Anti-il-4ra antibody, bispecific antibody, and pharmaceutical composition

Abstract

An anti-IL-4RA antibody, a bispecific antibody, and a pharmaceutical composition. The bispecific antibody is an anti-IL-4RA-anti-ST2 bispecific antibody. The anti-IL-4RA antibody and the bispecific antibody have superior affinity and specificity, and have good application prospects.

Description

Anti-IL-4RA antibodies, bispecific antibodies, and pharmaceutical compositions

[0001] Cross-references to related applications

[0002] This application is based on and claims priority to CN application number 202510072986.1, filed on January 16, 2025, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention belongs to the field of biomedicine and relates to an anti-IL-4RA antibody, a bispecific antibody, and a pharmaceutical composition. Specifically, the bispecific antibody is a bispecific antibody comprising the anti-IL-4RA antibody, and more specifically, it is an anti-IL-4RA-anti-ST2 bispecific antibody. Specifically, the pharmaceutical composition comprises the anti-IL-4RA antibody or the bispecific antibody. This invention also relates to the use of the anti-IL-4RA antibody or the bispecific antibody, particularly for pharmaceutical purposes. Background Technology

[0004] Interleukin-4 receptor (IL-4R) is a transmembrane receptor that exists in two different forms: one is type I IL-4R, which is composed of a high-affinity IL-4Rα subunit (i.e., IL-4RA in this invention) and a medium-affinity γC subunit. It binds to interleukin-4 (IL-4) and mediates IL-4-induced cell proliferation, activation and other biological functions; the other is type II IL-4R, which is composed of a high-affinity IL-4Rα subunit (IL-4RA) and an interleukin-13 receptor α subunit (IL-13Rα). Type II IL-4R is also a homologous functional receptor of interleukin-13 (IL-13) and can bind to IL-13 (Wang and Secombes, Cytokine. 2015. Vol. 75, No. 1, pp. 8-13 (2015)). IL-4R is expressed in T cells, B cells, hematopoietic stem cells, endothelial cells, epithelial cells, muscle, fibroblasts, hepatocytes, and brain tissue. After IL-4 binds to its receptor, IL-4RA works in conjunction with the γC subunit or the IL-13Rα subunit to activate a variety of non-receptor protein tyrosine kinases in the cytoplasm, further initiating downstream signal transduction pathways (Nelms et al., Annu Rev Immunol, Vol. 17, pp. 701-738 (1999); LaPorte et al., Cell, Vol. 132, No. 2, pp. 259-272 (2008)).

[0005] IL-4RA can bind with high affinity to IL-4 and IL-13 and is the main functional subunit of the aforementioned type I and type II IL4R. Inhibiting IL-4RA can effectively block the related biological functions mediated by IL-4 and IL-13 (Gessner et al., Immunobiology, Vol. 201, p. 285 (2000)).

[0006] IL-4 is a pleiotropic cytokine secreted by immune cells such as CD4+ T cell subsets, B cells, and mast cells; IL-13 is mainly produced by activated T cells (Th2 in mice) in the human body, and IL-13 has a variety of biological effects on monocytes (macrophages), B lymphocytes, NK cells, and vascular endothelial cells. In vitro studies have shown that IL-4 and IL-13 can exert corresponding effector functions in various cell types, such as T cells, B cells, eosinophils, mast cells, basophils, airway smooth muscle cells, respiratory epithelial cells, fibroblasts, and endothelial cells. These cells are the main effector cells leading to allergic diseases, such as allergic rhinitis, food allergies, and asthma (e.g., allergic asthma) (May et al., Cytokine, Vol. 75, No. 1, pp. 89-116 (2015)). In addition, airway smooth muscle cells, respiratory epithelial cells, fibroblasts, and endothelial cells are also involved in the pathogenesis, progression, and maintenance of chronic obstructive pulmonary disease (see Steinke et al., RespRes, Vol. 2, No. 66, pp. 66-70 (2001) and Willis-Karp et al., Immunol). Rev, Vol. 202, pp. 175-190, (2004)); Abnormal T cell and B cell function is the main mechanism of the occurrence of autoimmune diseases, and IL-4 participates in the occurrence of autoimmune diseases by binding to IL-4R on the surface of immune cells and activating downstream signaling pathways (May et al., Cytokine, Vol. 75, No. 1, pp. 89-116 (2015)).

[0007] The IL-4 / IL-13 pathway plays an important role in the pathology of asthma (Chatila et al., Trendsin Molecular Med, Vol. 10, No. 10, pp. 493-499, (2004)) and in other conditions listed elsewhere in this article. Airway hyperresponsiveness, excessive mucus secretion, and airway remodeling are key pathological features of asthma. Studies have confirmed that IL-4 and IL-13 are involved in the occurrence and maintenance of the above pathological processes (May et al., Cytokine, Vol. 75, No. 1, pp. 89-116 (2015)). IL-13 is considered a key cytokine that triggers airway hyperresponsiveness (AHR), and IL-4 is a major inducing factor for Th2 immune cell polarization and IgE production (Wynn et al., AnnuRev Immunol, Vol. 21, pp. 425-456, (2003)).

[0008] Atopic dermatitis, especially moderate to severe atopic dermatitis, is a serious chronic inflammatory skin disease characterized by intense itching, pronounced eczematous changes, and dry skin. Atopic dermatitis often begins in infancy and can persist throughout life for some patients. It can severely impact quality of life due to chronic recurrent eczematous rashes, severe itching, sleep deprivation, dietary restrictions, and psychosocial effects. IL-4 / IL-13 is considered a key driver of persistent intrinsic inflammation in atopic dermatitis (Malajian et al., Cytokine, Vol. 73, No. 2, pp. 311-318 (2015)). Clinical studies have demonstrated that the anti-human IL-4RA monoclonal antibody dupilumab is effective in treating moderate to severe atopic dermatitis (Beck et al., N. Engl. J. Med., Vol. 371, No. 2, pp. 130-139 (2014)), and it has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of moderate to severe atopic dermatitis.

[0009] Furthermore, basic medical research and clinical studies have confirmed that the IL-4, IL-13 and IL-4R pathways are involved in the occurrence and development of chronic obstructive pulmonary disease, sinusitis, and tumors. Inhibiting IL-4RA has the potential to treat chronic obstructive pulmonary disease (Jin Lin et al., Practical Medicine Journal, Vol. 30, No. 22, pp. 3543-3544 (2014)), sinusitis, pulmonary fibrosis, and tumors (May et al., Cytokine, Vol. 75, No. 1, pp. 89-116 (2015); Guo Changkuo et al., Chemistry of Life, Vol. 37, No. 3, pp. 413-418 (2017)). In adults with symptomatic chronic sinusitis and nasal polyps who were unresponsive to intranasal corticosteroid therapy, the anti-human IL-4RA monoclonal antibody Dupilumab, in combination with corticosteroids, significantly relieved symptoms and reduced nasal polyps (Bachert et al., JAMA, Vol. 315, No. 5, pp. 469-79 (2016)).

[0010] Anti-human IL-4RA antibody drugs have broad application prospects and can be used to treat allergic diseases such as allergic rhinitis, asthma, allergies, and atopic dermatitis. They can also be used for sinusitis, nasal polyps, chronic obstructive pulmonary disease, tissue fibrosis, tumors, and autoimmune diseases. Currently available anti-human IL-4RA antibody drugs have low affinity, and a high-affinity anti-human IL-4RA antibody is lacking. Therefore, developing antibody drugs with high affinity for human IL-4RA for the treatment of allergic diseases, resulting in better therapeutic effects and lower toxicity, is of great significance.

[0011] ST2 (growth-stimulating gene 2 protein, also known as IL1RL1, DER4, FIT-1, IL-33R, ST2L, ST2V, or T1) is a member of the interleukin-1 receptor family. It possesses a Toll / IL-1R domain, which contains approximately 160 amino acids and consists of five central β-sheets and five α-helices located on the cytoplasmic side of the protein (Tominaga SA putative protein of a growth-specific cDNA from BALB / c-3T3 cells is highly similar to the extracellular portion of mouse interleukin 1 receptor [J]. FEBS letters, 1989, 258(2): 301-304). ST2 is an important biomarker for heart failure and has also been found as a ligand for IL-33. The IL-33 / ST2 pathway is associated with various immune-mediated diseases, such as asthma and rheumatoid arthritis.

[0012] The ST2 gene product includes four subtypes: sST2, ST2L, ST2V, and ST2LV. sST2 is a soluble ST2 that can be secreted extracellularly and can also be induced to express in the retina, breast, and osteoblasts. ST2V is mainly expressed in the colon, stomach, small intestine, lungs, spleen, testes, and placenta, rather than in tissues such as the brain, heart, liver, kidneys, and skeletal muscle. Suzukawa et al. found that the receptor-type ST2L is mainly expressed on the surface of Th2 lymphocytes and mast cells, and some granulocytes such as basophils and eosinophils may express membrane-type ST2 molecules (Suzukawa M, Iikura M, Koketsu R, et al. An IL-1 cytokine member, IL-33, induces human basophil activation via its ST2 receptor[J]. The Journal of Immunology, 2008, 181(9): 5981-5989).

[0013] ST2 and IL-1 co-receptor protein (IL-1RAcP) form the IL-33 receptor complex (IL-33R1). IL-1RAcP binds to ST2 in a ligand-dependent manner, increasing the affinity of IL-33 for ST2. IL-33 transduces signals into the cell by binding to the IL-33 receptor complex on the cell membrane, recruiting downstream signaling molecules such as myeloid differentiation factor 88 (MyD88), IL-1 receptor-associated kinase (IRAK), and tumor necrosis factor receptor-associated factor 6 (TRAF6) and activating downstream mitogen-activated protein kinase (MAP-KK). MAP-KK, in turn, activates protein-1 (AP-1) via c-jun N-terminal kinase (JNK). TRAF6 can also activate the nuclear factor-κB (NF-κB) kinase inhibitor complex, leading to the release of NF-κB from the complex. NF-κB is the first responder to harmful cellular stimuli. There are many known activators of the NF-κB pathway, including: TNF-α, IL-1β, IL-2, IL-6, IL-8, IL-12, iNOS, COX2, chemokines, adhesion molecules, colony-stimulating factors, etc. (Kakkar R, Lee RT. The IL-33 / ST2 pathway: therapeutic target and novel biomarker[J]. Nature reviews Drug discovery, 2008, 7(10): 827-840).

[0014] It has been shown that dysregulation of the IL-33 / ST2 pathway can cause a variety of immune-mediated diseases, including asthma, rheumatoid arthritis, inflammatory bowel disease (Palmer G, Gabay C. Interleukin-33 biology with potential insights into human diseases[J]. Nature Reviews Rheumatology, 2011, 7(6):321), atopic dermatitis (Shimizu M, Matsuda A, Yanagisawa K, et al. Functional SNPs in the distal promoter of the ST2 gene are associated with atopic dermatitis[J]. Human molecular genetics, 2005, 14(19):2919-2927), and allergic rhinitis (Kamekura R, Kojima T, Takano K, et al. The role of IL-33 and its receptor ST2 in human nasal epithelium with allergic rhinitis[J]. Clinical & Experimental). Allergy, 2012, 42(2):218-228), nasal polyps and systemic sclerosis (Manetti M, Ibba-Manneschi L, Liakouli V, et al. The IL1-like cytokine IL-33 and its receptor ST2 are abnormally expressed in the affected skin and visceral organs of patients with systemic sclerosis[J]. Annals of the rheumatic diseases, 2010, 69(3):598-605).

[0015] Asthma is generally considered a Th2 cell-mediated disease, with T lymphocytes, eosinophils, and mast cells playing important roles in its pathogenesis. Several changes in the airway of asthma patients, such as hyperresponsiveness, inflammation, tissue remodeling, and damage to the airway epithelial cell layer, can lead to the release of damage-associated pattern molecules (DAMPs), including IL-33. Studies by Prefontaine et al. have found increased levels of IL-33 protein and mRNA in airway smooth muscle cells of subjects with severe respiratory asthma. D, Lajoie-Kadoch S, Foley S, et al. Increased expression of IL-33 in severe asthma: evidence of expression by airway smooth muscle cells[J]. The Journal of Immunology, 2009, 183(8): 5094-5103). In studies of the roles of IL-33 and ST2 in allergic airway inflammation using ST2 or IL-33-deficient mice, intranasal administration of IL-33 induced allergic inflammation, leading to increased levels of IL-5, IL-13, and eosinophil chemokines, increased mucus secretion, and infiltration of eosinophils and T lymphocytes. Conversely, ST2 knockout resulted in reduced airway inflammation and decreased IL-5 production. These studies indicate that the IL-33 / ST2 signaling pathway plays an important role in the development of allergic airway inflammation (Kondo Y, Yoshimoto T, Yasuda K, et al. Administration of IL-33 induces airway hyperresponsiveness and goblet cell hyperplasia in the lungs in the absence of adaptive immune system[J]. International immunology, 2008, 20(6):791-800).

[0016] Anti-ST2 antibody drugs have broad application prospects and can be used to treat diseases such as asthma, rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, allergic rhinitis, nasal polyps, and systemic sclerosis. Roche Pharmaceuticals From the drug development pipeline of Amgen The company has obtained authorization for the anti-ST2 antibody AMG282. AMG282 is currently in the mid-stage of clinical research and has completed clinical trials related to drug safety and effective dosage (NCT02170337, NCT01928368). AMG282 is expected to become a new generation of monoclonal antibody drugs for the treatment of asthma.

[0017] Currently, there is a need to develop new anti-IL-4RA antibodies and bispecific antibody drugs that simultaneously target IL-4RA and ST2. Summary of the Invention

[0018] Through in-depth research and creative work, the inventors have obtained a specific antibody (named 14A10) that specifically binds to human IL-4RA, and this antibody can effectively block the binding of human IL-4RA and IL-4. Furthermore, the inventors have creatively prepared humanized antibodies against human IL-4RA (named 14A10H42L25, 14A10H43L25, 14A10H49L25, and 14A10H50L25, respectively). These antibodies can effectively bind to human IL-4RA, blocking the binding of human IL-4RA to its ligands IL-4 or IL-13, and inhibiting the activation of downstream signaling pathways of human IL-4RA; they have the potential to be used in the preparation of drugs for the prevention and treatment of allergic rhinitis, asthma, allergies, atopic dermatitis, sinusitis, nodular prurigo, chronic urticaria, nasal polyps, chronic obstructive pulmonary disease, tissue fibrosis, and autoimmune diseases. Furthermore, the inventors have prepared an anti-IL-4RA-anti-ST2 bispecific antibody based on this. This provides the following invention:

[0019] One aspect of the present invention relates to an anti-IL-4RA antibody or an antigen-binding fragment thereof, said anti-IL-4RA antibody comprising a heavy chain variable region and a light chain variable region, said heavy chain variable region comprising complementarity-determining regions HCDR1 to HCDR3, and said light chain variable region comprising complementarity-determining regions LCDR1 to LCDR3, wherein:

[0020] HCDR1 contains the amino acid sequence shown in SEQ ID NO:5, HCDR2 contains the amino acid sequence shown in SEQ ID NO:96, and HCDR3 contains the amino acid sequence shown in SEQ ID NO:7; and

[0021] LCDR1 contains the amino acid sequence shown in SEQ ID NO:8, LCDR2 contains the amino acid sequence shown in SEQ ID NO:9, and LCDR3 contains the amino acid sequence shown in SEQ ID NO:10.

[0022] In some embodiments of the present invention, the anti-IL-4RA or its antigen-binding fragment, wherein...

[0023] HCDR1 contains the amino acid sequence shown in SEQ ID NO:5, HCDR2 contains the amino acid sequence shown in SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:6, and HCDR3 contains the amino acid sequence shown in SEQ ID NO:7; and

[0024] LCDR1 contains the amino acid sequence shown in SEQ ID NO:8, LCDR2 contains the amino acid sequence shown in SEQ ID NO:9, and LCDR3 contains the amino acid sequence shown in SEQ ID NO:10.

[0025] In some embodiments of the present invention, the anti-IL-4RA or its antigen-binding fragment, wherein...

[0026] HCDR1 contains the amino acid sequence shown in SEQ ID NO:5, HCDR2 contains the amino acid sequence shown in SEQ ID NO:23, and HCDR3 contains the amino acid sequence shown in SEQ ID NO:7; and

[0027] LCDR1 contains the amino acid sequence shown in SEQ ID NO:8, LCDR2 contains the amino acid sequence shown in SEQ ID NO:9, and LCDR3 contains the amino acid sequence shown in SEQ ID NO:10.

[0028] In some embodiments of the present invention, the anti-IL-4RA or its antigen-binding fragment, wherein...

[0029] HCDR1 contains the amino acid sequence shown in SEQ ID NO:5, HCDR2 contains the amino acid sequence shown in SEQ ID NO:24, and HCDR3 contains the amino acid sequence shown in SEQ ID NO:7; and

[0030] LCDR1 contains the amino acid sequence shown in SEQ ID NO:8, LCDR2 contains the amino acid sequence shown in SEQ ID NO:9, and LCDR3 contains the amino acid sequence shown in SEQ ID NO:10.

[0031] In some embodiments of the present invention, the anti-IL-4RA or its antigen-binding fragment, wherein...

[0032] HCDR1 contains the amino acid sequence shown in SEQ ID NO:5, HCDR2 contains the amino acid sequence shown in SEQ ID NO:6, and HCDR3 contains the amino acid sequence shown in SEQ ID NO:7; and

[0033] LCDR1 contains the amino acid sequence shown in SEQ ID NO:8, LCDR2 contains the amino acid sequence shown in SEQ ID NO:9, and LCDR3 contains the amino acid sequence shown in SEQ ID NO:10.

[0034] In some embodiments of the present invention, the anti-IL-4RA or its antigen-binding fragment, wherein HCDR1 to HCDR3 and LCDR1 to LCDR3 are determined according to the IMGT numbering system.

[0035] In some embodiments of the present invention, the anti-IL-4RA or its antigen-binding fragment, wherein...

[0036] The heavy chain variable region comprises an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:50 and SEQ ID NO:88, and the light chain variable region comprises an amino acid sequence selected from SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:52 and SEQ ID NO:90.

[0037] In some embodiments of the present invention, the anti-IL-4RA or its antigen-binding fragment thereof, wherein:

[0038] (1) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:2, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:4;

[0039] (2) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:13, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:4;

[0040] (3) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:15, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:4;

[0041] (4) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:17, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:4;

[0042] (5) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:19, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:4;

[0043] (6) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:50, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:4;

[0044] (7) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:88, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:4;

[0045] (8) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:2, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:21;

[0046] (9) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:13, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:21;

[0047] (10) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:15, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:21;

[0048] (11) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:17, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:21;

[0049] (12) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:19, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:21;

[0050] (13) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:50, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:21;

[0051] (14) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:88, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:21;

[0052] (15) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:2, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:52;

[0053] (16) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:13, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:52;

[0054] (17) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:15, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:52;

[0055] (18) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:17, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:52;

[0056] (19) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:19, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:52;

[0057] (20) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:50, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:52;

[0058] (21) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:88, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:52;

[0059] (22) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:2, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:90;

[0060] (23) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:13, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:90;

[0061] (24) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:15, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:90;

[0062] (25) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:17, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:90;

[0063] (26) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:19, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:90;

[0064] (27) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:50, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:90; or

[0065] (28) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:88, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:90.

[0066] In some embodiments of the present invention, the anti-IL-4RA antibody or its antigen-binding fragment comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is the human IgG heavy chain constant region, and the light chain constant region is the human Kappa chain constant region or the human Lambda chain constant region.

[0067] In some embodiments of the present invention, the anti-IL-4RA antibody or its antigen-binding fragment, wherein the heavy chain constant region is the human IgG1 heavy chain constant region and the light chain constant region is the human Kappa chain constant region;

[0068] Preferably, according to the EU numbering system, the human IgG1 heavy chain constant region contains the following mutations:

[0069] L234A and L235A,

[0070] L234A, L235A, and G237A, or

[0071] L234A, L235A, G237A, M428L, and N434S;

[0072] Preferably, the amino acid sequence of the constant region of the human IgG1 heavy chain is as shown in any of SEQ ID NOs:66-69;

[0073] Preferably, the amino acid sequence of the constant region of the human Kappa chain is shown in SEQ ID NO:72.

[0074] In this invention, unless otherwise specified, the letter before the site represents the amino acid before the mutation, and the letter after the site represents the amino acid after the mutation.

[0075] In some embodiments of the present invention, the anti-IL-4RA antibody or its antigen-binding fragment, wherein the heavy chain constant region is the human IgG4 heavy chain constant region and the light chain constant region is the human Kappa chain constant region;

[0076] Preferably, according to the EU numbering system, the human IgG4 heavy chain constant region contains the following mutations: M252Y, S254T, and T256E;

[0077] Preferably, the amino acid sequence of the constant region of the human IgG4 heavy chain is as shown in SEQ ID NO:70 or SEQ ID NO:71;

[0078] Preferably, the amino acid sequence of the constant region of the human Kappa chain is shown in SEQ ID NO:72;

[0079] Optionally, the amino acid sequence of the heavy chain of the anti-IL-4RA antibody is shown in SEQ ID NO:11, and the amino acid sequence of the light chain is shown in SEQ ID NO:12.

