The two polypeptide chains contain V from N-terminus to C-terminus H 1-LV H 2-C H 1 structure, where V H 1 is the heavy chain variable region that specifically binds to TSLP; V H 2 is the heavy chain variable region that specifically binds to LIGHT; L is a connecting peptide; C H 1 is the heavy chain constant region C H 1 domain; wherein the polypeptide chain further comprises an Fc, the Fc comprising a C H 2 and C H 3 domains; and The other two polypeptide chains contain V L 1-LV L 2-C L structure, where V L 1 is the light chain variable region that specifically binds to TSLP; V L 2 is the light chain variable region that specifically binds to LIGHT; L is a connecting peptide; C L is the light chain constant region.

6. The multispecific antibody according to any one of claims 4-5, comprising: (a) the amino acid sequence of SEQ ID NO: 37 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 37; and / or the amino acid sequence of SEQ ID NO: 38 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 38; (b) the amino acid sequence of SEQ ID NO: 39 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 39; and / or the amino acid sequence of SEQ ID NO: 40 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 40; (c) the amino acid sequence of SEQ ID NO: 41 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 41; and / or the amino acid sequence of SEQ ID NO: 42 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 42; or (d) the amino acid sequence of SEQ ID NO: 43 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 43; and / or the amino acid sequence of SEQ ID NO: 44 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:

44.

7. The multispecific antibody according to claim 6, comprising: (a) the amino acid sequence of SEQ ID NO: 57 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 57; and / or the amino acid sequence of SEQ ID NO: 38 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 38; (b) the amino acid sequence of SEQ ID NO: 58 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 58; and / or the amino acid sequence of SEQ ID NO: 40 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 40; (c) the amino acid sequence of SEQ ID NO: 59 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 59; and / or the amino acid sequence of SEQ ID NO: 42 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 42; or (d) the amino acid sequence of SEQ ID NO: 60 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 60; and / or the amino acid sequence of SEQ ID NO: 44 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:

44.

8. The multispecific antibody according to any one of claims 1 to 3, which has a Hetero H, CrossMab structure and comprises four polypeptide chains: in, A polypeptide chain from N-terminus to C-terminus contains V H 1-C H 1 structure, where V H 1 is the heavy chain variable region that specifically binds to LIGHT; C H 1 is the heavy chain constant region C H 1 domain; wherein the polypeptide chain further comprises an Fc, the Fc comprising a C H 2 and C H 3 domains; A polypeptide chain from N-terminus to C-terminus contains V L 1-C L , where V L 1 is the light chain variable region that specifically binds to LIGHT, C L is the light chain constant region; A polypeptide chain from N-terminus to C-terminus contains V H 2-C L , where V H 2 is the heavy chain variable region that specifically binds to TSLP; C L is a light chain constant region; wherein the polypeptide chain further comprises an Fc, the Fc comprising a C H 2 and C H 3 domains; and A polypeptide chain from N-terminus to C-terminus contains V L 2-C H 1, where V L 2 is the light chain variable region that specifically binds to TSLP, C H 1 is the heavy chain constant region C H 1 domain.

9. The multispecific antibody according to any one of claims 1 to 3, which has a Hetero H, CrossMab structure and comprises four polypeptide chains: in, A polypeptide chain from N-terminus to C-terminus contains V H 1-C H 1 structure, where V H 1 is the heavy chain variable region that specifically binds to TSLP; C H 1 is the heavy chain constant region C H 1 domain; wherein the polypeptide chain further comprises an Fc, the Fc comprising a C H 2 and C H 3 domains; A polypeptide chain from N-terminus to C-terminus contains V L 1-C L , where V L 1 is the light chain variable region that specifically binds to TSLP, C L is the light chain constant region; A polypeptide chain from N-terminus to C-terminus contains V H 2-C L , where V H 2 is the heavy chain variable region that specifically binds to LIGHT; C L is a light chain constant region; wherein the polypeptide chain further comprises an Fc, the Fc comprising a C H 2 and C H 3 domains; and A polypeptide chain from N-terminus to C-terminus contains V L 2-C H 1, where V L 2 is the light chain variable region that specifically binds to LIGHT, C H 1 is the heavy chain constant region C H 1 domain.

10. The multispecific antibody according to any one of claims 8 to 9, comprising: (a) the amino acid sequence of SEQ ID NO: 47 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 47; and / or the amino acid sequence of SEQ ID NO: 48 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 48; and / or the amino acid sequence of SEQ ID NO: 45 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 45; and / or the amino acid sequence of SEQ ID NO: 46 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 46; (b) the amino acid sequence of SEQ ID NO:47 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:47; and / or the amino acid sequence of SEQ ID NO:48 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:48; and / or the amino acid sequence of SEQ ID NO:49 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:49; and / or the amino acid sequence of SEQ ID NO:50 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:50; (c) the amino acid sequence of SEQ ID NO:51 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:51; and / or the amino acid sequence of SEQ ID NO:52 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:52; and / or the amino acid sequence of SEQ ID NO:53 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:53; and / or the amino acid sequence of SEQ ID NO:54 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:54; or (d) the amino acid sequence of SEQ ID NO:55 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:55; and / or the amino acid sequence of SEQ ID NO:56 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:56; and / or the amino acid sequence of SEQ ID NO:53 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:53; and / or the amino acid sequence of SEQ ID NO:54 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:

54.

11. The multispecific antibody according to claim 10, comprising: (a) the amino acid sequence of SEQ ID NO:46 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:46; and / or the amino acid sequence of SEQ ID NO:48 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:48; and / or the amino acid sequence of SEQ ID NO:61 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:61; and / or the amino acid sequence of SEQ ID NO:62 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:62; (b) the amino acid sequence of SEQ ID NO:48 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:48; and / or the amino acid sequence of SEQ ID NO:50 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:50; and / or the amino acid sequence of SEQ ID NO:62 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:62; and / or the amino acid sequence of SEQ ID NO:63 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:63; (c) the amino acid sequence of SEQ ID NO:52 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:52; and / or the amino acid sequence of SEQ ID NO:54 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:54; and / or the amino acid sequence of SEQ ID NO:64 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:64; and / or the amino acid sequence of SEQ ID NO:65 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:65; or (d) the amino acid sequence of SEQ ID NO:54 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:54; and / or the amino acid sequence of SEQ ID NO:56 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:56; and / or the amino acid sequence of SEQ ID NO:65 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:65; and / or the amino acid sequence of SEQ ID NO:66 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:

66.

12. A pharmaceutical composition comprising: An antibody or antigen-binding fragment that specifically recognizes LIGHT and an antibody or antigen-binding fragment that specifically recognizes TSLP, wherein: The antibody or antigen-binding fragment that specifically binds to LIGHT comprises: Heavy chain variable region (V H ), the V H comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 1; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and a light chain variable region (V L ), the V L comprising: a light chain complementarity determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 4; a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6; as well as, The antibody or antigen-binding fragment that specifically binds to TSLP comprises: (1) Heavy chain variable region (V H ), the V H comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 12; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 14; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16; and a light chain variable region (V L ), the V L comprising: a light chain complementarity determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 18; a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19; and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20; or (2) Heavy chain variable region (V H ), the V H comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 13; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 15; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17; and a light chain variable region (V L ), the V L It comprises: a light chain complementary determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO:21; a LC-CDR2 comprising the amino acid sequence of SEQ ID NO:22; and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO:

23.