[0080] In some embodiments of the present invention, the anti-IL-4RA antibody or its antigen-binding fragment, wherein,

[0081] The antibody includes a non-CDR region, and the non-CDR region is derived from human antibodies, mouse antibodies, or rabbit antibodies.

[0082] In some embodiments of the present invention, the anti-IL-4RA antibody or its antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragment, single-chain antibody (scFv), scFv-Fc, humanized antibody or chimeric antibody.

[0083] In some embodiments of the present invention, the anti-IL-4RA antibody or its antigen-binding fragment,

[0084] Its EC binding to the IL4-RA antigen on the surface of 293T-IL4Rα cells 50 Less than or equal to the ECG binding of the antibody Dupilumab to the IL4-RA antigen on the surface of 293T-IL4Rα cells 50 Preferably, the measurement is performed by fluorescence-activated cell sorting.

[0085] Its activity in blocking the upregulation of CD23 expression levels on the surface of B cells by IL-4 is greater than or equal to that of the antibody Dupilumab;

[0086] Its activity in blocking the upregulation of CD23 expression on the surface of B cells by IL-13 is greater than or equal to that of the antibody Dupilumab;

[0087] Its activity in blocking the upregulation of CD23 expression levels on the surface of monocytes by IL-4 is greater than or equal to that of the antibody Dupilumab; and / or

[0088] Its activity in blocking the upregulation of CD23 expression on the surface of monocytes by IL-13 is greater than or equal to that of the antibody Dupilumab.

[0089] In some embodiments of the present invention, the anti-IL-4RA antibody or its antigen-binding fragment is selected from (1) to (4) below:

[0090] (1) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:13, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:21; its heavy chain constant region is the human IgG4 heavy chain constant region, and its light chain constant region is the human Kappa chain constant region.

[0091] (2) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:15, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:21; its heavy chain constant region is the human IgG4 heavy chain constant region, and its light chain constant region is the human Kappa chain constant region.

[0092] (3) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:17, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:21; its heavy chain constant region is the human IgG4 heavy chain constant region, and its light chain constant region is the human Kappa chain constant region.

[0093] (4) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:19, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:21; its heavy chain constant region is the human IgG4 heavy chain constant region, and its light chain constant region is the human Kappa chain constant region.

[0094] In some embodiments of the present invention, the anti-IL-4RA antibody or its antigen-binding fragment is selected from (1) to (4) below:

[0095] (1) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:13, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:21; its heavy chain constant region is the human IgG1 heavy chain constant region, and its light chain constant region is the human Kappa chain constant region.

[0096] (2) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:15, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:21; its heavy chain constant region is the human IgG1 heavy chain constant region, and its light chain constant region is the human Kappa chain constant region.

[0097] (3) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:17, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:21; its heavy chain constant region is the human IgG1 heavy chain constant region, and its light chain constant region is the human Kappa chain constant region.

[0098] (4) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:19, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:21; its heavy chain constant region is the human IgG1 heavy chain constant region, and its light chain constant region is the human Kappa chain constant region.

[0099] In some embodiments of the present invention, the anti-IL-4RA antibody or its antigen-binding fragment is selected from antibodies 14A10H42L25, 14A10H43L25, 14A10H49L25 and 14A10H50L25.

[0100] Another aspect of the present invention relates to an isolated nucleic acid molecule that encodes the anti-IL-4RA antibody or its antigen-binding fragment as described in any one of the present invention.

[0101] The present invention also relates to a recombinant vector comprising the isolated nucleic acid molecules of the present invention.

[0102] The present invention also relates to a host cell containing the isolated nucleic acid molecules of the present invention, or the recombinant vector of the present invention.

[0103] Another aspect of the present invention relates to an antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof and a small molecule drug, wherein the antibody or antigen-binding fragment thereof is any one of the anti-IL-4RA antibodies or antigen-binding fragments thereof described in the present invention; preferably, the small molecule drug is a small molecule cytotoxic drug; more preferably, the small molecule drug is an anti-tumor chemotherapy drug.

[0104] Another aspect of the present invention relates to a pharmaceutical composition comprising an effective amount of any one of the anti-IL-4RA antibodies or antigen-binding fragments thereof or any one of the antibody-drug conjugates of the present invention; optionally, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients;

[0105] Preferably, the pharmaceutical composition is an injection, a nasal spray, or a nasal drop.

[0106] Another aspect of the present invention relates to a packaged product comprising the anti-IL-4RA antibody or its antigen-binding fragment as described in any one of the present invention, the antibody-drug conjugate as described in any one of the present invention, or the pharmaceutical composition of the present invention, and a packaging container;

[0107] Preferably, the packaging container is a syringe, injection pen, nasal spray, or dropper bottle;

[0108] Preferably, the packaged product further includes a product instruction manual.

[0109] Another aspect of the invention relates to a combination product comprising a first product and a second product in individually packaged form, wherein,

[0110] The first product comprises an anti-IL-4RA antibody or its antigen-binding fragment as described in any one of the present invention, an antibody-drug conjugate as described in any one of the present invention, or a pharmaceutical composition of the present invention;

[0111] The second product contains one or more glucocorticoids;

[0112] Preferably, the glucocorticoid is selected from one or more of prednisone, methylprednisolone, betamethasone, beclomethasone dipropionate, prednisolone, hydrocortisone, dexamethasone, and triamcinolone;

[0113] Preferably, the first product and the second product further comprise one or more pharmaceutically acceptable excipients.

[0114] Preferably, the combined product further includes a product instruction manual.

[0115] Another aspect of the present invention relates to the use of any anti-IL-4RA antibody or antigen-binding fragment thereof described in any one of the present invention, any antibody-drug conjugate described in any one of the present invention, or any pharmaceutical composition of the present invention in the preparation of a medicament for treating or preventing a disease, wherein the disease is one or more selected from allergic diseases, autoimmune diseases, inflammatory diseases, and tumors;

[0116] Preferably, the disease is mediated by IL-4 and / or IL-13;

[0117] Preferably, the disease is selected from one or more of the following: asthma, allergic rhinitis, sinusitis, nodular prurigo, chronic urticaria, nasal polyps, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, food allergy, rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, ankylosing spondylitis, eosinophilic esophagitis, pulmonary fibrosis, systemic lupus erythematosus, cardiovascular disease, and systemic sclerosis.

[0118] Preferably, the asthma is allergic asthma;

[0119] Preferably, the allergic rhinitis is seasonal allergic rhinitis (SAR);

[0120] Preferably, the sinusitis is chronic sinusitis;

[0121] Preferably, the chronic obstructive pulmonary disease is moderate to severe chronic obstructive pulmonary disease;

[0122] Preferably, the atopic dermatitis is moderate to severe atopic dermatitis;

[0123] Preferably, the single dose, calculated based on the anti-IL-4RA antibody or its antigen-binding fragment, is 0.1-100 mg per kilogram of body weight, more preferably 5-50 mg or 5-15 mg per kilogram of body weight;

[0124] Preferably, each patient is given a single dose of 0-1000 mg, 0-900 mg, 0-800 mg, 0-700 mg, 0-600 mg, 0-500 mg, 0-400 mg, 0-300 mg, 0-200 mg, 0-100 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or 1000 mg, calculated according to the anti-IL-4RA antibody or its antigen-binding fragment.

[0125] Preferably, the medication is administered once every 3 days, every 4 days, every 5 days, every 6 days, every 10 days, every week, every 2 weeks, or every 3 weeks;

[0126] Preferably, the administration method is intravenous infusion, intravenous injection, intradermal injection, subcutaneous injection, intramuscular injection, or nasal administration.

[0127] The anti-IL-4RA antibody or its antigen-binding fragment according to any one of the present invention, or the antibody-drug conjugate according to any one of the present invention, or the pharmaceutical composition of the present invention, is used to treat or prevent a disease, wherein the disease is one or more selected from allergic diseases, autoimmune diseases, inflammatory diseases, and tumors;

[0128] Preferably, the disease is mediated by IL-4 and / or IL-13;

[0129] Preferably, the disease is selected from one or more of the following: asthma, allergic rhinitis, sinusitis, nodular prurigo, chronic urticaria, nasal polyps, chronic obstructive pulmonary disease, pulmonary fibrosis, food allergy, rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, ankylosing spondylitis, eosinophilic esophagitis, pulmonary fibrosis, systemic lupus erythematosus, cardiovascular disease, and systemic sclerosis.

[0130] Preferably, the asthma is allergic asthma;

[0131] Preferably, the allergic rhinitis is seasonal allergic rhinitis;

[0132] Preferably, the sinusitis is chronic sinusitis;

[0133] Preferably, the chronic obstructive pulmonary disease is moderate to severe chronic obstructive pulmonary disease;

[0134] Preferably, the atopic dermatitis is moderate to severe atopic dermatitis;

[0135] Preferably, the single dose, calculated based on the anti-IL-4RA antibody or its antigen-binding fragment, is 0.1-100 mg per kilogram of body weight, more preferably 5-50 mg or 5-15 mg per kilogram of body weight;

[0136] Preferably, each patient is given a single dose of 0-1000 mg, 0-900 mg, 0-800 mg, 0-700 mg, 0-600 mg, 0-500 mg, 0-400 mg, 0-300 mg, 0-200 mg, 0-100 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or 1000 mg, calculated according to the anti-IL-4RA antibody or its antigen-binding fragment.

[0137] Preferably, the medication is administered once every 3 days, every 4 days, every 5 days, every 6 days, every 10 days, every week, every 2 weeks, or every 3 weeks;

[0138] Preferably, the administration method is intravenous infusion, intravenous injection, intradermal injection, subcutaneous injection, intramuscular injection, or nasal administration.

[0139] Another aspect of the present invention relates to a method of treating or preventing a disease, comprising the step of administering to a subject in need an effective amount of any one of the anti-IL-4RA antibodies or antigen-binding fragments thereof, any one of the antibody-drug conjugates or pharmaceutical compositions of the present invention, wherein the disease is one or more selected from allergic diseases, autoimmune diseases, inflammatory diseases and tumors;

[0140] Preferably, the disease is mediated by IL-4 and / or IL-13;

[0141] Preferably, the disease is selected from one or more of the following: asthma, allergic rhinitis, sinusitis, nodular prurigo, chronic urticaria, nasal polyps, chronic obstructive pulmonary disease, pulmonary fibrosis, food allergy, rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, ankylosing spondylitis, eosinophilic esophagitis, pulmonary fibrosis, systemic lupus erythematosus, cardiovascular disease, and systemic sclerosis.

[0142] Preferably, the asthma is allergic asthma;

[0143] Preferably, the allergic rhinitis is seasonal allergic rhinitis;

[0144] Preferably, the sinusitis is chronic sinusitis;

[0145] Preferably, the chronic obstructive pulmonary disease is moderate to severe chronic obstructive pulmonary disease;

[0146] Preferably, the atopic dermatitis is moderate to severe atopic dermatitis;

[0147] Preferably, the single dose, calculated based on the anti-IL-4RA antibody or its antigen-binding fragment, is 0.1-100 mg per kilogram of body weight, more preferably 5-50 mg or 5-15 mg per kilogram of body weight;

[0148] Preferably, each patient is given a single dose of 0-1000 mg, 0-900 mg, 0-800 mg, 0-700 mg, 0-600 mg, 0-500 mg, 0-400 mg, 0-300 mg, 0-200 mg, 0-100 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or 1000 mg, calculated according to the anti-IL-4RA antibody or its antigen-binding fragment.

[0149] Preferably, the medication is administered once every 3 days, every 4 days, every 5 days, every 6 days, every 10 days, every week, every 2 weeks, or every 3 weeks;

[0150] Preferably, the administration method is intravenous infusion, intravenous injection, intradermal injection, subcutaneous injection, intramuscular injection, or nasal administration.

[0151] Another aspect of the present invention relates to a bispecific antibody comprising:

[0152] Targeting the first protein functional region of IL-4RA, and

[0153] Targeting a second protein functional region that is different from the target of IL-4RA;

[0154] in:

[0155] The first protein functional region contains any one of the anti-IL-4RA antibodies or its antigen-binding fragments as described in any one of the present invention.

[0156] In some embodiments of the present invention, the bispecific antibody is wherein the target, which is different from IL-4RA, is ST2.

[0157] In some embodiments of the present invention, the bispecific antibody is an anti-IL-4RA-anti-ST2 bispecific antibody.

[0158] In some embodiments of the present invention, the bispecific antibody is a bispecific antibody in the form of IgG-scFv.

[0159] In some embodiments of the present invention, the bispecific antibody is a tetravalent bispecific antibody in the form of IgG-scFv.

[0160] In some embodiments of the present invention, the bispecific antibody is wherein the second protein functional region comprises an anti-ST2 antibody or an antigen-binding fragment thereof.

[0161] In some embodiments of the present invention, the bispecific antibody, wherein the anti-ST2 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising complementarity-determining regions HCDR1 to HCDR3, and the light chain variable region comprising complementarity-determining regions LCDR1 to LCDR3, wherein:

[0162] HCDR1 contains the amino acid sequence shown in SEQ ID NO:29, HCDR2 contains the amino acid sequence shown in SEQ ID NO:30, and HCDR3 contains the amino acid sequence shown in SEQ ID NO:31; and

[0163] LCDR1 contains the amino acid sequence shown in SEQ ID NO:32, LCDR2 contains the amino acid sequence shown in SEQ ID NO:33, and LCDR3 contains the amino acid sequence shown in SEQ ID NO:34.

[0164] In some embodiments of the present invention, the bispecific antibodies, wherein HCDR1 to HCDR3 and LCDR1 to LCDR3 of anti-IL-4RA or its antigen-binding fragment and anti-ST2 or its antigen-binding fragment are determined according to the IMGT numbering system.

[0165] In some embodiments of the present invention, the bispecific antibody, wherein,

[0166] The heavy chain variable region of the anti-ST2 antibody comprises an amino acid sequence selected from SEQ ID NO:26, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:49, and SEQ ID NO:92; and

[0167] The light chain variable region of the anti-ST2 antibody comprises an amino acid sequence selected from SEQ ID NO:28, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:51 and SEQ ID NO:93.

[0168] In some embodiments of the present invention, the bispecific antibody, wherein the anti-ST2 antibody:

[0169] (1) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:26, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:28;

[0170] (2) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:37, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:28;

[0171] (3) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:39, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:28;

[0172] (4) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:41, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:28;

[0173] (5) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:43, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:28;

[0174] (6) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:49, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:28;

[0175] (7) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:92, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:28;

[0176] (8) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:26, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:45;

[0177] (9) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:37, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:45;

[0178] (10) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:39, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:45;

[0179] (11) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:41, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:45;

[0180] (12) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:43, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:45;

[0181] (13) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:49, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:45;

[0182] (14) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:92, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:45;

[0183] (15) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:26, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:47;

[0184] (16) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:37, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:47;

[0185] (17) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:39, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:47;

[0186] (18) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:41, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:47;

[0187] (19) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:43, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:47;

[0188] (20) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:49, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:47;

[0189] (21) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:92, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:47;

[0190] (22) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:26, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:51;

[0191] (23) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:37, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:51;

[0192] (24) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:39, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:51;

[0193] (25) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:41, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:51;

[0194] (26) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:43, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:51;

[0195] (27) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:49, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:51;

[0196] (28) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:92, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:51;

[0197] (29) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:26, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:93;

[0198] (30) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:37, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:93;

[0199] (31) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:39, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:93;

[0200] (32) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:41, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:93;

[0201] (33) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:43, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:93;

[0202] (34) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:49, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:93; or

[0203] (35) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:92, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:93.

[0204] This invention also relates to a bispecific antibody, comprising:

[0205] Targeting the first protein functional region of IL-4RA, and

[0206] Targeting the second protein functional region of ST2;

[0207] in:

[0208] The first protein functional region contains any one of the anti-IL-4RA antibodies or its antigen-binding fragments as described in any one of the present invention;

[0209] The second protein functional region contains an anti-ST2 antibody or its antigen-binding fragment.

[0210] This invention also relates to a bispecific antibody, comprising:

[0211] Targeting the first protein functional region of IL-4RA, and

[0212] Targeting the second protein functional region of ST2;

[0213] in:

[0214] The first protein functional region contains any one of the anti-IL-4RA antibodies or its antigen-binding fragments as described in any one of the present invention;

[0215] The second protein functional region contains an anti-ST2 antibody or its antigen-binding fragment;

[0216] The anti-IL-4RA antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises complementarity-determining regions HCDR1 to HCDR3, and the light chain variable region comprises complementarity-determining regions LCDR1 to LCDR3, wherein: HCDR1 comprises the amino acid sequence shown in SEQ ID NO:5, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:6, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:7; and LCDR1 comprises the amino acid sequence shown in SEQ ID NO:8, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:9, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:10.

[0217] The anti-ST2 antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises complementarity-determining regions HCDR1 to HCDR3, and the light chain variable region comprises complementarity-determining regions LCDR1 to LCDR3. HCDR1 comprises the amino acid sequence shown in SEQ ID NO:29, HCDR2 comprises the amino acid sequence shown in SEQ ID NO:30, and HCDR3 comprises the amino acid sequence shown in SEQ ID NO:31. Furthermore, LCDR1 comprises the amino acid sequence shown in SEQ ID NO:32, LCDR2 comprises the amino acid sequence shown in SEQ ID NO:33, and LCDR3 comprises the amino acid sequence shown in SEQ ID NO:34.

[0218] In some embodiments of the present invention, the bispecific antibody, wherein the anti-IL-4RA antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region of any one of the preceding items (1) to (28), and the anti-ST2 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region of any one of the preceding items (1) to (35).

[0219] In some embodiments of the present invention, the bispecific antibody is wherein the first protein functional region is directly connected to the second protein functional region or is connected through a linker.

[0220] Preferably, the linker is selected from the amino acid sequences of SEQ ID NOs:53, 54 and 76-78;

[0221] Preferably, the first protein functional region and the second protein functional region are independently one, two, or more.

[0222] In some embodiments of the present invention, the bispecific antibody is a bispecific antibody in the form of IgG-scFv.

[0223] In some embodiments of the present invention, the bispecific antibody is wherein the first protein functional region is an immunoglobulin against IL-4RA, and the second protein functional region is a single-chain antibody against ST2.

[0224] Preferably, the anti-ST2 single-chain antibody comprises two molecules, which are respectively linked to the N-terminus or C-terminus of the two heavy chains of the anti-IL-4RA immunoglobulin.

[0225] Preferably, the bispecific antibody is a bispecific antibody in the form of IgG-scFv;

[0226] Preferably, the bispecific antibody is a tetravalent bispecific antibody in the form of IgG-scFv.

[0227] In some embodiments of the present invention, the bispecific antibody, wherein,

[0228] The bispecific antibody includes:

[0229] Targeting the first protein functional region of IL-4RA, and

[0230] Targeting the second protein functional region of ST2;

[0231] The first protein has one functional region, and the second protein has two functional regions;

[0232] Wherein, the first protein functional region is an immunoglobulin against IL-4RA, and the second protein functional region is a single-chain antibody against ST2;

[0233] The amino acid sequence of the heavy chain variable region of the anti-IL-4RA immunoglobulin is shown in SEQ ID NO:17, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:21.

[0234] The amino acid sequence of the heavy chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:49, and the amino acid sequence of the light chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:51; or, the amino acid sequence of the heavy chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:43, and the amino acid sequence of the light chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:45.

[0235] The anti-ST2 single-chain antibody is respectively linked to the C-terminus of the two heavy chains of the anti-IL-4RA immunoglobulin;

[0236] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the anti-ST2 single-chain antibody and the light chain variable region of the anti-ST2 single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different;

[0237] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO:53 and SEQ ID NO:54.

[0238] In some embodiments of the present invention, the bispecific antibody, wherein,

[0239] The bispecific antibody includes:

[0240] Targeting the first protein functional region of IL-4RA, and

[0241] Targeting the second protein functional region of ST2;

[0242] The first protein has one functional region, and the second protein has two functional regions;

[0243] Wherein, the first protein functional region is an immunoglobulin against IL-4RA, and the second protein functional region is a single-chain antibody against ST2;

[0244] The amino acid sequence of the heavy chain variable region of the anti-IL-4RA immunoglobulin is shown in SEQ ID NO:13, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:21; and

[0245] i) The amino acid sequence of the heavy chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:37, and the amino acid sequence of the light chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:45.

[0246] ii) The amino acid sequence of the heavy chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:37, and the amino acid sequence of the light chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:47;

[0247] iii) The amino acid sequence of the heavy chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:39, and the amino acid sequence of the light chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:47;

[0248] iv) The amino acid sequence of the heavy chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:41, and the amino acid sequence of the light chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:45.

[0249] v) The amino acid sequence of the heavy chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:41, and the amino acid sequence of the light chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:47.

[0250] vi) The amino acid sequence of the heavy chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:43, and the amino acid sequence of the light chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:45.

[0251] vii) The amino acid sequence of the heavy chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:43, and the amino acid sequence of the light chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:47; or,

[0252] viii) The amino acid sequence of the heavy chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:49, and the amino acid sequence of the light chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:51.

[0253] The anti-ST2 single-chain antibody is respectively linked to the C-terminus of the two heavy chains of the anti-IL-4RA immunoglobulin;

[0254] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the anti-ST2 single-chain antibody and the light chain variable region of the anti-ST2 single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different;

[0255] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO:53 and SEQ ID NO:54.

[0256] In some embodiments of the present invention, the bispecific antibody, wherein,

[0257] The bispecific antibody includes:

[0258] Targeting the first protein functional region of IL-4RA, and

[0259] Targeting the second protein functional region of ST2;

[0260] The first protein has one functional region, and the second protein has two functional regions;

[0261] Wherein, the first protein functional region is an immunoglobulin against IL-4RA, and the second protein functional region is a single-chain antibody against ST2;

[0262] The amino acid sequence of the heavy chain variable region of the anti-IL-4RA immunoglobulin is shown in SEQ ID NO:15, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:21; and

[0263] i) The amino acid sequence of the heavy chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:37, and the amino acid sequence of the light chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:45.

[0264] ii) The amino acid sequence of the heavy chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:37, and the amino acid sequence of the light chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:47;

[0265] iii) The amino acid sequence of the heavy chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:39, and the amino acid sequence of the light chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:47;

[0266] iv) The amino acid sequence of the heavy chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:41, and the amino acid sequence of the light chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:45.

[0267] v) The amino acid sequence of the heavy chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:41, and the amino acid sequence of the light chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:47.

[0268] vi) The amino acid sequence of the heavy chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:43, and the amino acid sequence of the light chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:45.

[0269] vii) The amino acid sequence of the heavy chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:43, and the amino acid sequence of the light chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:47; or,

[0270] viii) The amino acid sequence of the heavy chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:49, and the amino acid sequence of the light chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:51.

[0271] The anti-ST2 single-chain antibody is respectively linked to the C-terminus of the two heavy chains of the anti-IL-4RA immunoglobulin;

[0272] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the anti-ST2 single-chain antibody and the light chain variable region of the anti-ST2 single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different;

[0273] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO:53 and SEQ ID NO:54.

[0274] In some embodiments of the present invention, the bispecific antibody, wherein,

[0275] The bispecific antibody includes:

[0276] Targeting the first protein functional region of IL-4RA, and

[0277] Targeting the second protein functional region of ST2;

[0278] The first protein has one functional region, and the second protein has two functional regions;

[0279] Wherein, the first protein functional region is an immunoglobulin against IL-4RA, and the second protein functional region is a single-chain antibody against ST2;

[0280] The amino acid sequence of the heavy chain variable region of the anti-IL-4RA immunoglobulin is shown in SEQ ID NO:17, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:21; and

[0281] i) The amino acid sequence of the heavy chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:37, and the amino acid sequence of the light chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:45.

[0282] ii) The amino acid sequence of the heavy chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:37, and the amino acid sequence of the light chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:47;

[0283] iii) The amino acid sequence of the heavy chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:39, and the amino acid sequence of the light chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:47;

[0284] iv) The amino acid sequence of the heavy chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:41, and the amino acid sequence of the light chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:45.

[0285] v) The amino acid sequence of the heavy chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:41, and the amino acid sequence of the light chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:47.

[0286] vi) The amino acid sequence of the heavy chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:43, and the amino acid sequence of the light chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:45.

[0287] vii) The amino acid sequence of the heavy chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:43, and the amino acid sequence of the light chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:47; or,

[0288] viii) The amino acid sequence of the heavy chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:49, and the amino acid sequence of the light chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:51.

[0289] The anti-ST2 single-chain antibody is respectively linked to the C-terminus of the two heavy chains of the anti-IL-4RA immunoglobulin;

[0290] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the anti-ST2 single-chain antibody and the light chain variable region of the anti-ST2 single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different;

[0291] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO:53 and SEQ ID NO:54.

[0292] In some embodiments of the present invention, the bispecific antibody, wherein,

[0293] The bispecific antibody includes:

[0294] Targeting the first protein functional region of IL-4RA, and

[0295] Targeting the second protein functional region of ST2;

[0296] The first protein has one functional region, and the second protein has two functional regions;

[0297] Wherein, the first protein functional region is an immunoglobulin against IL-4RA, and the second protein functional region is a single-chain antibody against ST2;

[0298] The amino acid sequence of the heavy chain variable region of the anti-IL-4RA immunoglobulin is shown in SEQ ID NO:19, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:21; and

[0299] i) The amino acid sequence of the heavy chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:37, and the amino acid sequence of the light chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:45.

[0300] ii) The amino acid sequence of the heavy chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:37, and the amino acid sequence of the light chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:47;

[0301] iii) The amino acid sequence of the heavy chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:39, and the amino acid sequence of the light chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:47;

[0302] iv) The amino acid sequence of the heavy chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:41, and the amino acid sequence of the light chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:45.

[0303] v) The amino acid sequence of the heavy chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:41, and the amino acid sequence of the light chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:47.

[0304] vi) The amino acid sequence of the heavy chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:43, and the amino acid sequence of the light chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:45.

[0305] vii) The amino acid sequence of the heavy chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:43, and the amino acid sequence of the light chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:47; or,

[0306] viii) The amino acid sequence of the heavy chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:49, and the amino acid sequence of the light chain variable region of the single-chain antibody against ST2 is shown in SEQ ID NO:51.

[0307] The anti-ST2 single-chain antibody is respectively linked to the C-terminus of the two heavy chains of the anti-IL-4RA immunoglobulin;

[0308] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the anti-ST2 single-chain antibody and the light chain variable region of the anti-ST2 single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different;

[0309] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO:53 and SEQ ID NO:54.

[0310] In some embodiments of the present invention, the bispecific antibody, wherein the anti-IL-4RA immunoglobulin comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region is the human IgG heavy chain constant region, and the light chain constant region is the human Kappa chain constant region or the human Lambda chain constant region.

[0311] In some embodiments of the present invention, the bispecific antibody comprises a heavy chain constant region of human IgG1 and a light chain constant region of human Kappa chain of the anti-IL-4RA immunoglobulin.

[0312] Preferably, according to the EU numbering system, the human IgG1 heavy chain constant region contains the following mutations:

[0313] L234A and L235A,

[0314] L234A, L235A, and G237A, or

[0315] L234A, L235A, G237A, M428L, and N434S;

[0316] Preferably, the amino acid sequence of the constant region of the human IgG1 heavy chain is as shown in any of SEQ ID NOs:66-69;

[0317] Preferably, the amino acid sequence of the constant region of the human Kappa chain is shown in SEQ ID NO:72.

[0318] In some embodiments of the present invention, the bispecific antibody comprises a heavy chain constant region of human IgG4 heavy chain constant region and a light chain constant region of human Kappa chain constant region of anti-IL-4RA immunoglobulin.

[0319] Preferably, according to the EU numbering system, the human IgG4 heavy chain constant region contains the following mutations: M252Y, S254T, and T256E;

[0320] Preferably, the amino acid sequence of the constant region of the human IgG4 heavy chain is as shown in SEQ ID NO:70 or SEQ ID NO:71;

[0321] Preferably, the amino acid sequence of the constant region of the human Kappa chain is shown in SEQ ID NO:72.

[0322] In some embodiments of the present invention, the bispecific antibody, wherein,

[0323] The bispecific antibody includes:

[0324] Targeting the first protein functional region of IL-4RA, and

[0325] Targeting the second protein functional region of ST2;

[0326] The first protein has one functional region, and the second protein has two functional regions;

[0327] Wherein, the first protein functional region is an immunoglobulin against IL-4RA, and the second protein functional region is a single-chain antibody against ST2;

[0328] The amino acid sequence of the heavy chain of the anti-IL-4RA immunoglobulin is shown in SEQ ID NO:85 or SEQ ID NO:86, and the amino acid sequence of its light chain is shown in SEQ ID NO:64.

[0329] The amino acid sequence of the heavy chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:49, and the amino acid sequence of the light chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:51.

[0330] The anti-ST2 single-chain antibody is respectively linked to the C-terminus of the two heavy chains of the anti-IL-4RA immunoglobulin;

[0331] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the anti-ST2 single-chain antibody and the light chain variable region of the anti-ST2 single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different;

[0332] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO:53 and SEQ ID NO:54.

[0333] In some embodiments of the present invention, the bispecific antibody, wherein,

[0334] The bispecific antibody includes:

[0335] Targeting the first protein functional region of IL-4RA, and

[0336] Targeting the second protein functional region of ST2;

[0337] The first protein has one functional region, and the second protein has two functional regions;

[0338] Wherein, the first protein functional region is an immunoglobulin against IL-4RA, and the second protein functional region is a single-chain antibody against ST2;

[0339] The amino acid sequence of the heavy chain of the anti-IL-4RA immunoglobulin is shown in SEQ ID NO:85 or SEQ ID NO:86, and the amino acid sequence of its light chain is shown in SEQ ID NO:64.

[0340] The amino acid sequence of the heavy chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:43, and the amino acid sequence of the light chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:45.

[0341] The anti-ST2 single-chain antibody is respectively linked to the C-terminus of the two heavy chains of the anti-IL-4RA immunoglobulin;

[0342] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the anti-ST2 single-chain antibody and the light chain variable region of the anti-ST2 single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different;

[0343] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO:53 and SEQ ID NO:54.

[0344] In some embodiments of the present invention, the bispecific antibody, wherein,

[0345] The bispecific antibody includes:

[0346] Targeting the first protein functional region of IL-4RA, and

[0347] Targeting the second protein functional region of ST2;

[0348] The first protein has one functional region, and the second protein has two functional regions;

[0349] Wherein, the first protein functional region is an immunoglobulin against IL-4RA, and the second protein functional region is a single-chain antibody against ST2;

[0350] The amino acid sequence of the heavy chain of the anti-IL-4RA immunoglobulin is shown in SEQ ID NO:85 or SEQ ID NO:86, and the amino acid sequence of its light chain is shown in SEQ ID NO:64.

[0351] The amino acid sequence of the heavy chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:37, and the amino acid sequence of the light chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:45.

[0352] The anti-ST2 single-chain antibody is respectively linked to the C-terminus of the two heavy chains of the anti-IL-4RA immunoglobulin;

[0353] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the anti-ST2 single-chain antibody and the light chain variable region of the anti-ST2 single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different;

[0354] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO:53 and SEQ ID NO:54.

[0355] In some embodiments of the present invention, the bispecific antibody, wherein,

[0356] The bispecific antibody includes:

[0357] Targeting the first protein functional region of IL-4RA, and

[0358] Targeting the second protein functional region of ST2;

[0359] The first protein has one functional region, and the second protein has two functional regions;

[0360] Wherein, the first protein functional region is an immunoglobulin against IL-4RA, and the second protein functional region is a single-chain antibody against ST2;

[0361] The amino acid sequence of the heavy chain of the anti-IL-4RA immunoglobulin is shown in SEQ ID NO:85 or SEQ ID NO:86, and the amino acid sequence of its light chain is shown in SEQ ID NO:64.

[0362] The amino acid sequence of the heavy chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:37, and the amino acid sequence of the light chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:47.

[0363] The anti-ST2 single-chain antibody is respectively linked to the C-terminus of the two heavy chains of the anti-IL-4RA immunoglobulin;

[0364] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the anti-ST2 single-chain antibody and the light chain variable region of the anti-ST2 single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different;

[0365] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO:53 and SEQ ID NO:54.

[0366] In some embodiments of the present invention, the bispecific antibody, wherein,

[0367] The bispecific antibody includes:

[0368] Targeting the first protein functional region of IL-4RA, and

[0369] Targeting the second protein functional region of ST2;

[0370] The first protein has one functional region, and the second protein has two functional regions;

[0371] Wherein, the first protein functional region is an immunoglobulin against IL-4RA, and the second protein functional region is a single-chain antibody against ST2;

[0372] The amino acid sequence of the heavy chain of the anti-IL-4RA immunoglobulin is shown in SEQ ID NO:85 or SEQ ID NO:86, and the amino acid sequence of its light chain is shown in SEQ ID NO:64.

[0373] The amino acid sequence of the heavy chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:39, and the amino acid sequence of the light chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:47.

[0374] The anti-ST2 single-chain antibody is respectively linked to the C-terminus of the two heavy chains of the anti-IL-4RA immunoglobulin;

[0375] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the anti-ST2 single-chain antibody and the light chain variable region of the anti-ST2 single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different;

[0376] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO:53 and SEQ ID NO:54.

[0377] In some embodiments of the present invention, the bispecific antibody, wherein,

[0378] The bispecific antibody includes:

[0379] Targeting the first protein functional region of IL-4RA, and

[0380] Targeting the second protein functional region of ST2;

[0381] The first protein has one functional region, and the second protein has two functional regions;

[0382] Wherein, the first protein functional region is an immunoglobulin against IL-4RA, and the second protein functional region is a single-chain antibody against ST2;

[0383] The amino acid sequence of the heavy chain of the anti-IL-4RA immunoglobulin is shown in SEQ ID NO:85 or SEQ ID NO:86, and the amino acid sequence of its light chain is shown in SEQ ID NO:64.

[0384] The amino acid sequence of the heavy chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:41, and the amino acid sequence of the light chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:45.

[0385] The anti-ST2 single-chain antibody is respectively linked to the C-terminus of the two heavy chains of the anti-IL-4RA immunoglobulin;

[0386] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the anti-ST2 single-chain antibody and the light chain variable region of the anti-ST2 single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different;

[0387] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO:53 and SEQ ID NO:54.

[0388] In some embodiments of the present invention, the bispecific antibody, wherein,

[0389] The bispecific antibody includes:

[0390] Targeting the first protein functional region of IL-4RA, and

[0391] Targeting the second protein functional region of ST2;

[0392] The first protein has one functional region, and the second protein has two functional regions;

[0393] Wherein, the first protein functional region is an immunoglobulin against IL-4RA, and the second protein functional region is a single-chain antibody against ST2;

[0394] The amino acid sequence of the heavy chain of the anti-IL-4RA immunoglobulin is shown in SEQ ID NO:85 or SEQ ID NO:86, and the amino acid sequence of its light chain is shown in SEQ ID NO:64.

[0395] The amino acid sequence of the heavy chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:41, and the amino acid sequence of the light chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:47.

[0396] The anti-ST2 single-chain antibody is respectively linked to the C-terminus of the two heavy chains of the anti-IL-4RA immunoglobulin;

[0397] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the anti-ST2 single-chain antibody and the light chain variable region of the anti-ST2 single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different;

[0398] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO:53 and SEQ ID NO:54.

[0399] In some embodiments of the present invention, the bispecific antibody, wherein,

[0400] The bispecific antibody includes:

[0401] Targeting the first protein functional region of IL-4RA, and

[0402] Targeting the second protein functional region of ST2;

[0403] The first protein has one functional region, and the second protein has two functional regions;

[0404] Wherein, the first protein functional region is an immunoglobulin against IL-4RA, and the second protein functional region is a single-chain antibody against ST2;

[0405] The amino acid sequence of the heavy chain of the anti-IL-4RA immunoglobulin is shown in SEQ ID NO:85 or SEQ ID NO:86, and the amino acid sequence of its light chain is shown in SEQ ID NO:64.

[0406] The amino acid sequence of the heavy chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:43, and the amino acid sequence of the light chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:47.

[0407] The anti-ST2 single-chain antibody is respectively linked to the C-terminus of the two heavy chains of the anti-IL-4RA immunoglobulin;

[0408] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the anti-ST2 single-chain antibody and the light chain variable region of the anti-ST2 single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different;

[0409] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO:53 and SEQ ID NO:54.

[0410] In some embodiments of the present invention, the bispecific antibody is wherein the first protein functional region is a single-chain antibody against IL-4RA, and the second protein functional region is an immunoglobulin against ST2.

[0411] Preferably, the anti-IL-4RA single-chain antibody comprises two molecules, which are respectively linked to the N-terminus or C-terminus of the two heavy chains of the anti-ST2 immunoglobulin.

[0412] Preferably, the bispecific antibody is a bispecific antibody in the form of IgG-scFv;

[0413] Preferably, the bispecific antibody is a tetravalent bispecific antibody in the form of IgG-scFv.

[0414] In some embodiments of the present invention, the bispecific antibody comprises a heavy chain constant region of human IgG heavy chain constant region and a light chain constant region of human Kappa chain constant region or human Lambda chain constant region of anti-ST2 immunoglobulin.

[0415] In some embodiments of the present invention, the bispecific antibody comprises a heavy chain constant region of human IgG1 and a light chain constant region of human Kappa chain of the anti-ST2 immunoglobulin.

[0416] Preferably, according to the EU numbering system, the human IgG1 heavy chain constant region contains the following mutations:

[0417] L234A and L235A,

[0418] L234A, L235A, and G237A, or

[0419] L234A, L235A, G237A, M428L, and N434S;

[0420] Preferably, the amino acid sequence of the constant region of the human IgG1 heavy chain is as shown in any of SEQ ID NOs:66-69;

[0421] Preferably, the amino acid sequence of the constant region of the human Kappa chain is shown in SEQ ID NO:72.

[0422] In some embodiments of the present invention, the bispecific antibody comprises a heavy chain constant region of human IgG4 and a light chain constant region of human Kappa chain of the anti-ST2 immunoglobulin.

[0423] Preferably, according to the EU numbering system, the human IgG4 heavy chain constant region contains the following mutations: M252Y, S254T, and T256E;

[0424] Preferably, the amino acid sequence of the constant region of the human IgG4 heavy chain is as shown in SEQ ID NO:70 or SEQ ID NO:71;

[0425] Preferably, the amino acid sequence of the constant region of the human Kappa chain is shown in SEQ ID NO:72.

[0426] In some embodiments of the present invention, the bispecific antibody, wherein,

[0427] The bispecific antibody includes:

[0428] Targeting the first protein functional region of IL-4RA, and

[0429] Targeting the second protein functional region of ST2;

[0430] The first protein has two functional regions, and the second protein has one functional region;

[0431] Wherein, the first protein functional region is a single-chain antibody against IL-4RA, and the second protein functional region is an immunoglobulin against ST2;

[0432] The amino acid sequence of the heavy chain variable region of the anti-IL-4RA single-chain antibody is shown in SEQ ID NO:17, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:21; or, the amino acid sequence of the heavy chain variable region of the anti-IL-4RA single-chain antibody is shown in SEQ ID NO:50, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:52.

[0433] The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:43, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:45.

[0434] The anti-IL-4RA single-chain antibody is respectively linked to the C-terminus of the two heavy chains of the anti-ST2 immunoglobulin;

[0435] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the anti-IL-4RA single-chain antibody and the light chain variable region of the anti-IL-4RA single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different;

[0436] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO:53 and SEQ ID NO:54.

[0437] In some embodiments of the present invention, the bispecific antibody, wherein,

[0438] The bispecific antibody includes:

[0439] Targeting the first protein functional region of IL-4RA, and

[0440] Targeting the second protein functional region of ST2;

[0441] The first protein has two functional regions, and the second protein has one functional region;

[0442] Wherein, the first protein functional region is a single-chain antibody against IL-4RA, and the second protein functional region is an immunoglobulin against ST2;

[0443] The amino acid sequence of the heavy chain variable region of the anti-IL-4RA single-chain antibody is shown in SEQ ID NO:13, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:21; and

[0444] i) The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:37, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:45.

[0445] ii) The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:37, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:47;

[0446] iii) The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:39, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:47;

[0447] iv) The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:41, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:45.

[0448] v) The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:41, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:47.

[0449] vi) The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:43, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:45.

[0450] vii) The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:43, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:47; or,

[0451] viii) The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:49, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:51.

[0452] The single-chain antibody of IL-4RA is respectively linked to the C-terminus of the two heavy chains of the anti-ST2 immunoglobulin.

[0453] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the anti-IL-4RA single-chain antibody and the light chain variable region of the anti-IL-4RA single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different;

[0454] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO:53 and SEQ ID NO:54.

[0455] In some embodiments of the present invention, the bispecific antibody, wherein,

[0456] The bispecific antibody includes:

[0457] Targeting the first protein functional region of IL-4RA, and

[0458] Targeting the second protein functional region of ST2;

[0459] The first protein has two functional regions, and the second protein has one functional region;

[0460] Wherein, the first protein functional region is a single-chain antibody against IL-4RA, and the second protein functional region is an immunoglobulin against ST2;

[0461] The amino acid sequence of the heavy chain variable region of the anti-IL-4RA single-chain antibody is shown in SEQ ID NO:15, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:21; and

[0462] i) The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:37, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:45.