13. A method for treating and / or preventing a disease or condition in an individual in need thereof, comprising administering to the individual an effective amount of an antibody or antigen-binding fragment that specifically recognizes LIGHT and an antibody or antigen-binding fragment that specifically recognizes TSLP, wherein: The antibody or antigen-binding fragment that specifically binds to LIGHT comprises: Heavy chain variable region (V H ), the V H comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 1; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and a light chain variable region (V L ), the V L comprising: a light chain complementarity determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 4; a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 6; as well as, The antibody or antigen-binding fragment that specifically binds to TSLP comprises: (1) Heavy chain variable region (V H ), the V H comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 12; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 14; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 16; and a light chain variable region (V L ), the V L comprising: a light chain complementarity determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 18; a LC-CDR2 comprising the amino acid sequence of SEQ ID NO: 19; and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 20; or (2) Heavy chain variable region (V H ), the V H comprising: a heavy chain complementarity determining region (HC-CDR) 1 comprising the amino acid sequence of SEQ ID NO: 13; HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 15; and HC-CDR3 comprising the amino acid sequence of SEQ ID NO: 17; and a light chain variable region (V L ), the V L It comprises: a light chain complementary determining region (LC-CDR) 1 comprising the amino acid sequence of SEQ ID NO:21; a LC-CDR2 comprising the amino acid sequence of SEQ ID NO:22; and a LC-CDR3 comprising the amino acid sequence of SEQ ID NO:

23.

14. The pharmaceutical composition according to claim 12 or the method according to claim 13, wherein: The antibody or antigen-binding fragment that specifically binds to LIGHT comprises: V H , comprising the amino acid sequence of SEQ ID NO: 8 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 8; and V L , comprising the amino acid sequence of SEQ ID NO: 11 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 11; as well as, The antibody or antigen-binding fragment that specifically binds to TSLP comprises: (1)V H , comprising the amino acid sequence of SEQ ID NO: 24 or a variant thereof, said variant having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 24; and V L , comprising the amino acid sequence of SEQ ID NO: 26 or a variant thereof, said variant having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 26; or (2)V H , comprising the amino acid sequence of SEQ ID NO: 25 or a variant thereof, said variant having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 25; and V L , comprising the amino acid sequence of SEQ ID NO: 27 or a variant thereof, wherein the variant has at least 80% sequence identity with the amino acid sequence of SEQ ID NO:

27.

15. The method according to any one of claims 13-14, wherein an antibody or antigen-binding fragment specific for LIGHT and an antibody or antigen-binding fragment specifically recognizing TSLP are administered to the individual simultaneously or sequentially.

16. An isolated nucleic acid molecule encoding the multispecific antibody of any one of claims 1-11.

17. A vector comprising the nucleic acid molecule of claim 16.

18. An isolated host cell comprising the multispecific antibody of any one of claims 1-11, the nucleic acid molecule of claim 16, or the vector of claim 17.

19. A method for preparing the multispecific antibody according to any one of claims 1 to 11, comprising: a) culturing the host cell of claim 18 under conditions effective to express the antibody; and b) obtaining the expressed antibody from the host cell.

20. A pharmaceutical composition comprising the multispecific antibody of any one of claims 1-11, the nucleic acid molecule of claim 16, the vector of claim 17, the isolated host cell of claim 18, or the multispecific antibody produced by the method of claim 19, and a pharmaceutically acceptable carrier or excipient.

21. Use of the multispecific antibody of any one of claims 1-11, the nucleic acid molecule of claim 16, the vector of claim 17, the host cell of claim 18, the multispecific antibody produced by the method of claim 19, or the pharmaceutical composition of claim 12, 14, or 20 in the preparation of a medicament for treating a disease or condition in an individual in need thereof.

22. The use according to claim 21, wherein the disease or condition comprises a disease or condition associated with an inflammatory, respiratory or autoimmune disease.

23. The method of claim 22, wherein the disease or condition comprises asthma, lupus nephritis, IgA nephropathy, type 1 diabetes, inflammatory bowel disease (Crohn's disease, ulcerative colitis), eosinophilic esophagitis, adult respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis, COVID19, airway inflammation, lung disease, bronchiolitis, inflammatory disease, allergic reaction, lupus, atopic dermatitis, arthritis, herpes (e.g., dermatitis herpetiformis), chronic idiopathic urticaria, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune uveitis, transplant rejection, allograft rejection, graft-versus-host disease (GVHD), psoriasis, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia.

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