[0463] ii) The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:37, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:47;

[0464] iii) The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:39, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:47;

[0465] iv) The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:41, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:45.

[0466] v) The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:41, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:47.

[0467] vi) The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:43, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:45.

[0468] vii) The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:43, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:47; or,

[0469] viii) The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:49, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:51.

[0470] The single-chain antibody of IL-4RA is respectively linked to the C-terminus of the two heavy chains of the anti-ST2 immunoglobulin.

[0471] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the anti-IL-4RA single-chain antibody and the light chain variable region of the anti-IL-4RA single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different;

[0472] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO:53 and SEQ ID NO:54.

[0473] In some embodiments of the present invention, the bispecific antibody, wherein,

[0474] The bispecific antibody includes:

[0475] Targeting the first protein functional region of IL-4RA, and

[0476] Targeting the second protein functional region of ST2;

[0477] The first protein has two functional regions, and the second protein has one functional region;

[0478] Wherein, the first protein functional region is a single-chain antibody against IL-4RA, and the second protein functional region is an immunoglobulin against ST2;

[0479] The amino acid sequence of the heavy chain variable region of the anti-IL-4RA single-chain antibody is shown in SEQ ID NO:17, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:21; and

[0480] i) The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:37, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:45.

[0481] ii) The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:37, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:47;

[0482] iii) The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:39, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:47;

[0483] iv) The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:41, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:45.

[0484] v) The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:41, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:47.

[0485] vi) The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:43, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:45.

[0486] vii) The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:43, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:47; or,

[0487] viii) The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:49, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:51.

[0488] The single-chain antibody of IL-4RA is respectively linked to the C-terminus of the two heavy chains of the anti-ST2 immunoglobulin.

[0489] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the anti-IL-4RA single-chain antibody and the light chain variable region of the anti-IL-4RA single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different;

[0490] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO:53 and SEQ ID NO:54.

[0491] In some embodiments of the present invention, the bispecific antibody, wherein,

[0492] The bispecific antibody includes:

[0493] Targeting the first protein functional region of IL-4RA, and

[0494] Targeting the second protein functional region of ST2;

[0495] The first protein has two functional regions, and the second protein has one functional region;

[0496] Wherein, the first protein functional region is a single-chain antibody against IL-4RA, and the second protein functional region is an immunoglobulin against ST2;

[0497] The amino acid sequence of the heavy chain variable region of the anti-IL-4RA single-chain antibody is shown in SEQ ID NO:19, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:21; and

[0498] i) The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:37, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:45.

[0499] ii) The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:37, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:47;

[0500] iii) The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:39, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:47;

[0501] iv) The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:41, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:45.

[0502] v) The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:41, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:47.

[0503] vi) The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:43, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:45.

[0504] vii) The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:43, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:47; or,

[0505] viii) The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:49, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:51.

[0506] The single-chain antibody of IL-4RA is respectively linked to the C-terminus of the two heavy chains of the anti-ST2 immunoglobulin.

[0507] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the anti-IL-4RA single-chain antibody and the light chain variable region of the anti-IL-4RA single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different;

[0508] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO:53 and SEQ ID NO:54.

[0509] In some embodiments of the present invention, the bispecific antibody, wherein,

[0510] The bispecific antibody includes:

[0511] Targeting the first protein functional region of IL-4RA, and

[0512] Targeting the second protein functional region of ST2;

[0513] The first protein has two functional regions, and the second protein has one functional region;

[0514] Wherein, the first protein functional region is a single-chain antibody against IL-4RA, and the second protein functional region is an immunoglobulin against ST2;

[0515] The amino acid sequence of the heavy chain variable region of the anti-IL-4RA single-chain antibody is shown in SEQ ID NO:50, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:52; and

[0516] i) The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:37, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:45.

[0517] ii) The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:37, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:47;

[0518] iii) The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:39, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:47;

[0519] iv) The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:41, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:45.

[0520] v) The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:41, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:47.

[0521] vi) The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:43, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:45.

[0522] vii) The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:43, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:47; or,

[0523] viii) The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:49, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:51.

[0524] The single-chain antibody of IL-4RA is respectively linked to the C-terminus of the two heavy chains of the anti-ST2 immunoglobulin.

[0525] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the anti-IL-4RA single-chain antibody and the light chain variable region of the anti-IL-4RA single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different;

[0526] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO:53 and SEQ ID NO:54.

[0527] In some embodiments of the present invention, the bispecific antibody, wherein,

[0528] The bispecific antibody includes:

[0529] Targeting the first protein functional region of IL-4RA, and

[0530] Targeting the second protein functional region of ST2;

[0531] The first protein has two functional regions, and the second protein has one functional region;

[0532] Wherein, the first protein functional region is a single-chain antibody against IL-4RA, and the second protein functional region is an immunoglobulin against ST2;

[0533] The amino acid sequence of the heavy chain variable region of the single-chain antibody against IL-4RA is shown in SEQ ID NO:17, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:21.

[0534] The amino acid sequence of the heavy chain of the anti-ST2 immunoglobulin is shown in SEQ ID NO:87, and the amino acid sequence of the light chain of the anti-ST2 immunoglobulin is shown in SEQ ID NO:65.

[0535] The anti-IL-4RA single-chain antibody is respectively linked to the C-terminus of the two heavy chains of the anti-ST2 immunoglobulin;

[0536] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the anti-IL-4RA single-chain antibody and the light chain variable region of the anti-IL-4RA single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different;

[0537] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO:53 and SEQ ID NO:54.

[0538] In some embodiments of the present invention, the bispecific antibody, wherein,

[0539] The bispecific antibody includes:

[0540] Targeting the first protein functional region of IL-4RA, and

[0541] Targeting the second protein functional region of ST2;

[0542] The first protein has two functional regions, and the second protein has one functional region;

[0543] Wherein, the first protein functional region is a single-chain antibody against IL-4RA, and the second protein functional region is an immunoglobulin against ST2;

[0544] The amino acid sequence of the heavy chain variable region of the single-chain antibody against IL-4RA is shown in SEQ ID NO:50, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:52.

[0545] The amino acid sequence of the heavy chain of the anti-ST2 immunoglobulin is shown in SEQ ID NO:87, and the amino acid sequence of the light chain of the anti-ST2 immunoglobulin is shown in SEQ ID NO:65.

[0546] The anti-IL-4RA single-chain antibody is respectively linked to the C-terminus of the two heavy chains of the anti-ST2 immunoglobulin;

[0547] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the anti-IL-4RA single-chain antibody and the light chain variable region of the anti-IL-4RA single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different;

[0548] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO:53 and SEQ ID NO:54.

[0549] In some embodiments of the present invention, the bispecific antibody, wherein,

[0550] The bispecific antibody includes:

[0551] Targeting the first protein functional region of IL-4RA, and

[0552] Targeting the second protein functional region of ST2;

[0553] The first protein has two functional regions, and the second protein has one functional region;

[0554] Wherein, the first protein functional region is a single-chain antibody against IL-4RA, and the second protein functional region is an immunoglobulin against ST2;

[0555] The amino acid sequence of the heavy chain variable region of the single-chain antibody against IL-4RA is shown in SEQ ID NO:13, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:21.

[0556] The amino acid sequence of the heavy chain of the anti-ST2 immunoglobulin is shown in SEQ ID NO:87, and the amino acid sequence of the light chain of the anti-ST2 immunoglobulin is shown in SEQ ID NO:65.

[0557] The anti-IL-4RA single-chain antibody is respectively linked to the C-terminus of the two heavy chains of the anti-ST2 immunoglobulin;

[0558] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the anti-IL-4RA single-chain antibody and the light chain variable region of the anti-IL-4RA single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different;

[0559] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO:53 and SEQ ID NO:54.

[0560] In some embodiments of the present invention, the bispecific antibody, wherein,

[0561] The bispecific antibody includes:

[0562] Targeting the first protein functional region of IL-4RA, and

[0563] Targeting the second protein functional region of ST2;

[0564] The first protein has two functional regions, and the second protein has one functional region;

[0565] Wherein, the first protein functional region is a single-chain antibody against IL-4RA, and the second protein functional region is an immunoglobulin against ST2;

[0566] The amino acid sequence of the heavy chain variable region of the single-chain antibody against IL-4RA is shown in SEQ ID NO:15, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:21.

[0567] The amino acid sequence of the heavy chain of the anti-ST2 immunoglobulin is shown in SEQ ID NO:87, and the amino acid sequence of the light chain of the anti-ST2 immunoglobulin is shown in SEQ ID NO:65.

[0568] The anti-IL-4RA single-chain antibody is respectively linked to the C-terminus of the two heavy chains of the anti-ST2 immunoglobulin;

[0569] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the anti-IL-4RA single-chain antibody and the light chain variable region of the anti-IL-4RA single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different;

[0570] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO:53 and SEQ ID NO:54.

[0571] In some embodiments of the present invention, the bispecific antibody, wherein,

[0572] The bispecific antibody includes:

[0573] Targeting the first protein functional region of IL-4RA, and

[0574] Targeting the second protein functional region of ST2;

[0575] The first protein has two functional regions, and the second protein has one functional region;

[0576] Wherein, the first protein functional region is a single-chain antibody against IL-4RA, and the second protein functional region is an immunoglobulin against ST2;

[0577] The amino acid sequence of the heavy chain variable region of the single-chain antibody against IL-4RA is shown in SEQ ID NO:19, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:21.

[0578] The amino acid sequence of the heavy chain of the anti-ST2 immunoglobulin is shown in SEQ ID NO:87, and the amino acid sequence of the light chain of the anti-ST2 immunoglobulin is shown in SEQ ID NO:65.

[0579] The anti-IL-4RA single-chain antibody is respectively linked to the C-terminus of the two heavy chains of the anti-ST2 immunoglobulin;

[0580] The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the anti-IL-4RA single-chain antibody and the light chain variable region of the anti-IL-4RA single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different;

[0581] Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO:53 and SEQ ID NO:54.

[0582] In some embodiments of the present invention, the bispecific antibody is a tetramer formed by two identical first peptide chains and two identical second peptide chains, wherein...

[0583] The first peptide chain comprises an amino acid sequence selected from SEQ ID NOs:55-63, and the second peptide chain comprises an amino acid sequence shown in SEQ ID NO:64;

[0584] Preferably, the first peptide chain and the second peptide chain, and / or the two first peptide chains are independently connected by one or more (e.g., 2, 3, 4 or 5) disulfide bonds.

[0585] In some embodiments of the present invention, the bispecific antibody is a tetramer formed by two identical first peptide chains and two identical second peptide chains, wherein...

[0586] The first peptide chain comprises an amino acid sequence selected from SEQ ID NOs:55-63, and the second peptide chain comprises the amino acid sequence shown in SEQ ID NO:65;

[0587] Preferably, the first peptide chain and the second peptide chain, and / or the two first peptide chains are independently connected by one or more (e.g., 2, 3, 4 or 5) disulfide bonds.

[0588] In some embodiments of the present invention, the bispecific antibody is selected from the bispecific antibodies L4RT04(LS), L4RT01, L4RT02, L4RT03, L4RT04, TL4R01, TL4R02, TL4R03 and TL4R04 of the present invention.

[0589] Another aspect of the invention relates to an isolated nucleic acid molecule that encodes the bispecific antibody described in any one of the inventions.

[0590] Another aspect of the invention relates to a recombinant vector comprising the isolated nucleic acid molecules of the invention.

[0591] Another aspect of the invention relates to a host cell comprising the isolated nucleic acid molecule of the invention or the recombinant vector of the invention.

[0592] Another aspect of the present invention relates to an antibody-drug conjugate comprising a bispecific antibody and a small molecule drug, wherein the bispecific antibody is any one of the bispecific antibodies described in the present invention; preferably, the small molecule drug is a small molecule cytotoxic drug; more preferably, the small molecule drug is an anti-tumor chemotherapy drug.

[0593] Another aspect of the present invention relates to a pharmaceutical composition comprising an effective amount of any one of the bispecific antibodies or any one of the antibody-drug conjugates of the present invention; optionally, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0594] Preferably, the pharmaceutical composition is an injection, a nasal spray, or a nasal drop.

[0595] Another aspect of the present invention relates to a packaged product comprising the bispecific antibody, the antibody-drug conjugate, or the pharmaceutical composition of any one of the present invention, as described in any one of the present invention, and a packaging container;

[0596] Preferably, the packaging container is a syringe, injection pen, nasal spray, or dropper bottle;

[0597] Preferably, the packaged product further includes a product instruction manual.

[0598] Another aspect of the invention relates to a combination product comprising a first product and a second product in individually packaged form, wherein,

[0599] The first product comprises a bispecific antibody, an antibody-drug conjugate, or a pharmaceutical composition as described in any one of the present invention.

[0600] The second product contains one or more glucocorticoids;

[0601] Preferably, the glucocorticoid is selected from one or more of prednisone, methylprednisolone, betamethasone, beclomethasone dipropionate, prednisolone, hydrocortisone, dexamethasone, and triamcinolone;

[0602] Preferably, the first product and the second product further comprise one or more pharmaceutically acceptable excipients.

[0603] Preferably, the combined product further includes a product instruction manual.

[0604] Another aspect of the present invention relates to the use of any bispecific antibody, antibody-drug conjugate, or pharmaceutical composition described in any one of the present invention in the preparation of a medicament for treating or preventing a disease, wherein the disease is one or more selected from allergic diseases, autoimmune diseases, inflammatory diseases, and tumors;

[0605] Preferably, the disease is selected from one or more of the following: asthma, allergic rhinitis, sinusitis, nodular prurigo, chronic urticaria, nasal polyps, chronic obstructive pulmonary disease, pulmonary fibrosis, food allergy, rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, ankylosing spondylitis, eosinophilic esophagitis, pulmonary fibrosis, systemic lupus erythematosus, cardiovascular disease, and systemic sclerosis.

[0606] Preferably, the asthma is allergic asthma;

[0607] Preferably, the allergic rhinitis is seasonal allergic rhinitis;

[0608] Preferably, the sinusitis is chronic sinusitis;

[0609] Preferably, the chronic obstructive pulmonary disease is moderate to severe chronic obstructive pulmonary disease;

[0610] Preferably, the atopic dermatitis is moderate to severe atopic dermatitis;

[0611] Preferably, the single-dose dose based on the bispecific antibody is 0.1-100 mg per kilogram of body weight, more preferably 5-50 mg or 5-15 mg per kilogram of body weight;

[0612] Preferably, each patient is given a single dose of 0-1000 mg, 0-900 mg, 0-800 mg, 0-700 mg, 0-600 mg, 0-500 mg, 0-400 mg, 0-300 mg, 0-200 mg, 0-100 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or 1000 mg, calculated according to the bispecific antibody.

[0613] Preferably, the medication is administered once every 3 days, every 4 days, every 5 days, every 6 days, every 10 days, every week, every 2 weeks, or every 3 weeks;

[0614] Preferably, the administration method is intravenous infusion, intravenous injection, intradermal injection, subcutaneous injection, intramuscular injection, or nasal administration.

[0615] The bispecific antibody, antibody-drug conjugate, or pharmaceutical composition according to any one of the present invention is used to treat or prevent a disease, wherein the disease is one or more selected from allergic diseases, autoimmune diseases, inflammatory diseases, and tumors.

[0616] Preferably, the disease is selected from one or more of the following: asthma, allergic rhinitis, sinusitis, nodular prurigo, chronic urticaria, nasal polyps, chronic obstructive pulmonary disease, pulmonary fibrosis, food allergy, rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, ankylosing spondylitis, eosinophilic esophagitis, pulmonary fibrosis, systemic lupus erythematosus, cardiovascular disease, and systemic sclerosis.

[0617] Preferably, the asthma is allergic asthma;

[0618] Preferably, the allergic rhinitis is seasonal allergic rhinitis;

[0619] Preferably, the sinusitis is chronic sinusitis;

[0620] Preferably, the chronic obstructive pulmonary disease is moderate to severe chronic obstructive pulmonary disease;

[0621] Preferably, the atopic dermatitis is moderate to severe atopic dermatitis;

[0622] Preferably, the single-dose dose based on the bispecific antibody is 0.1-100 mg per kilogram of body weight, more preferably 5-50 mg or 5-15 mg per kilogram of body weight;

[0623] Preferably, each patient is given a single dose of 0-1000 mg, 0-900 mg, 0-800 mg, 0-700 mg, 0-600 mg, 0-500 mg, 0-400 mg, 0-300 mg, 0-200 mg, 0-100 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or 1000 mg, calculated according to the bispecific antibody.

[0624] Preferably, the medication is administered once every 3 days, every 4 days, every 5 days, every 6 days, every 10 days, every week, every 2 weeks, or every 3 weeks;

[0625] Preferably, the administration method is intravenous infusion, intravenous injection, intradermal injection, subcutaneous injection, intramuscular injection, or nasal administration.

[0626] Another aspect of the present invention relates to a method of treating or preventing a disease, comprising the step of administering to a subject in need an effective amount of any one of the bispecific antibodies, antibody-drug conjugates, or pharmaceutical compositions of the present invention, wherein the disease is one or more selected from allergic diseases, autoimmune diseases, inflammatory diseases, and tumors.

[0627] Preferably, the disease is selected from one or more of the following: asthma, allergic rhinitis, sinusitis, nodular prurigo, chronic urticaria, nasal polyps, chronic obstructive pulmonary disease, pulmonary fibrosis, food allergy, rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, ankylosing spondylitis, eosinophilic esophagitis, pulmonary fibrosis, systemic lupus erythematosus, cardiovascular disease, and systemic sclerosis.

[0628] Preferably, the asthma is allergic asthma;

[0629] Preferably, the allergic rhinitis is seasonal allergic rhinitis;

[0630] Preferably, the sinusitis is chronic sinusitis;

[0631] Preferably, the chronic obstructive pulmonary disease is moderate to severe chronic obstructive pulmonary disease;

[0632] Preferably, the atopic dermatitis is moderate to severe atopic dermatitis;

[0633] Preferably, the single-dose dose based on the bispecific antibody is 0.1-100 mg per kilogram of body weight, more preferably 5-50 mg or 5-15 mg per kilogram of body weight;

[0634] Preferably, each patient is given a single dose of 0-1000 mg, 0-900 mg, 0-800 mg, 0-700 mg, 0-600 mg, 0-500 mg, 0-400 mg, 0-300 mg, 0-200 mg, 0-100 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or 1000 mg, calculated according to the bispecific antibody.

[0635] Preferably, the medication is administered once every 3 days, every 4 days, every 5 days, every 6 days, every 10 days, every week, every 2 weeks, or every 3 weeks;

[0636] Preferably, the administration method is intravenous infusion, intravenous injection, intradermal injection, subcutaneous injection, intramuscular injection, or nasal administration.

[0637] In some embodiments, the diseases described in this invention are IL-4 / IL-4RA, IL-13 / IL-4RA, and / or IL-33 / ST2 mediated diseases.

[0638] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used in this invention are all conventional procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0639] As used herein, the term "antigen-binding region" refers to a portion of a protein or protein that specifically binds to a designated antigen. For example, this portion of an antibody containing amino acid residues that interact with the antigen and confer specificity and affinity to the antigen is called an "antigen-binding region." Antigen-binding regions typically include one or more "complementarity-determining regions" (CDRs). Some antigen-binding regions also include one or more "fragment regions" (FRs). A CDR is an amino acid sequence that contributes to antigen-binding specificity and affinity. The assignment of amino acids to the regions or domains follows known antibody numbering systems, such as the Kabat numbering system, Martin numbering system, Chothia numbering system, AHo numbering system, or IMGT numbering system (Dondelinger, Mathieu et al. Understanding the Significance and Implications of Antibody Numbering and Antigen-Binding Surface / Residue Definition. Frontiers in Immunology vol.9 2278. 16 Oct. 2018). Based on known antibody numbering systems, those skilled in the art can determine the CDR sequences of the heavy and light chains based on given antibody heavy and light chain sequences.

[0640] As used herein, the term "antibody" refers to any intact immunoglobulin of the same type or its antigen-binding fragment that can compete with an intact antibody for specific binding to a target antigen, and includes, for example, chimeric antibodies, humanized antibodies, fully humanized antibodies, and bispecific antibodies or their antigen-binding fragments. Such "antibodies" are a class of antigen-binding proteins. Intact antibodies typically contain at least two full-length heavy chains and two full-length light chains, but in some cases, may include fewer chains, such as antibodies naturally occurring in camelids that may contain only heavy chains. Antibodies or their antigen-binding fragments may be derived from a single source or may be "chimeric," meaning that different portions of the antibody may be derived from two different sources further described below. Antibodies or their antigen-binding fragments may be produced in hybridomas using recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Unless otherwise indicated, the term "antibody" includes, in addition to antibodies containing two full-length heavy chains and two full-length light chains, its derivatives, variants, and fragments.

[0641] As used herein, the term "antibody" or "immunoglobulin chain (heavy chain or light chain)" and its "antigen-binding fragment" (or simply "fragment") comprise a portion of an antibody that lacks at least some of the amino acids present in the full-length chain of the antibody but is capable of specifically binding an antigen (in any way, this portion is obtained or synthesized). Such fragments are biologically active because they specifically bind to a target antigen and can compete with other antibodies or their antigen-binding fragments for specific binding to a given epitope. In one aspect, such fragments will retain at least one CDR present in the full-length light chain or heavy chain of the antibody, and in some embodiments will comprise a single heavy chain and / or light chain or a portion thereof. These biologically active fragments can be produced by recombinant DNA technology or can be produced, for example, by enzymatic or chemical cleavage of an intact antibody. Immunofunctional immunoglobulin fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, domain antibodies, and single-chain antibodies, and can be derived from any mammalian source, including but not limited to humans, mice, rats, camels, or rabbits. It is also envisioned that the functional portions of the antibodies disclosed herein, such as one or more CDRs, can be covalently bound to a second protein or small molecule to generate a therapeutic agent that targets a specific target in the body, thereby possessing bifunctional therapeutic properties or having an extended serum half-life, such as a fusion protein.

[0642] As used herein, the terms “full-length antibody,” “full-length antibody,” “intact antibody,” and “whole antibody” are used interchangeably to refer to an antibody having a structure substantially similar to that of a natural antibody or a heavy chain having an Fc region as defined herein.

[0643] The term "light chain" includes full-length light chains and segments thereof with sufficient variable region sequences to confer binding specificity. Full-length light chains include a variable region domain (VL) and a constant region domain (CL). The variable region domain of the light chain is located at the amino terminus of the polypeptide. Light chains include κ chains and λ chains.

[0644] The term "heavy chain" includes the full-length heavy chain and its segments with sufficient variable region sequences to confer binding specificity. The full-length heavy chain includes a variable region domain (VH) and three constant region domains (CH1, CH2, and CH3). The VH domain is located at the amino terminus of the polypeptide, and the CH domain is located at the carboxyl terminus, with CH3 being closest to the carboxyl terminus of the polypeptide. The heavy chain can have any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 isotypes), IgA (including IgA1 and IgA2 isotypes), IgM, and IgE.

[0645] As used in this invention, the term "Fab fragment" consists of a light chain and a CH1 group, as well as a variable region of a heavy chain. The heavy chain of the Fab molecule cannot form disulfide bonds with another heavy chain molecule.

[0646] As used in this invention, the term "Fc" region contains two heavy chain segments comprising the CH1 and CH2 domains of the antibody. The two heavy chain segments are held together by two or more disulfide bonds and by hydrophobic interactions through the CH3 domain.

[0647] As used in this invention, the term "Fab' fragment" comprises a portion of a light chain and a portion of a heavy chain (containing a VH domain and a CH1 domain, as well as a portion of the region between the CH1 and CH2 domains) so that interchain disulfide bonds can be formed between the two heavy chains of two Fab' fragments to form an F(ab')2 molecule.

[0648] As used in this invention, the term "F(ab')2 fragment" comprises two light chains and two heavy chains containing portions of a constant region between CH1 and CH2 domains, so as to form interchain disulfide bonds between the two heavy chains. The F(ab')2 fragment thus consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.

[0649] As used in this invention, the term "Fv region" includes variable regions from both the heavy and light chains, but lacks constant regions.

[0650] As used in this invention, the term “Fd” fragment refers to an antibody fragment consisting of VH and CH1 domains (Ward et al., Nature 341:544-546 (1989)).

[0651] As used in this invention, the term “dAb” fragment (Ward et al., Nature 341:544-546 (1989)) consists of a VH domain.

[0652] As used herein, the term "Fab'-SH" is the name given to Fab' herein, wherein one or more cysteine ​​residues of a constant domain carry a free thiol group.

[0653] As used in this invention, the term "Fab / c" fragment is an antibody cleavage intermediate formed by the digestion of immunoglobulins by pepsin. It has the advantages of both Fab and Fc regions, namely, strong diffusion ability, slow in vivo metabolic clearance, and high affinity (Liu Jianjun, Journal of Cellular and Molecular Immunology, 1989(4):29-29).

[0654] As used herein, the term "single-chain antibody" is an Fv molecule in which the variable regions of the heavy and light chains are linked by flexible linkers to form a single polypeptide chain (which forms the antigen-binding region) (see, for example, Bird et al., Science. 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA. 90:5879-5883 (1988)). Single-chain antibodies are described in detail in International Patent Application Publication No. WO 88 / 01649 and U.S. Patents USP 4,946,778 and USP 5,260,203 (the disclosures of which are incorporated herein by reference).

[0655] As used herein, the term "domain antibody" is an immunofunctional immunoglobulin fragment containing only a variable region of the heavy chain or a variable region of the light chain. In some cases, two or more VH regions are covalently linked via peptide linkers to generate multivalent domain antibodies (particularly bivalent domain antibodies). The two VH regions of a bivalent domain antibody can target the same or different antigens.

[0656] As used herein, the term "bivalent antigen-binding protein" or "bivalent antibody" comprises two antigen-binding sites. In some cases, the two binding sites have the same antigen specificity. A bivalent antibody can be bispecific.

[0657] As used in this invention, the terms "multispecific antigen-binding protein" or "multispecific antibody" are antigen-binding proteins or antibodies that target more than one antigen or epitope.

[0658] As used herein, the terms "bispecific," "dual-specific," or "bifunctional" antigen-binding protein or antibody refer to hybrid antigen-binding proteins or antibodies that each have two distinct antigen-binding sites. A bispecific antibody is a multispecific antigen-binding protein or antibody and can be produced by a variety of methods, including, but not limited to, hybridoma fusion or Fab' fragment linkage. See, for example, Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 79: 315-321; Kostelny et al., 1992, J. Immunol. 148: 1547-1553. The two binding sites of a bispecific antigen-binding protein or antibody will bind two distinct epitopes, which are located on the same or different protein targets.

[0659] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" refer to an antibody or a fragment of an antibody derived from a group of highly homologous antibody molecules, i.e., a group of identical antibody molecules except for the possibility of spontaneous natural mutations. Monoclonal antibodies exhibit high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically comprise at least two or more different antibodies that typically recognize different epitopes on an antigen. Monoclonal antibodies are generally obtained using hybridoma techniques first reported by Kohler et al. (Nature, 256:495, 1975), but can also be obtained using recombinant DNA techniques (see, for example, USP 4,816,567).

[0660] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained by replacing all or part of the CDR region of a human immunoglobulin (receptor antibody) with the CDR region of a non-human antibody (donor antibody), wherein the donor antibody can be a non-human antibody (e.g., mouse, rat, or rabbit) with the expected specificity, affinity, or reactivity. Furthermore, some amino acid residues of the frame region (FR) of the receptor antibody may also be replaced by amino acid residues of the corresponding non-human antibody, or by amino acid residues of other antibodies, to further improve or optimize the antibody's performance. For more detailed information on humanized antibodies, please refer to, for example, Jones et al., Nature, 321:522 525 (1986); Reichmann et al., Nature, 332:323 329 (1988); Presta, Curr. Op. Struct. Biol., 2:593 596 (1992); and Clark, Immunol. Today 21:397 402 (2000).

[0661] As used herein, the term "epitope" refers to a site on an antigen that is specifically bound by an immunoglobulin or antibody. "Epitope" is also referred to in the art as an "antigenic determinant." An epitope or antigenic determinant typically consists of chemically active surface groups of a molecule, such as amino acids or carbohydrate or sugar side chains, and usually has specific three-dimensional structural features and specific charge characteristics. For example, an epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a unique spatial conformation, which can be "linear" or "conformal." See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, Ed. (1996). In a linear epitope, all points of interaction between the protein and the interacting molecule (e.g., an antibody) are linear along the primary amino acid sequence of the protein. In a conformational epitope, points of interaction are separated by protein amino acid residues.

[0662] The terms “peptide” or “protein” are used interchangeably herein to refer to polymers of amino acid residues. The term is also used for amino acid polymers in which one or more amino acid residues are analogs or simulants of corresponding naturally occurring amino acids, and for naturally occurring amino acid polymers. The term may also include amino acid polymers that have been modified, for example by adding sugar residues to form glycoproteins, or phosphorylated amino acid polymers. Peptides and proteins may be produced by naturally occurring cells and non-recombinant cells; or by genetically engineered or recombinant cells, and comprise molecules having the amino acid sequence of a natural protein, or molecules having one or more amino acids with a natural sequence that have been omitted, added, and / or substituted.

[0663] The terms “peptide” and “protein” specifically include antibodies, such as anti-human IL-4RA antibodies (also known as IL-4RA antibodies), IL-4RA binding proteins, antibodies or sequences with one or more amino acid deletions, additions and / or substitutions of antigen-binding proteins.

[0664] The term "peptide fragment" refers to a polypeptide that has an amino-terminal deletion, a carboxyl-terminal deletion, and / or an internal deletion compared to a full-length protein. Such fragments may also contain modified amino acids compared to a full-length protein. In some embodiments, the fragment length is from about 5 to 500 amino acids. For example, the fragment length may be at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids. Useful peptide fragments include immunofunctional fragments of antibodies, including binding domains. In the case of human IL-4RA antibodies, useful fragments include, but are not limited to, CDR regions, variable domains of the heavy or light chain, portions of the antibody chain, or variable domains that exactly include two CDRs.

[0665] The terms “human IL-4RA,” “hIL-4RA,” “human IL-4 receptor A,” and “human IL-4 receptor α subunit” are used interchangeably and refer to the human interleukin-4 receptor α subunit. IL-4 and IL-13 are the primary endogenous agonists of IL-4RA. Unless otherwise specified or clearly understood from the context in which the terms are used, “IL-4RA” refers to human IL-4RA.

[0666] The terms “human ST2” and “IL1RL1” are used interchangeably and refer to the human growth-stimulating gene 2 protein. Unless otherwise specified or clearly understood from the context in which the terms are used, “ST2” refers to human ST2.

[0667] A “derivative” of a polypeptide is a polypeptide that is chemically modified in a manner different from that of an insertion, deletion, or substitution variant, such as by conjugation of another chemical part (e.g., an antigen-binding protein or antibody), such as a polypeptide conjugated with PEG.

[0668] As used herein, the terms "separated" or "isolated" refer to substances obtained artificially from their natural state. If a substance or component is found in nature as a "separated" substance, it may be due to an alteration of its natural environment, the separation of the substance from its natural environment, or both. For example, a certain unseparated polynucleotide or polypeptide may naturally exist in the body of a living animal, and a high-purity identical polynucleotide or polypeptide separated from this natural state is called a separated substance. The terms "separated" or "isolated" do not exclude the presence of artificial or synthetic substances, nor do they exclude the presence of other impurities that do not affect the activity of the substance.

[0669] As used in this invention, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0670] As used in this invention, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0671] As used in this invention, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its targeted antigen. In some embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific to an antigen) means that the antibody binds to less than about 10 -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or lower affinity (K) D () binds to the antigen.

[0672] As used in this invention, the term "K" D "K" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, used to describe the binding affinity between the antibody and the antigen. Among several parameters measured by molecular binding kinetics, K is... D The value is the dissociation equilibrium constant, a parameter in antibody drug research that characterizes the strength of the affinity between the test antibody and the target antigen molecule. It is calculated using the formula K.D = kdis / kon, the smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. kon, the binding rate constant, equals the rate at which the antigen-antibody complex forms; a smaller kon indicates a faster antibody-antigen binding speed. kdis, the dissociation rate constant, equals the rate at which the antibody dissociates from the antigen-antibody complex; a smaller kdis indicates a slower rate of antibody detachment from the antigen, and a more stable antibody-antigen binding. Typically, antibodies bind at a rate less than approximately 10... -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or a smaller dissociation equilibrium constant (K) D The binding antigen (e.g., IL-4RA protein) is measured, for example, using surface plasmon resonance (SPR) in a BIACORE instrument or a Fortebio molecular interaction instrument.

[0673] As used in this invention, the terms "monoclonal antibody" and "monoclonal antibody" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably; and the terms "peptide" and "protein" have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0674] As used in this invention, the terms “hybridoma” and “hybridoma cell line” are used interchangeably, and when referring to the terms “hybridoma” and “hybridoma cell line”, they also include subclones and progeny cells of the hybridoma.

[0675] As used herein, the term “pharmaceutically acceptable carrier and / or excipient” means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art and includes, but is not limited to: pH adjusters, buffers, viscosity reducers, surfactants, adjuvants, ionic strength enhancers, protectants, etc.

[0676] As used herein, the term "carrier" refers to a diluent, adjuvant, excipient, or medium that is administered with the therapeutic agent. Such drug carriers can be sterile liquids, such as water and oils (including oils of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc.). Water is the preferred carrier when the drug composition is administered intravenously. Saline solutions, as well as aqueous solutions of dextran and glycerol, can also be used as liquid carriers, particularly for injectable solutions. Suitable drug excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol, etc. If desired, the composition may also contain small amounts of wetting agents or emulsifiers, or pH buffers, such as acetates, citrates, or phosphates. It is also envisioned that the composition include antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and osmolarity regulators such as sodium chloride or dextran. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The composition can be formulated into suppositories using conventional binders and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable drug carriers are described in EW Martin's Remington's Pharmaceutical Sciences, which is incorporated herein by reference. Such compositions would contain a therapeutically effective amount of an antigen-binding polypeptide (preferably in a purified form) and a suitable amount of carrier to provide a form suitable for administration to the patient. The formulation should be suitable for the route of administration. Parenteral formulations can be packaged in ampoules made of glass or plastic, disposable syringes, or multi-dose vials.

[0677] As used in this article, the terms “percentage sequence identity” and “percentage sequence homology” are used interchangeably.

[0678] As used herein, the terms “similarity” or “sequence similarity” and “identity” refer to the relationship between the sequences of two or more protein or polypeptide molecules, as determined by alignment and comparison of sequences. “Percentage identity” refers to the percentage of identical residues among the amino acids in the molecules being compared and can be calculated based on the size of the smallest molecule to be compared. To perform these calculations, gaps in the alignment (if any) must be resolved using a specific mathematical model or computer program (i.e., an “algorithm”). When applied to polypeptides, the term “bulk identity” means that two peptide sequences, when optimally aligned using, for example, programs such as GAP or BESTFIT, with the default gap weights provided by the programs, share at least 70%, 75%, or 80% sequence identity, at least 90% or 95% sequence identity, and at least 97%, 98%, or 99% sequence identity. In some cases, dissimilar residue sites differ due to conserved amino acid substitutions. A “conserved amino acid substitution” is a substitution in which an amino acid residue is replaced by another amino acid residue having a side chain R group possessing similar chemical properties (e.g., charge or aqueousity). Generally, conserved amino acid substitutions will not substantially alter the functional properties of a protein. Where two or more amino acid sequences differ from each other due to a conserved substitution, the percentage sequence identity can be increased to correct for the conserved nature of the substitution. Methods for making this adjustment are well known to those skilled in the art. See, for example, Pearson, Methods Mol. Biol. 243:307-31 (1994). Examples of amino acid groups having side chains with similar chemical properties include: 1) aliphatic hydroxyl side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine ​​and methionine. The conserved amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine.

[0679] Alternatively, a conservative permutation is any change that has a positive value in the PAM250 log-likelihood matrix as disclosed in Gonnet et al., Science 256:1443-45 (1992) (incorporated hereby by reference). A “moderately conservative” permutation is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0680] Sequence analysis software is commonly used to measure peptide sequence identity. Protein analysis software uses measures of similarity assigned to different substitutions, deletions, and other modifications, including conserved amino acid substitutions, to match sequences. For example, GCG includes programs such as "Gap" and "Bestfit," which (using default parameters specified by the program) can be used to determine sequence homology or sequence identity between closely related peptides (e.g., homologous peptides from different biological species) or between wild-type proteins and their mutant proteins. See, for example, GCG Version 6.1 (University of Wisconsin, WI). Peptide sequences can also be compared using FASTA with default or recommended parameters; see GCG Version 6.10. FASTA (e.g., FASTA2 and FASTA3) provides alignment and percentage sequence identity of the best overlapping region between the challenge sequence and the search sequence (Pearson, Methods Enzymol. 183:63-98 (1990): Pearson, Methods Mol. Biol. 132:185-219 (2000)). When comparing sequences with databases containing a large number of sequences from different organisms, another preferred algorithm is the computer program BLAST, specifically blastp or tblastn (using the default parameters provided by the program). See, for example, Altschul et al., Mol. Biol. 215:403-410 (1990); Altschul et al., Nucleic Acids Res. 25:3389-402 (1997).

[0681] In this invention, unless otherwise specified, the term "comprising" may refer only to the included object, or it may include one or more other optional elements in addition to the included object. For example, including SEQ ID NO:2 may refer to SEQ ID NO:2 itself, or it may further include other amino acid sequences, and after including other amino acid sequences, it has the same or similar function as SEQ ID NO:2, or can achieve the effects of this invention.

[0682] In this invention, unless otherwise specified, the terms "first" (e.g., first protein functional region, first linker, or first peptide chain) and "second" (e.g., second protein functional region, second linker, or second peptide chain) are used for distinguishing references or clarifying expressions, and do not have a typical meaning of order.

[0683] Beneficial effects of the invention

[0684] This invention achieves one or more of the following technical effects:

[0685] (1) The anti-IL-4RA antibody or its antigen-binding fragment of the present invention can specifically bind to IL-4RA and has high affinity.

[0686] (2) The bispecific antibody of the present invention can specifically bind to IL-4RA and ST2 with high affinity.

[0687] (3) In the bispecific antibody of the present invention, the anti-IL-4RA antibody and the anti-ST2 antibody have a synergistic effect.

[0688] (4) The bispecific antibody of the present invention can effectively block the binding of IL-33 to ST2.

[0689] (5) The bispecific antibody of the present invention can effectively block the binding of IL-4 / IL-13 to IL-4RA.

[0690] (6) The bispecific antibody of the present invention can effectively inhibit the proliferation of TF-1 cells induced by IL-4.

[0691] (7) The bispecific antibody of the present invention can effectively inhibit the upregulation of CD23 expression level in B cells by IL-4 / IL-13.

[0692] (8) The bispecific antibody of the present invention can effectively inhibit the secretion of cytokines such as IL-6 and / or IL-8 caused by one, two or three of IL-4, IL-13 and IL-33.

[0693] (9) The bispecific antibody of the present invention has shown excellent in vivo efficacy in animal models such as mouse models of rhinitis and mouse models of asthma.

[0694] (10) The anti-IL-4RA antibody of the present invention or its antigen-binding fragment or the bispecific antibody of the present invention can effectively treat or prevent the indications described in the present invention. Attached Figure Description

[0695] Figure 1A: ELISA results of the binding activity of anti-IL-4RA antibodies 14A10H42L25, 14A10H43L25, 14A10H49L25, and 14A10H50L25 to antigen IL4RA-mFc.

[0696] Figure 1B: ELISA results of the binding activity of anti-IL-4RA antibodies 14A10H49L25 and 14A10H50L25 to antigen IL4RA-mFc.

[0697] Figure 1C: Competitive ELISA results of antibodies 14A10H42L25 and 14A10H43L25 blocking the binding activity of IL4-N-his to antigen IL4RA-hFc.

[0698] Figure 1D: Competitive ELISA results of antibodies 14A10H49L25 and 14A10H50L25 blocking the binding activity of IL4-N-his to antigen IL4RA-hFc.

[0699] Figure 2: Binding activity of anti-IL-4RA antibody to 293T-IL4Rα cell surface antigen.

[0700] Figure 3A: Anti-IL-4RA antibody inhibits IL-4-induced proliferation of TF-1 cells. Compared with isotype control, *p<0.05, **p<0.01, ***p<0.001.

[0701] Figure 3B: Anti-IL-4RA antibody inhibits IL-13-induced proliferation of TF-1 cells. ***p<0.001 compared to isotype control.

[0702] Figure 4A: Anti-IL-4RA antibody inhibits the upregulation of CD23 expression in human B cells by IL-4.

[0703] Figure 4B: Anti-IL-4RA antibody inhibits the upregulation of CD23 expression in human B cells by IL-13.

[0704] Figure 4C: Anti-IL-4RA antibody inhibits the upregulation of CD23 expression in human monocytes by IL-4.

[0705] Figure 4D: Anti-IL-4RA antibody inhibits the upregulation of CD23 expression in human monocytes by IL-13.

[0706] Figure 5A: Results of indirect ELISA assay of the binding activity of L4RT01, L4RT02, L4RT03, L4RT04, 14A10H49L25 to antigen IL4RA-mFc.

[0707] Figure 5B: Results of indirect ELISA assay for the binding activity of TL4R01, TL4R02, TL4R03, TL4R04, 14A10H49L25 with antigen IL4RA-mFc.

[0708] Figure 5C: Results of indirect ELISA assay for the binding activity of L4RT04(LS) to antigen IL4RA-mFc.

[0709] Figure 5D: Results of indirect ELISA assay of the binding activity of L4RT01, L4RT02, L4RT03, L4RT04, 3H11H46L7 to antigen ST2-mFc.

[0710] Figure 5E: Results of indirect ELISA assay for the binding activity of TL4R01, TL4R02, TL4R03, TL4R04, 3H11H46L7 with antigen ST2-mFc.

[0711] Figure 5F: Results of indirect ELISA assay for the binding activity of L4RT04(LS) to antigen ST2-mFc.

[0712] Figure 6A: Results of competitive ELISA assay of the activity of L4RT01, L4RT02, L4RT03, L4RT04, 14A10H49L25 in competitive binding of IL4RA-hFc with IL4-N-his.

[0713] Figure 6B: Results of competitive ELISA assay of the activity of TL4R01, TL4R02, TL4R03, TL4R04, 14A10H49L25 in competitive binding of IL4RA-hFc with IL4-N-his.

[0714] Figure 6C: Results of competitive ELISA assay for the activity of L4RT04(LS) and IL4-N-his in competitively binding to IL4RA-hFc.

[0715] Figure 6D: Results of competitive ELISA assay of the activity of L4RT01, L4RT02, L4RT03, L4RT04, 3H11H46L7 in competitive binding to ST2-mFc with IL33(112-270)-Nhis-biotin.

[0716] Figure 6E: Results of competitive ELISA assay of the activity of TL4R01, TL4R02, TL4R03, TL4R04, 3H11H46L7 and IL33(112-270)-Nhis-biotin in competitively binding ST2-mFc.

[0717] Figure 6F: Results of competitive ELISA assay for the activity of L4RT04(LS) and IL33(112-270)-Nhis-biotin in competitively binding ST2-mFc.

[0718] Figure 7A: Detection results of kinetic parameters of binding between humanized antibody TL4R04 and IL4RA-His.

[0719] Figure 7B: Detection results of kinetic parameters of binding between humanized antibody L4RT03 and IL4RA-His.

[0720] Figure 7C: Detection results of kinetic parameters of binding between humanized antibody L4RT04(LS) and IL4RA-His.

[0721] Figure 7D: Detection results of kinetic parameters of binding between humanized antibody Dupilumab and IL4RA-His.

[0722] Figure 7E: Detection results of kinetic parameters of binding between humanized antibody TL4R04 and ST2-His.

[0723] Figure 7F: Detection results of kinetic parameters of binding between humanized antibody L4RT03 and ST2-His.

[0724] Figure 7G: Detection results of kinetic parameters of binding between humanized antibody L4RT04(LS) and ST2-His.

[0725] Figure 7H: Detection results of kinetic parameters of binding between humanized antibody AMG282 and ST2-His.

[0726] Figure 8A: FACS detection of the binding activity of L4RT04, TL4R04, 3H11H46L7(hG1WT), AMG282 and ST2 on the cell membrane surface of 293T-ST2-Luc.

[0727] Figure 8B: FACS detection of the binding activity of L4RT04(LS), AMG282 and ST2 on the surface of 293T-ST2 cells.

[0728] Figure 8C: FACS detection of the binding activity of Dupilumab, 14A10H49L25, TL4R04, L4RT04 to IL-4RA on the cell membrane surface of 293T-IL4Rα.

[0729] Figure 8D: FACS detection of the binding activity of Dupilumab, L4RT04(LS) to IL-4RA on the cell membrane surface of 293T-IL4Rα.

[0730] Figure 9A: 3H11H45L7(hG1WT), 3H11H46L7(hG1WT), TL4R01, L4RT03, TL4R04, and L4RT04 block the binding of IL-33 to ST2. Compared with the isotype control, *p<0.05, **p<0.01, ***p<0.001.

[0731] Figure 9B: L4RT04(LS) blocks the binding of IL-33 to ST2. Compared with the isotype control, *p<0.05, **p<0.01, ***p<0.001.

[0732] Figure 10A: 14A10H49L25, L4RT03, L4RT04, TL4R04, and Dupilumab block the binding of IL-13 to IL-4RA. Compared with the isotype control, ***p<0.001.

[0733] Figure 10B: L4RT04(LS) and Dupilumab block the binding of IL-13 to IL-4RA. Compared with the isotype control, ***p<0.001.

[0734] Figure 10C: L4RT04, TL4R04, and Dupilumab block the binding of IL-4 to IL-4RA. Compared with the isotype control, ***p<0.001.

[0735] Figure 10D: L4RT04(LS), 14A10H49L25, and Dupilumab block the binding of IL-4 to IL-4RA. Compared with the isotype control, **p<0.01, ***p<0.001.

[0736] Figure 11A: 3H11H46L7(hG1WT), L4RT03, TL4R04, L4RT04, and AMG282 inhibited IL-6 secretion in the IL-33 system. Compared with the isotype control, *p<0.05, **p<0.01, ***p<0.001.

[0737] Figure 11B: L4RT04(LS) and AMG282 inhibit IL-6 secretion in the IL-33 system. Compared with the isotype control, ***p<0.001.

[0738] Figure 12A: 14A10H49L25, L4RT03, L4RT04, TL4R01, TL4R04, and Dupilumab inhibit IL-4-induced TF-1 cell proliferation.

[0739] Figure 12B: L4RT04 (LS) and Dupilumab inhibit IL-4-induced TF-1 cell proliferation.

[0740] Figure 13A: Dupilumab, 14A10H49L25, L4RT04 and L4RT04 (LS) inhibit the upregulation of CD23 expression in B cells by IL-4.

[0741] Figure 13B: Dupilumab, 14A10H49L25, L4RT04 and L4RT04 (LS) inhibit the upregulation of CD23 expression in B cells by IL-13.

[0742] Figure 14: Dupilumab, L4RT04, and TL4R04 inhibit CCL26 secretion in the IL-4 system. ***p<0.001 compared with isotype control.

[0743] Figure 15: L4RT04(LS), L4RT04, Dupilumab, AMG282, 14A10H49L25, 3H11H46L7, and the combination of 14A10H49L25+3H11H46L7 and Dupilumab+AMG282 inhibited IL-6 secretion in the IL-4+IL-33 system. Compared with the isotype control, *p<0.05, **p<0.01, ***p<0.001.

[0744] Figure 16: Inhibition of IL-6 secretion in the IL-13+IL-33 system by L4RT04(LS), L4RT04, Dupilumab, AMG282, 14A10H49L25, 3H11H46L7, and the combination of 14A10H49L25+3H11H46L7 and Dupilumab+AMG282. Compared with isotype control, *p<0.05, **p<0.01, ***p<0.001.

[0745] Figure 17: The anti-IL-4RA-anti-ST2 bispecific antibody inhibited IL-6 secretion induced by co-stimulation of IL-4, IL-13, and IL-33. *p<0.05, **p<0.01

[0746] Information on some sequences involved in this invention is shown in Table A below.

[0747] Table A: Information on partial sequences involved in this invention Detailed Implementation

[0748] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0749] The 293T-IL4Rα cell line was constructed by Zhongshan Kangfang Biopharmaceutical Co., Ltd. The 293T-IL4Rα cell line was obtained by infecting HEK293T cells with a virus. The virus preparation used 3rd Generation Lentiviral Systems, see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini LJ Virol. 1998. 72(11): 8463-8471. The lentiviral expression vector used was pCDH-huIL4RaFL-GFP-Puro (where huIL4Ra, Genebank ID: NP000409.1; vector pCDH-CMV-MCS-EF1-GFP-Puro was purchased from Youbao Biotechnology, product number: VT8070).

[0750] The 293T-ST2-Luc cell line was constructed by Zhongshan Kangfang Biopharmaceutical Co., Ltd. The 293T-ST2-Luc cell line was obtained by viral infection of HEK293T cells. The virus preparation used 3rd Generation Lentiviral Systems, see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini LJ Virol. 1998. 72(11): 8463-8471. The lentiviral expression vectors used were pCDH-NFκB-Luc2P-hygro (where the vector pCDH-Hygro was modified based on pCDH-CMV-MCS-EF1-Puro (purchased from Youbao Biotechnology, product number: VT1480)) and pCDH-CMV-IL1RL1-FL (where IL1RL1 is ST2, and its amino acid sequence is shown in SEQ ID NO: 74; where the vector pCDH-CMV-Puro was purchased from Youbao Biotechnology, product number: VT1480).

[0751] The 293T-STAT6Luc cell line was constructed by Zhongshan Kangfang Biopharmaceutical Co., Ltd. The 293T-STAT6Luc cell line was obtained by viral infection of HEK293T cells. The virus preparation used 3rd Generation Lentiviral Systems, see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini LJ Virol. 1998. 72(11): 8463-8471. The lentiviral expression vector used was plenti6.3 / V5-hSTAT6-BSD (where hSTAT6 is from Genebank). ID:NP_001171549.1; vector pCDH-CMV-MCS-EF1-Puro was purchased from UBO Biotechnology, product number: 1480) and pCDH-hSTAT6RE-Luc2P-hygro (where the vector pCDH-Hygro was obtained by modifying pCDH-CMV-MCS-EF1-Puro (purchased from UBO Biotechnology, product number: VT1480)).

[0752] The A549-ST2 cell line was constructed by Zhongshan Kangfang Biopharmaceutical Co., Ltd. The A549-ST2 cell line was obtained by viral infection of A549 cells. The virus preparation used 3rd Generation Lentiviral Systems, see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini LJ Virol. 1998. 72(11): 8463-8471. The lentiviral expression vector used was pCDH-CMV-IL1RL1-FL (where IL1RL1 is ST2, and its amino acid sequence is shown in SEQ ID NO: 74; the vector pCDH-CMV-Puro was purchased from Youbao Biotechnology, product number: VT1480).

[0753] In the experimental examples of the present invention, the isotype control antibodies used, namely hIgG1, hIgG1(DM), and hIgG4, are all antibodies targeting human anti-egg lysosome (HEL). The variable region sequences of these antibodies are derived from Acierno et al.'s paper "Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies" (Acierno et al. J Mol Biol. 2007; 374(1):130-46.). The constant region fragment of hIgG1 uses Ig gamma-1 chain C region, ACCESSION: P01857-1 as the heavy chain constant region, and Ig kappa chain C region, ACCESSION: P01834 as the light chain constant region. hIgG1(DM) has L234A and L235A mutations in the heavy chain constant region of hIgG1 (according to the EU numbering system). The heavy chain constant region of hIgG4 uses Ig gamma-4 chain C region. Region ACCESSION: P01861.1 is used as the heavy chain constant region and the S228P mutation is introduced to improve stability; Ig kappa chain C region ACCESSION: P01834 is the light chain constant region; hIgG1, hIgG1(DM) and hIgG4 were all prepared in the laboratory of Zhongshan Kangfang Biopharmaceutical Co., Ltd.

[0754] In the following embodiments of the present invention, the positive control antibody Dupilumab is used:

[0755] The heavy chain amino acid sequence is SEQ ID NO:79.

[0756] The light chain amino acid sequence is SEQ ID NO:80.

[0757] In the following embodiments of the present invention, the positive control antibody AMG282 is used:

[0758] The heavy chain amino acid sequence is SEQ ID NO:81.

[0759] The light chain amino acid sequence is SEQ ID NO:82.

[0760] Preparation Example 1: Preparation and Sequence Analysis of Antibody 14A10 against Human IL-4RA

[0761] 1. Preparation of hybridoma cell line 14A10

[0762] The immunogen IL-4RA-mFc used to prepare the anti-IL-4RA antibody was a fusion protein of the mature human IL-4RA peptide (Genbank ID: NP_001244336.1) and the mFc tag (SEQ ID NO: 6) (synthesized by Zhongshan Kangfang Biopharmaceutical Co., Ltd.), and human hematologic leukemia cells TF-1 (ATCC, CRL-2003) were used to immunize BALB / c mice (purchased from Guangdong Medical Experimental Animal Center). Following existing techniques (e.g., Stewart, SJ, “Monoclonal Antibody Production”, in Basic Methods in antibody Production and Characterization, Eds. G. Howard and DRBethell, Boca Raton: CRC Press, 2000), spleen cells from antigen-immunized BALB / c mice (purchased from Guangdong Medical Experimental Animal Center) were fused with mouse myeloma cells to form hybridoma cells. Using IL-4RA-hFc protein (IL-4RA as described above, hFc being a purified human IgG Fc tag, specifically the Ig gamma-1 chain C region, Genbank ID: P01857, positions 114-330) as the antigen, ELISA plates were coated with hybridoma cells to obtain hybridoma cells that secreted antibodies specifically binding to IL-4RA-hFc. The hybridoma cells obtained from indirect ELISA were then screened using competitive ELISA to identify hybridoma cell lines that secreted monoclonal antibodies that competitively bind to IL-4RA-hFc with the ligand IL4-N-his (IL4 NCBI Gene ID: AAH70123.1). These lines were then subjected to limiting dilution to obtain hybridoma cell lines that stably secreted anti-human IL-4RA antibodies. The monoclonal antibodies secreted by these hybridoma cell lines were named 14A10.

[0763] 2. Preparation of anti-IL-4RA antibody 14A10

[0764] The hybridoma cell line prepared above was cultured in a cell culture incubator (5% CO2, 37℃) using CD medium (Chemical Defined Medium containing 1% penicillin and streptomycin). After 7 days, the cell culture supernatant was collected and purified by high-speed centrifugation, vacuum filtration through a microporous membrane, and HiTrap protein A HP column to obtain antibody 14A10.

[0765] 3. Sequence analysis of anti-IL-4RA antibody 14A10

[0766] mRNA was extracted from the hybridoma cell line obtained in step 1 according to the method of the cultured bacterial total RNA extraction kit (Tiangen, catalog number DP430).

[0767] According to Invitrogen III. First-Strand Synthesis System for RT-PCR Kit Instructions: Synthesize cDNA and perform PCR amplification.

[0768] The PCR amplification products were directly cloned using TA. For specific procedures, please refer to the instructions of the pEASY-T1 Cloning Kit (Transgen CT101).

[0769] The TA clone product was directly sequenced, and the sequencing results are as follows:

[0770] The nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO:1, and the amino acid sequence it encodes is shown in SEQ ID NO:2.

[0771] The nucleic acid sequence of the light chain variable region is shown in SEQ ID NO:3, and the amino acid sequence it encodes is shown in SEQ ID NO:4.

[0772] According to the IMGT numbering system:

[0773] The sequence of heavy chain CDR1 (HCDR1) is shown in SEQ ID NO:5, the sequence of heavy chain CDR2 (HCDR2) is shown in SEQ ID NO:6, and the sequence of heavy chain CDR3 (HCDR3) is shown in SEQ ID NO:7.

[0774] The sequence of light chain CDR1 (LCDR1) is shown in SEQ ID NO:8, the sequence of light chain CDR2 (LCDR2) is shown in SEQ ID NO:9, and the sequence of light chain CDR3 (LCDR3) is shown in SEQ ID NO:10.

[0775] Preparation Example 2: Design and Preparation of Anti-IL-4RA Chimeric Antibody 14A10 (CH)

[0776] Based on the variable region sequence of antibody 14A10 obtained in Preparation Example 1, a chimeric antibody was designed. The chimeric antibody's heavy chain constant region adopted the human Ig gamma-4 chain C region; the light chain constant region adopted the human Ig kappa chain C region, resulting in chimeric antibody 14A10(CH). The amino acid sequence of the 14A10(CH) heavy chain variable region is shown in SEQ ID NO:88, and the amino acid sequence of the 14A10(CH) light chain variable region is shown in SEQ ID NO:90; their coding sequences are shown in SEQ ID NO:89 and SEQ ID NO:91, respectively. The amino acid sequence of the 14A10(CH) heavy chain is shown in SEQ ID NO:11, and the amino acid sequence of the 14A10(CH) light chain is shown in SEQ ID NO:12; their coding sequences are shown in SEQ ID NO:35 and SEQ ID NO:36, respectively.

[0777] The 14A10(CH) heavy chain cDNA and light chain cDNA were cloned into the pUC57simple vector (provided by GenScript) to obtain pUC57simple-14A10(CH)H and pUC57simple-14A10(CH)L, respectively. Following the standard techniques described in *Molecular Cloning: A Laboratory Manual (Third Edition)*, the full-length heavy and light chain genes synthesized by EcoRI and HindIII restriction enzyme digestion were subcloned into the expression vector pcDNA3.1 to obtain expression plasmids pcDNA3.1-14A10(CH)H and pcDNA3.1-14A10(CH)L. Further sequencing analysis of the heavy / light chain genes in the recombinant expression plasmids was then performed. Subsequently, the gene combination pcDNA3.1-14A10(CH)H / pcDNA3.1-14A10(CH)L, containing the corresponding light and heavy chain recombinant plasmids, was designed and co-transfected into 293F cells. The culture medium was then collected for purification. After sequencing verification, an endotoxin-free expression plasmid was prepared and transiently transfected into HEK293 cells for antibody expression. After 7 days of culture, the cell culture medium was collected, and affinity purification using a Protein A column was performed to obtain the chimeric antibody 14A10(CH).

[0778] The three heavy chain CDRs (HCDR1-HCDR3) and three light chain CDRs (LCDR1-LCDR3) of the chimeric antibody 14A10 (CH) are identical to those of antibody 14A10.

[0779] Preparation Example 3: Design and Preparation of Humanized Anti-human IL-4RA

[0780] 1. Design of anti-IL4RA humanized antibodies

[0781] Based on the mouse sequence of hybridoma antibody 14A10 obtained in Preparation Example 1, antibody humanization design and mutation optimization were carried out based on a computer-simulated structural model, resulting in the variable region sequences of antibodies 14A10H42L25, 14A10H43L25, 14A10H49L25 and 14A10H50L25, as shown in Table 1 below.

[0782] Table 1

[0783] The constant region of the antibody heavy chain is the Ig gamma-4chain C region, as shown in SEQ ID NO:70; the constant region of the light chain is the Ig kappa chain C region, as shown in SEQ ID NO:72.

[0784] According to the IMGT numbering system:

[0785] The HCDR1 (SEQ ID NO:5), HCDR3 (SEQ ID NO:7), and LCDR1-LCDR3 (SEQ ID NOs:8-10) of antibodies 14A10H42L25, 14A10H43L25, 14A10H49L25, and 14A10H50L25 are identical to the HCDR1, HCDR3, and LCDR1-LCDR3 of antibody 14A10.

[0786] The HCDR2 of 14A10H42L25 and 14A10H49L25 is the same: INPSSSGRN (SEQ ID NO:23).

[0787] The HCDR2 of 14A10H43L25 and 14A10H50L25 is the same: INPGSGRN (SEQ ID NO:24).

[0788] 2. Preparation of humanized antibodies

[0789] The cDNA of the heavy chain variable region and the cDNA of the light chain variable region of 14A10H42L25, 14A10H43L25, 14A10H49L25 and 14A10H50L25 were cloned into the pUC57 simple vector (provided by Genscript Biotech Co., Ltd.) to obtain pUC57simple-14A10H42, pUC57simple-14A10L25, pUC57simple-14A10H43, pUC57simple-14A10H49 and pUC57simple-14A10H50, respectively. Following the standard techniques described in *Molecular Cloning: A Laboratory Manual (Second Edition)*, variable region fragments were obtained by enzyme digestion and subcloning into pcDNA3.1 vectors containing the corresponding heavy chain constant region fragments and light chain constant region fragments. (For heavy chain constant region fragments, the fragments were cloned into vector pcDNA3.1 by restriction enzyme digestion (HindIII & EcoRI); for light chain constant region fragments, the fragments were cloned into vector pcDNA3.1 by restriction enzyme digestion (HindIII & EcoRI)). The resulting fragments were pcDNA3.1-14A10H42, pcDNA3.1-14A10L25, pcDNA3.1-14A10H43, pcDNA3.1-14A10H49, and pcDNA3.1-14A10H50. Subsequently, the recombinant plasmids containing the corresponding light and heavy chains (pcDNA3.1-14A10H42 and pcDNA3.1-14A10L25, pcDNA3.1-14A10H43 and pcDNA3.1-14A10L25, pcDNA3.1-14A10H49 and pcDNA3.1-14A10L25, pcDNA3.1-14A10H50 and pcDNA3.1-114A10L25) were transfected into 293F cells, and the culture medium was collected for purification. After sequencing verification, endotoxin-free expression plasmids were prepared and transiently transfected into HEK293 cells for antibody expression. After 7 days of culture, the cell culture medium was collected and affinity purified using a Protein A column to obtain humanized antibodies 14A10H42L25, 14A10H43L25, 14A10H49L25, and 14A10H50L25.

[0790] Preparation Example 4: Preparation and Sequence Analysis of Anti-ST2 Antibody

[0791] 1. Preparation of hybridoma cell lines

[0792] The antigen used to prepare anti-ST2 antibodies is human ST2-ECD-His (sequence shown in SEQ ID NO:75). Spleen cells from immunized mice were fused with mouse myeloma cells to create hybridoma cells. Using human ST2-ECD-His as the antigen, hybridoma cells were screened using an indirect ELISA method to obtain hybridoma cells capable of secreting antibodies that specifically bind to ST2. The selected hybridoma cells were then subjected to limiting dilution to obtain stable hybridoma cell lines. The monoclonal antibodies secreted by these hybridoma cell lines were named 3H11.

[0793] 2. Preparation of anti-ST2 antibody 3H11

[0794] The hybridoma cell line prepared above was cultured in a cell culture incubator (5% CO2, 37℃) using CD medium (Chemical Defined Medium containing 1% penicillin and streptomycin). After 7 days, the cell culture supernatant was collected, and the antibody 3H11 was obtained by high-speed centrifugation, vacuum filtration through a microporous membrane, and purification using a HiTrap protein A HP column.

[0795] 3. Sequence analysis of the anti-ST2 antibody 3H11

[0796] mRNA was extracted from the hybridoma cell line obtained in step 1 according to the method of the cultured bacterial total RNA extraction kit (Tiangen, catalog number DP430).

[0797] According to Invitrogen III. First-Strand Synthesis System for RT-PCR Kit Instructions: Synthesize cDNA and perform PCR amplification.

[0798] The PCR amplification products were directly cloned using TA. For specific procedures, please refer to the instructions of the pEASY-T1 Cloning Kit (Transgen CT101).

[0799] The TA clone product was directly sequenced, and the sequencing results are as follows:

[0800] The nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO:25, and the amino acid sequence it encodes is shown in SEQ ID NO:26.

[0801] The nucleic acid sequence of the light chain variable region is shown in SEQ ID NO:27, and the amino acid sequence it encodes is shown in SEQ ID NO:28.

[0802] According to the IMGT numbering system:

[0803] The sequence of heavy chain CDR1 (HCDR1) is shown in SEQ ID NO:29, the sequence of heavy chain CDR2 (HCDR2) is shown in SEQ ID NO:30, and the sequence of heavy chain CDR3 (HCDR3) is shown in SEQ ID NO:31.

[0804] The sequence of light chain CDR1 (LCDR1) is shown in SEQ ID NO:32, the sequence of light chain CDR2 (LCDR2) is shown in SEQ ID NO:33, and the sequence of light chain CDR3 (LCDR3) is shown in SEQ ID NO:34.

[0805] Preparation Example 5: Design and Preparation of Anti-ST2 Chimeric Antibody 3H11(CH)

[0806] Based on the variable region sequence of antibody 3H11 obtained in Preparation Example 4, a chimeric antibody 3H11(CH) was designed. Its heavy chain constant region adopted the human Ig gamma-1 chain C region; its light chain constant region was the human Ig kappa chain C region. A point mutation (L234A) was introduced at position 234 of the heavy chain constant region, and a point mutation (L235A) was introduced at position 235. The amino acid sequence of the 3H11(CH) heavy chain is shown in SEQ ID NO:83, and the amino acid sequence of the 3H11(CH) light chain is shown in SEQ ID NO:84. The amino acid sequence of the variable region of the 3H11(CH) heavy chain is shown in SEQ ID NO:92, and the amino acid sequence of the variable region of the 3H11(CH) light chain is shown in SEQ ID NO:93.

[0807] The 3H11(CH) heavy chain cDNA and light chain cDNA were cloned into the pUC57simple vector (provided by GenScript) to obtain pUC57simple-3H11(CH)H and pUC57simple-3H11(CH)L, respectively. Following the standard techniques described in *Molecular Cloning: A Laboratory Manual (3rd Edition)*, the full-length heavy and light chain genes synthesized by EcoRI and HindIII restriction enzyme digestion were subcloned into the expression vector pcDNA3.1 to obtain expression plasmids pcDNA3.1-3H11(CH)H and pcDNA3.1-3H11(CH)L. Further sequencing analysis of the heavy / light chain genes in the recombinant expression plasmids was performed. Subsequently, the gene combination pcDNA3.1-3H11(CH)H / pcDNA3.1-3H11(CH)L, containing the corresponding light and heavy chain recombinant plasmids, was co-transfected into 293F cells, and the culture medium was collected for purification. After sequencing verification, an endotoxin-free expression plasmid was prepared and transiently transfected into HEK293 cells for antibody expression. After 7 days of culture, the cell culture medium was collected and the chimeric antibody was obtained by affinity purification using a Protein A column.

[0808] Preparation Example 6: Design and preparation of humanized antibodies and mutants against ST2

[0809] 1. Design of light and heavy chains of humanized antibodies against human ST2: 3H11H4L7, 3H11H4L8, 3H11H40L8, 3H11H45L7, 3H11H45L8, 3H11H46L7, and 3H11H46L8

[0810] Based on the sequence of hybridoma antibody 3H11 obtained in Preparation Example 4, antibody humanization design and mutation optimization were carried out based on a computer-simulated structural model. The variable region sequences of antibodies 3H11H4L7, 3H11H4L8, 3H11H40L8, 3H11H45L7, 3H11H45L8, 3H11H46L7 and 3H11H46L8 were designed, as shown in Table 2 below.

[0811] Table 2

[0812] The heavy chain constant region of the antibodies all adopts the human Ig gamma-4 chain C region; the light chain constant region is the human Ig kappa chain C region; and point mutations were introduced at position 252 (M252Y) from methionine to tyrosine, position 254 (S254T) from serine to threonine, and position 256 (T256E) from threonine to glutamic acid, thereby obtaining humanized antibodies 3H11H4L7, 3H11H4L8, 3H11H40L8, 3H11H45L7, 3H11H45L8, 3H11H46L7, and 3H11H46L8 carrying these three mutation sites; the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:71, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:72.

[0813] 2. Preparation of humanized antibodies 3H11H4L7, 3H11H4L8, 3H11H40L8, 3H11H45L7, 3H11H45L8, 3H11H46L7, and 3H11H46L8

[0814] The heavy chain cDNA and light chain cDNA of 3H11H4L7, the heavy chain cDNA and light chain cDNA of 3H11H4L8, the heavy chain cDNA and light chain cDNA of 3H11H40L8, the heavy chain cDNA and light chain cDNA of 3H11H45L7, the heavy chain cDNA and light chain cDNA of 3H11H45L8, the heavy chain cDNA and light chain cDNA of 3H11H46L7, and the heavy chain cDNA and light chain cDNA of 3H11H46L8 were cloned into the pUC57simple vector (provided by GenScript) to obtain pUC57simple-3H11H4 and pUC57simple-3H11L7, respectively.

[0815] pUC57simple-3H11H4, pUC57simple-3H11L8;

[0816] pUC57simple-3H11H40, pUC57simple-3H11L8;

[0817] pUC57simple-3H11H45, pUC57simple-3H11L7;

[0818] pUC57simple-3H11H45, pUC57simple-3H11L8;

[0819] pUC57simple-3H11H46, pUC57simple-3H11L7; and

[0820] pUC57simple-3H11H46, pUC57simple-3H11L8.

[0821] Following the standard techniques described in *Molecular Cloning: A Laboratory Manual (Third Edition)*, the full-length heavy and light chain genes synthesized by EcoRI and HindIII restriction enzyme digestion were subcloned into the expression vector pcDNA3.1 to obtain expression plasmids pcDNA3.1-3H11H4, pcDNA3.1-3H11L7, pcDNA3.1-3H11L8, pcDNA3.1-3H11H40, pcDNA3.1-3H11H8, pcDNA3.1-3H11H45, and pcDNA3.1-3H11H46. Further sequencing analysis was performed on the heavy / light chain genes of the recombinant expression plasmids. Subsequently, gene combinations designed with corresponding light and heavy chain recombinant plasmids (pcDNA3.1-3H11H4 / pcDNA3.1-3H11L7, pcDNA3.1-3H11H4 / pcDNA3.1-3H11L8, pcDNA3.1-3H11H40 / pcDNA3.1-3H11L8, pcDNA3.1-3H11H45 / pcDNA3.1-3H11L7, pcDNA3.1-3H11H45 / pcDNA3.1-3H11L8, pcDNA3.1-3H11H46 / pcDNA3.1-3H11L7, pcDNA3.1-3H11H46 / pcDNA3.1-3H11L8) were co-transfected into 293F cells, and the culture medium was collected for purification. After sequencing verification, an endotoxin-free expression plasmid was prepared and transiently transfected into HEK293 cells for antibody expression. After 7 days of culture, the cell culture medium was collected and affinity purified using a Protein A column to obtain humanized antibodies.

[0822] Preparation Example 7: Design and preparation of humanized antibodies 3H11H45L7 (G1WT) and 3H11H46L7 (G1WT)

[0823] The heavy chain constant region of humanized antibodies 3H11H45L7 and 3H11H46L7 was replaced with the human Ig gamma-1 chain C region (SEQ ID NO: 66), and the light chain constant region was replaced with the human Ig kappa chain C region (SEQ ID NO: 72), thereby obtaining humanized antibodies 3H11H45L7(G1WT) and 3H11H46L7(G1WT). The preparation method of antibodies 3H11H45L7(G1WT) and 3H11H46L7(G1WT) is described in Preparation Example 6.

[0824] Preparation Example 8: Design and preparation of anti-IL-4RA-anti-ST2 bispecific antibody

[0825] 1. Sequence Design

[0826] The anti-IL-4RA-anti-ST2 bispecific antibodies L4RT04(LS), L4RT01, L4RT02, L4RT03, L4RT04, TL4R01, TL4R02, TL4R03, and TL4R04 in this invention follow a Morrison pattern (IgG-scFv), meaning that the scFv fragment of another antibody is linked to the C-terminus of both heavy chains of an IgG antibody. The constant regions of the heavy chains all utilize the human Ig gamma-1 chain C region; the constant regions of the light chains all utilize the human Ig kappa chain C region. The main component design of the anti-IL-4RA-anti-ST2 bispecific antibodies is shown in Table 3 below.

[0827] The amino acid sequence of linker (G4S)3 is shown in SEQ ID NO:53, and the amino acid sequence of linker (G4S)4 is shown in SEQ ID NO:54.

[0828] The bispecific antibodies designed in Table 3 above all have a point mutation introduced at position 234 (L234A) of leucine to alanine, at position 235 (L235A) of leucine to alanine, and at position 237 (G237A) of glycine to alanine, thus obtaining bispecific antibodies containing the above mutation sites. In addition, a point mutation was further introduced at position 428 (M428L) of methionine to leucine and at position 434 (N434S) of aspartic acid to serine in the heavy chain of the bispecific antibody L4RT04(LS).

[0829] The amino acid sequences of the "heavy chain of the immunoglobulin moiety + linker + scFv moiety" (equivalent to the heavy chain of the bispecific antibody) of L4RT04(LS), L4RT01, L4RT02, L4RT03, L4RT04, TL4R01, TL4R02, TL4R03 and TL4R04 are shown in SEQ ID NO:55 to SEQ ID NO:63, respectively; wherein:

[0830] The amino acid sequence of the heavy chain of the immunoglobulin moiety of L4RT04(LS) is shown in SEQ ID NO:85;

[0831] The amino acid sequences of the heavy chain of the immunoglobulin moiety of L4RT01, L4RT02, L4RT03 and L4RT04 are all as shown in SEQ ID NO:86.

[0832] The amino acid sequences of the heavy chain of the immunoglobulin moiety of TL4R01, TL4R02, TL4R03 and TL4R04 are all shown in SEQ ID NO:87.

[0833] The amino acid sequences of the "light chains of the immunoglobulin moiety" (equivalent to the light chains of bispecific antibodies) of L4RT04(LS), L4RT01, L4RT02, L4RT03 and L4RT04 are shown in SEQ ID NO:64.

[0834] The amino acid sequences of the "light chains of the immunoglobulin moiety" (equivalent to the light chains of bispecific antibodies) of TL4R01, TL4R02, TL4R03 and TL4R04 are shown in SEQ ID NO:65.

[0835] Example 1: Detection of antibody-antigen binding activity using ELISA method

[0836] 1. The binding activity of antibodies 14A10H42L25, 14A10H43L25, 14A10H49L25, and 14A10H50L25 to antigen IL4RA-mFc was detected by indirect ELISA.

[0837] IL4RA-mFc was coated onto ELISA plates and incubated. After blocking, the target antibodies were added, and after incubation and washing, goat anti-human IgG Fc and HRP (purchased from Jackson ImmunoResearch Inc., catalog number: 109-035-098) were added. After incubation and washing, a colorimetric reaction was performed using TMB (Neogen, 308177). After the colorimetric reaction was terminated, the absorbance at 450 nm was measured using an ELISA reader. The data were analyzed and processed using SoftMax Pro 6.2.1 software.

[0838] The results are shown in Tables 4 and 5, and Figures 1A and 1B. Antibodies 14A10H42L25, 14A10H43L25, 14A10H49L25, and 14A10H50L25, along with the positive control antibody Dupilumab targeting the same site, can all effectively bind to the antigen human IL4RA-mFc, and their binding efficiency is dose-dependent.

[0839] Table 4: Binding activities of 14A10H42L25, 14A10H43L25 and Dupilumab with antigen IL4RA-mFc

[0840] Table 5: Binding activities of 14A10H50L25, 14A10H49L25 and Dupilumab with antigen IL4RA-mFc

[0841] 2. The competitive ELISA method was used to detect the blocking activity of antibodies 14A10H42L25, 14A10H43L25, 14A10H49L25, and 14A10H50L25 in the binding of IL4-N-his to antigen IL4RA-hFc.

[0842] The extracellular matrix (EC5) of 14A10H42L25, 14A10H43L25, 14A10H49L25, and 14A10H50L25 was determined by ELISA to competitively bind human IL4RA-hFc to the ligand human IL4-N-His of the target antigen. 50 (Half-number effect concentration) was used to investigate the activity of 14A10H42L25, 14A10H43L25, 14A10H49L25, and 14A10H50L25 in blocking the binding of the target antigen ligand to human IL4RA-hFc.

[0843] After coating and blocking human IL4RA-hFc in the ELISA plate, the test antibody was added, followed by an equal volume of human IL4-N-His (synthesized by Zhongshan Kangfang Biopharmaceutical Co., Ltd.). The mixture was incubated, washed, and then mouse anti-his and HRP (purchased from Kangwei Century Biotechnology Co., Ltd., catalog number: CW0285A) were added and incubated. After washing, TMB (Neogen, 308177) was used for color development, and the process was terminated. The ELISA plate was immediately placed in a microplate reader, and the OD values ​​of each well were read at a wavelength of 450 nm. The data were analyzed using SoftMax Pro 6.2.1 software. Specific detection results are listed in Tables 6 and 7.

[0844] Plotting antibody concentration on the x-axis and absorbance on the y-axis using a 4-parameter fitting curve yields the corresponding ECG blocking effect of the detected antibody. 50 As shown in Figures 1C and 1D, 14A10H42L25, 14A10H43L25, 14A10H49L25, 14A10H50L25, and their corresponding target positive control antibody Dupilumab can all effectively block the binding of the ligand human IL4-N-His to the antigen human IL4RA-hFc, and the blocking efficiency shows a dose-dependent relationship.

[0845] Table 6: Activity assay results of 14A10H42L25, 14A10H43L25 and Dupilumab competing with human IL4-N-His for binding to human IL4RA-hFc

[0846] Table 7: Activity assay results of 14A10H50L25, 14A10H49L25 and Dupilumab competing with human IL4-N-His for binding to human IL4RA-hFc

[0847] Example 2: FACS detection of the binding activity of anti-IL-4RA antibody to IL4-RA antigen on the surface of 293T-IL4Rα cells

[0848] 293T-IL4Rα cells (constructed by Kangfang Biotechnology) were collected by digestion with 0.05% trypsin (Gibco, catalog number: 25300-120), centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. Cells were resuspended in DMEM + 10% FBS, and transferred at 300,000 cells / well to conical 96-well plates. 100 μl of 1% PBSA (i.e., PBS + 1% BSA, BSA (Sigma, catalog number: V900933-1KG)) was added to each well, and the supernatant was discarded after centrifugation. Experimental antibodies such as Dupilumab (Regeneron, batch number: 7L615F) were diluted to working concentrations of 0.000004 nM, 0.00004 nM, 0.0004 nM, 0.004 nM, 0.044 nM, 0.222 nM, 1.11 nM, 3.3 nM, and 10 nM. Add the diluted antibody to the corresponding wells, and set up an isotype control (hIgG4), a negative control (cells + AF647 secondary antibody group), and a blank control (293T-IL4Rα cells only). Incubate on ice for 60 min. Add 100 μl of 1% PBSA, centrifuge at 350 x g for 5 min, discard the supernatant, and wash twice with an appropriate amount of 1% PBSA. Add 50 μl / well of secondary antibody Alexa Fluor 647Mouse Anti-Human IgG Fc (Southern Biotech, catalog number: 9040-31, 1:300 dilution), and incubate on ice in the dark for 30 min. Add 100 μl of 1% PBSA, centrifuge at 350 x g for 5 min, discard the supernatant, and wash twice with an appropriate amount of 1% PBSA. Resuspend the cells in 200 μl of 1% PBSA before testing.

[0849] The experimental results are shown in Figure 2 and Table 8.

[0850] Table 8: Binding activity of anti-IL-4RA antibody to 293T-IL4Rα cell surface antigen

[0851] The results showed that 14A10(CH), 14A10H42L25, 14A10H43L25, 14A10H49L25 and 14A10H50L25 could all effectively bind to the IL4-RA antigen on the surface of 293T-IL4Rα cells, and the binding ability of 14A10H42L25, 14A10H43L25 and 14A10(CH) was comparable to or even slightly better than that of the control antibody Dupilumab.

[0852] Example 3: Cellular biological activity analysis of anti-IL-4RA antibody

[0853] The cellular biological activities of 14A10H49L25, 14A10H50L25, and 14A10H43L25 in blocking the proliferation of human IL-4 and IL-13-induced TF-1 cells were detected using a cell proliferation assay. The specific steps are as follows:

[0854] TF-1 cells (purchased from the American Type Culture Collection, catalog number: CRL-2003) were seeded in RPMI 1640 + 10% FBS + 2.5 g / L glucose (Sigma, catalog number: G7528-250G) + 2 ng / mL GM-CSF (Peprotech, catalog number: 300-03) and cultured normally. On the day of the experiment, TF-1 cells were collected by centrifugation, resuspended in culture medium without GM-CSF, counted, and seeded at 20,000 cells per well into 96-well plates. A blank control (group with only TF-1 cells), a negative control (IL-4 group or IL-13 group), an isotype control group (hIgG4), and an experimental antibody group were set up. According to the experimental design, experimental antibodies (final concentrations of 0.004 nM, 0.02 nM, 0.1 nM, 0.5 nM, 2.5 nM, or 50 nM) or isotype control antibody hIgG4 (final concentration of 50 nM) were added to the cells. Two replicates were set for each treatment, and the cells were pre-incubated at 37°C and 5% CO2 for 30 min. After pre-incubation, human IL-4 (Peprotech, catalog number: 200-04, final concentration of 0.25 nM) or human IL-13 (Sino Biological, catalog number: 10369-HNAC-20, final concentration of 0.8 nM) were added, and the cells were incubated at 37°C and 5% CO2 for 72 h. After 72 h, CCK8 reagent was added according to the instructions of the CCK8 kit (purchased from Dojin, catalog number: CK04), mixed well, and the OD values ​​were read at 450 nm and 600 nm using a microplate reader.

[0855] The test results are shown in Figures 3A and 3B. The results showed that both human IL-4 and IL-13 could effectively promote the proliferation of TF-1 cells; compared with the isotype control antibody (hIgG4), 14A10H49L25, 14A10H50L25, and 14A10H43L25 could specifically inhibit the proliferation of TF-1 cells induced by IL-4 and IL-13 in a dose-dependent manner.

[0856] Example 4: Anti-IL-4RA antibody inhibits the upregulation of CD23 expression in PBMC cells

[0857] 1. Anti-IL-4RA antibody inhibits the upregulation of CD23 expression in B cells.

[0858] Using Ficoll-Paque TM Plus (GE, catalog number: 17-1440-02) isolated PBMCs from peripheral blood donated by healthy volunteers and cryopreserved. PBMCs were resuscitated and cultured overnight at 37°C in a 5% FBS-containing RPMI 1640 complete medium. PBMCs were routinely collected and added at 300,000 cells per well to very low adsorption round-bottom 96-well plates (Corning, catalog number: 7007). Blank control (group with only PBMCs and secondary antibody), negative control (group with IL-4 or IL-13 added to the blank control), isotype control (hIgG4), and experimental antibody group were set up. According to the experimental design, experimental antibodies such as Dupilumab (Regeneron, batch number: 7L615F) (final concentrations of 25 nM, 2.5 nM, and 0.25 nM) or isotype control hIgG4 (final concentration of 25 nM) were added to the cells, and pre-incubated for 30 min. After 30 minutes, add human IL-4 (Peprotech, catalog number: 200-04, final concentration: 100 pM) or human IL-13 (Sino Biological, catalog number: 10369-HNAC-20, final concentration: 100 ng / mL) and incubate for 2 days. After 2 days, transfer each group of PBMCs to 96-well flow cytometry plates and wash once with 1% PBSA (i.e., PBS + 1% BSA). Add Mouse IgG Isotype Control (Thermofisher, catalog number: 10400C, diluted 20 times with 1% PBSA) and incubate on ice for 20 minutes. After washing once with an appropriate amount of 1% PBSA, add FITC anti-human CD3 Antibody (Biolegend, catalog number: 344804, 1:100 dilution) and Brilliant Violet 421. TMA mixture of secondary antibodies, namely anti-human CD19 Antibody (Biolegend, catalog number: 302234, 1:100 dilution) and PE anti-human CD23 Antibody (Biolegend, catalog number: 338508, 1:50 dilution), was incubated on ice in the dark for 30 min. After washing once with 1% PBSA, the cells were resuspended and analyzed.

[0859] The test results are shown in Figures 4A and 4B.

[0860] The results showed that human IL-4 and human IL-13 upregulated the expression level of CD23 on the surface of B cells in human PBMCs. 14A10(CH), 14A10H42L25, 14A10H43L25, 14A10H49L25, and 14A10H50L25 specifically bound to human IL-4RA, effectively blocking the upregulation of CD23 expression on the surface of B cells by IL-4 and IL-13. The activity of 14A10H49L25 and 14A10H42L25 in blocking the upregulation of CD23 expression on the surface of B cells by IL-4 was slightly stronger than that of the control antibody Dupilumab.

[0861] 2. Anti-IL-4RA antibody inhibits the upregulation of CD23 expression in human monocytes.

[0862] Using Ficoll-Paque TMPlus (GE, catalog number: 17-1440-02) isolated PBMCs from peripheral blood donated by healthy volunteers and cryopreserved. PBMCs were resuscitated and cultured overnight at 37°C in a 5% FBS-containing RPMI 1640 complete medium. PBMCs were routinely collected and added at 300,000 cells per well to very low adsorption round-bottom 96-well plates (Corning, catalog number: 7007). Blank control (group with only PBMCs and secondary antibody), negative control (group with IL-4 or IL-13 added to the blank control), isotype control (hIgG4), and experimental antibody group were set up. According to the experimental design, experimental antibodies such as Dupilumab (Regeneron) (Regeneron, batch number: 7L615F) (final concentrations of 25 nM, 2.5 nM, and 0.25 nM) or isotype control hIgG4 (final concentration of 25 nM) were added to the cells, and pre-incubated for 30 min. After 30 min, add human IL-4 (Peprotech, catalog number: 200-04, final concentration: 100 pM) or human IL-13 (Sino Biological, catalog number: 10369-HNAC-20, final concentration: 100 ng / mL) and incubate for 2 days. After 2 days, transfer each group of PBMCs to a 96-well flow cytometry plate and wash once with 1% PBSA (i.e., PBS + 1% BSA). Add Mouse IgG Isotype Control (Thermofisher, catalog number: 10400C, diluted 20 times with 1% PBSA) and incubate on ice for 20 min. After washing once with an appropriate amount of 1% PBSA, add FITC anti-human CD3 Antibody (Biolegend, catalog number: 344804, 1:100 dilution) and Alexa Fluor. R A mixture of 700 anti-human CD14 Antibody (Biolegend, catalog number: 367114, 1:100 dilution) and PE anti-human CD23 Antibody (Biolegend, catalog number: 338508, 1:50 dilution) was incubated on ice in the dark for 30 min. After washing once with 1% PBSA, the sample was analyzed.

[0863] The test results are shown in Figures 4C to 4D.

[0864] The results showed that human IL-4 and human IL-13 upregulated the expression level of CD23 on the surface of monocytes in human PBMCs. 14A10(CH), 14A10H42L25, 14A10H43L25, 14A10H49L25, and 14A10H50L25 specifically bound to human IL-4RA, effectively blocking the upregulation of CD23 expression on the surface of monocytes by IL-4 and IL-13. The activity of 14A10H49L25 in blocking the upregulation of CD23 expression on the surface of monocytes by IL-4 was stronger than that of the control antibody Dupilumab.

[0865] Example 5: ELISA method for determining the binding activity of anti-IL-4RA-anti-ST2 bispecific antibody to antigen.

[0866] 1. The binding activity of L4RT01, L4RT02, L4RT03, L4RT04, TL4R01, TL4R02, TL4R03, TL4R04, and L4RT04 (LS) to the antigen IL4RA-mFc was determined by indirect ELISA. The specific method is as follows:

[0867] IL4RA-mFc was coated onto ELISA plates and incubated. After blocking, the target antibodies were added, followed by incubation and washing. Goat anti-human IgG Fc and HRP (purchased from Jackson ImmunoResearch Inc., catalog number: 109-035-098) were then added. After incubation and washing, a colorimetric reaction was performed using TMB (Neogen, 308177). After the colorimetric reaction was terminated, the absorbance at 450 nm was measured using an ELISA reader. Data were analyzed using SoftMax Pro 6.2.1 software.

[0868] The detection results are shown in Tables 9, 10, and 11, and Figures 5A to 5C. The results show that L4RT01, L4RT02, L4RT03, L4RT04, TL4R01, TL4R02, TL4R03, TL4R04, and L4RT04(LS) can effectively bind to the human IL4RA-mFc antigen, and the binding efficiency is dose-dependent. The binding efficiency (EC5) of antibodies L4RT01, L4RT02, L4RT03, L4RT04, TL4R01, TL4R02, TL4R03, TL4R04, and L4RT04(LS) was obtained through absorbance quantitative analysis and curve simulation calculation. 50 The values ​​are 0.027 nM, 0.035 nM, 0.032 nM, 0.035 nM, 0.053 nM, 0.085 nM, 0.041 nM, 0.043 nM, and 0.03 nM, respectively.

[0869] Table 11: ELISA detection of the binding of L4RT04(LS) to antigen IL-4RA-mFc

[0870] 2. The binding activity of L4RT01, L4RT02, L4RT03, L4RT04, TL4R01, TL4R02, TL4R03, TL4R04, and L4RT04 (LS) to the antigen ST2-mFc was determined by indirect ELISA. The specific method is as follows:

[0871] ST2-mFc was coated onto ELISA plates and incubated. After blocking, the target antibodies were added, followed by incubation and washing. Goat anti-human IgG Fc and HRP (purchased from Jackson ImmunoResearch Inc., catalog number: 109-035-098) were then added. After incubation and washing, a colorimetric reaction was performed using TMB (Neogen, 308177). After the colorimetric reaction was terminated, the absorbance at 450 nm was measured using an ELISA reader. Data were analyzed using SoftMax Pro 6.2.1 software.

[0872] The test results are shown in Tables 12, 13, and 14, as well as Figures 5D to 5F.

[0873] Table 14: ELISA detection of the binding of L4RT04(LS) to antigen ST2-mFc

[0874] The results showed that L4RT01, L4RT02, L4RT03, L4RT04, TL4R01, TL4R02, TL4R03, TL4R04, and L4RT04(LS) could effectively bind to the human ST2-mFc antigen, and the binding efficiency was dose-dependent. Quantitative absorbance analysis of the bound antibodies and curve simulation calculations yielded the EC50 binding efficiencies of antibodies L4RT01, L4RT02, L4RT03, L4RT04, TL4R01, TL4R02, TL4R03, TL4R04, and L4RT04(LS). 50 The values ​​are 0.040 nM, 0.064 nM, 0.079 nM, 0.067 nM, 0.046 nM, 0.051 nM, 0.058 nM, 0.098 nM, and 0.073 nM, respectively.

[0875] Example 6: Determination of competitive binding activity of anti-IL-4RA-anti-ST2 bispecific antibody using a competitive ELISA method

[0876] 1. Competitive ELISA method was used to determine the activity of anti-IL-4RA-anti-ST2 bispecific antibody and IL4-N-his in competitively binding to IL4RA-hFc.

[0877] After coating and blocking human IL4RA-hFc in an ELISA plate, the target antibody was added, followed by an equal volume of human IL4-N-His (synthesized by Zhongshan Kangfang Biopharmaceutical Co., Ltd.). The mixture was incubated, washed, and then mouse anti-his and HRP (purchased from Kangwei Century Biotechnology Co., Ltd., catalog number: CW0285A) were added and incubated. After washing, TMB (Neogen, 308177) was used for color development, and the process was terminated. The ELISA plate was immediately placed in an ELISA reader, and the OD values ​​of each well were read at a wavelength of 450 nm. The data were analyzed using SoftMax Pro 6.2.1 software. The OD values ​​for each dose are shown in Tables 15, 16, and 17. Quantitative analysis of the absorbance of the bound antibody was performed, and a curve was used to simulate the antibody's blocking efficiency of human IL4RA-hFc's binding to its ligand IL4-N-his to obtain the competitive binding EC50. 50 .

[0878] The results are shown in Figures 6A to 6C. The results show that L4RT01, L4RT02, L4RT03, L4RT04, TL4R01, TL4R02, TL4R03, TL4R04, L4RT04(LS), and Dupilumab effectively blocked the binding of antigen IL4RA-hFc to IL4-N-his, and the blocking efficiency showed a dose-dependent relationship. L4RT01, L4RT02, L4RT03, L4RT04, TL4R01, TL4R02, TL4R03, TL4R04, and L4RT04(LS) blocked the EC50 of IL4RA-hFc binding to IL4-N-his. 50 The values ​​are 1.918 nM, 1.786 nM, 2.191 nM, 2.066 nM, 3.136 nM, 2.906 nM, 1.845 nM, 2.179 nM, and 2.616 nM, respectively.

[0879] Table 17: Activity assay results of L4RT04(LS) competing with IL4-N-his for binding to IL4RA-hFc

[0880] 2. Competitive ELISA method was used to determine the activity of anti-IL-4RA-anti-ST2 bispecific antibody and IL33(112-270)-Nhis-biotin in competitively binding to ST2-mFc.

[0881] After coating and blocking human ST2-mFc in an ELISA plate, the target antibody was added, followed by an equal volume of human IL33(112-270)-Nhis-biotin (synthesized by Zhongshan Kangfang Biopharmaceutical Co., Ltd.). The mixture was incubated, washed, and then SA-HRP (KPL, 14-30-00) was added and incubated. After washing, TMB (Neogen, 308177) was used for color development, and the process was terminated. The ELISA plate was immediately placed in an ELISA reader, and the OD values ​​of each well were read at 450 nm. The data were analyzed using SoftMax Pro 6.2.1 software. The OD values ​​for each dose are shown in Tables 18, 19, and 20. Quantitative analysis of the absorbance of the bound antibody was performed, and a curve was used to simulate the antibody's blocking efficiency of ST2-mFc's binding to its ligand IL33(112-270)-Nhis-biotin to obtain the competitive binding EC50. 50 .

[0882] The results are shown in Figures 6D to 6F. The results show that L4RT01, L4RT02, L4RT03, L4RT04, TL4R01, TL4R02, TL4R03, TL4R04, L4RT04(LS), and AMG282 effectively blocked the binding of antigen ST2-mFc to IL33(112-270)-Nhis-biotin, and the blocking efficiency showed a dose-dependent relationship. L4RT01, L4RT02, L4RT03, L4RT04, TL4R01, TL4R02, TL4R03, TL4R04, and L4RT04(LS) blocked the EC50 of ST2-mFc binding to IL33(112-270)-Nhis-biotin. 50 The values ​​are 5.074 nM, 4.198 nM, 2.002 nM, 13.94 nM, 3.774 nM, 3.576 nM, 3.920 nM, 10.47 nM, and 15.1 nM, respectively.

[0883] Table 20: Activity assay results of L4RT04(LS) and IL33(112-270)-Nhis-biotin competing for ST2-mFc binding.

[0884] Example 7: Determination of kinetic parameters of humanized antibodies

[0885] 1. Determination of kinetic parameters of binding between humanized antibodies L4RT03, L4RT04 (LS), TL4R04 and IL4RA-His

[0886] The sample dilution buffer was PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4. A 5 μg / ml sample was immobilized on the AHC2 sensor at a height of 1 nm. The sensor was equilibrated in the buffer for 60 s. The immobilized sample was then bound to IL4RA-His at a concentration of 1.25–20 nM (two-fold dilution) for 200 s. The antibody was then dissociated in the buffer for 300 s. The sensor was regenerated using 10 mM glycine at pH 1.7 for 5 s, repeated four times. The sample plate was vibrated at 1000 rpm, the detection temperature was 30 °C, and the frequency was 5.0 Hz. Data were analyzed using a 1:1 model to obtain the affinity constant. Data acquisition software was Fortebio Data Acquisition 12.0, and data analysis software was Fortebio Data Analysis HT 12.0.

[0887] The results are shown in Table 21 and Figures 7A to 7D.

[0888] Table 21: Kinetic parameters of binding between humanized antibodies L4RT03, L4RT04 (LS), TL4R04 and IL4RA-His

[0889] KD is the affinity constant; kon is the antigen-antibody binding rate; kdis is the antigen-antibody dissociation rate; KD = kdis / kon.

[0890] The results showed that the humanized antibodies L4RT03, L4RT04(LS), and TL4R04 all had good affinity for the antigen IL4RA-His.

[0891] 2. Determination of kinetic parameters of binding between humanized antibodies L4RT03, L4RT04 (LS), TL4R04 and ST2-His

[0892] The sample dilution buffer was PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4. 1 μg / ml of sample was immobilized on the AHC2 sensor at a height of 0.5 nm. The sensor was equilibrated in the buffer for 60 s. The immobilized sample was then bound to ST2-His at a concentration of 200–2.469 nM (three-fold dilution) for 100 s. The antibody dissociated in the buffer for 150 s. The sensor was regenerated using 10 mM glycine at pH 1.7 for 5 s, repeated 5 times. The sample plate was vibrated at 1000 rpm, the detection temperature was 30 °C, and the frequency was 5.0 Hz. Data were analyzed using a 1:1 model to obtain the affinity constant. Data acquisition software was Fortebio Data Acquisition 12.0, and data analysis software was Fortebio Data Analysis HT 12.0.

[0893] The results are shown in Table 22 and Figures 7E to 7H.

[0894] Table 22: Kinetic parameters of binding between humanized antibodies L4RT03, L4RT04 (LS), TL4R04 and ST2-His

[0895] KD is the affinity constant; kon is the antigen-antibody binding rate; kdis is the antigen-antibody dissociation rate; KD = kdis / kon.

[0896] The results showed that the humanized antibodies L4RT03, L4RT04(LS), and TL4R04 all had good affinity for the antigen ST2-His.

[0897] Example 8: FACS detection of the binding activity of anti-IL-4RA-anti-ST2 bispecific antibody to antigen IL-4RA / ST2.

[0898] 1. FACS (Fluorescence-Activated Cell Sorting) detection of the binding activity of anti-IL-4RA-anti-ST2 bispecific antibody to ST2 on the surface of 293T-ST2-Luc cell membrane.

[0899] 293T-ST2-Luc cells (constructed by Kangfang Biotechnology) in logarithmic growth phase were collected at a ratio of 3 × 10⁻⁶. 5Cells were transferred to 96-well flow cytometry plates, and 100 μL of 1% PBSA was added. The plates were centrifuged at 350 × g for 5 min, and the supernatant was discarded. Antibodies such as AMG282 diluted with 1% PBSA were added (the final concentrations of the experimental antibodies in Figure 8A were 900 nM, 300 nM, 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, 1.23 nM, 0.123 nM, 0.0123 nM, and 0.00123 nM; the final concentrations of the experimental antibodies in Figure 8B were 100 nM, 33.3 nM, and 11.1 nM). The concentrations of hIgG1 were 3.7 nM, 1.23 nM, 0.123 nM, 0.0123 nM, 0.00123 nM, 0.000123 nM, and 0.0000123 nM. Simultaneously, blank control and isotype control hIgG1 groups were set up (the final hIgG1 concentration in Figure 8A was 900 nM; the final hIgG1 concentration in Figure 8B was 100 nM). After gentle mixing, the mixture was incubated on ice for 40 min. An appropriate amount of 1% PBSA was added, and the mixture was centrifuged at 350 × g for 5 min. The supernatant was discarded, and the mixture was washed three times with 1% PBSA. Alexa was added at a 300-fold dilution. 647-labeled mouse anti-human IgG secondary antibody (Southern Biotech, catalog number: 9040-31) was used to resuspend the cells and incubate them on ice in the dark for 0.5 h. An appropriate amount of 1% PBSA was added, and the cells were centrifuged at 350×g for 5 min. The supernatant was discarded, and the cells were washed twice with 1% PBSA. The cell pellet was resuspended in 200 μL of 1% PBSA and then analyzed.

[0900] The experimental results are shown in Tables 23 and 24, and Figures 8A and 8B.

[0901] Table 23: FACS detection of the binding activity of L4RT04, TL4R04, 3H11H46L7(hG1WT), AMG282 and ST2 on the cell membrane surface of 293T-ST2-Luc.

[0902] Table 24: FACS detection of the binding activity of L4RT04(LS), AMG282 and ST2 on the cell membrane surface of 293T-ST2-Luc cells.

[0903] The results showed that L4RT04, TL4R04, L4RT04(LS), 3H11H46L7(hG1WT), and AMG282 could all specifically bind to ST2 on the surface of the 293T-ST2-Luc cell membrane.

[0904] 2. FACS detection of the binding activity of anti-IL-4RA-anti-ST2 bispecific antibody to IL-4RA on the cell membrane surface of 293T-IL4Rα cells.

[0905] Collect 293T-IL4Rα cells (constructed by Kangfang Biotechnology) in logarithmic growth phase, at a ratio of 3 × 10⁻⁶. 5 Transfer cells per well to a 96-well flow cytometry plate, add 100 μL of 1% PBSA, centrifuge for 5 min, and discard the supernatant. Add experimental antibodies such as Dupilumab (Regeneron, batch number: 7L615F) diluted with 1% PBSA (final concentrations of experimental antibodies in Figure 8C are 10 nM, 3.3 nM, 1.11 nM, 0.222 nM, 0.044 nM, 0.004 nM, 0.0004 nM, 0.00004 nM and 0.000004 nM; final concentrations of experimental antibodies in Figure 8D are 10 nM, 3.3 nM, 1.11 nM, 0.222 nM, 0.044 nM, 0.004 nM, 0.0004 nM and 0.00004 nM). At the same time, set up blank control and isotype control group (hIgG1 and hIgG4 final concentrations of 10 nM). After gentle mixing, incubate on ice for 1 h. Add an appropriate amount of 1% PBSA, centrifuge at 350×g for 5 min, discard the supernatant, and wash twice with 1% PBSA. Add 300-fold diluted Alexa... Resuspend the cells in 647-labeled mouse anti-human IgG secondary antibody (Southern Biotech, catalog number: 9040-31) and incubate on ice in the dark for 0.5 h. Add an appropriate amount of 1% PBSA, centrifuge at 350×g for 5 min, discard the supernatant, and wash twice with 1% PBSA. Resuspend the cell pellet in 200 μL of 1% PBSA and then analyze the cells.

[0906] The experimental results are shown in Tables 25 and 26, and Figures 8C and 8D.

[0907] Table 25: FACS detection of the binding activity of Dupilumab, 14A10H49L25, TL4R04, L4RT04 to IL-4RA on the cell membrane surface of 293T-IL4Rα

[0908] Table 26: FACS detection of the binding activity of Dupilumab, L4RT04(LS) to IL-4RA on the cell membrane surface of 293T-IL4Rα

[0909] The results showed that 14A10H49L25, TL4R04, L4RT04, L4RT04(LS), and Dupilumab could all specifically bind to IL-4RA on the surface of the 293T-IL4Rα cell membrane.

[0910] Example 9: Reporter gene assay to detect the blocking of IL-33-ST2 binding by anti-IL-4RA-anti-ST2 bispecific antibody.

[0911] The experiment included a blank control (293T-ST2-Luc cell group), an NF-κB pathway positive control (cells + TNF-α group for pathway validation), a negative control (cells + IL-33 group), an isotype control, and an experimental antibody group, with two replicates for each sample. The final concentrations of the experimental antibodies were 300 nM, 100 nM, 33.33 nM, 11.11 nM, 3.7 nM, 1.23 nM, 0.123 nM, and 0.0123 nM (see Figure 9A) or 300 nM, 100 nM, 33.33 nM, 11.11 nM, 3.7 nM, 1.23 nM, 0.123 nM, 0.0123 nM, 0.00123 nM, and 0.000123 nM (see Figure 9B). The final concentration of the isotype control antibody hIgG1 was 300 nM. The final concentration of the positive reagent for verifying the NF-κB pathway, TNF-α (Peprotech, catalog number: 300-01A), was 10 ng / mL, and the final concentration of IL-33 was 1 pM.

[0912] 293T-ST2-Luc cells (constructed by Kangfang Biotechnology) were collected via routine digestion, centrifuged at 170xg for 5 min, and the supernatant was discarded. The cells were resuspended in DMEM complete medium (DMEM + 10% FBS) at a ratio of 1*10. 4 Cells were seeded per well in a 96-well black plate (Costar, model: 3916). Diluted antibody or TNF-α was added to the corresponding wells and incubated for 30 min. Diluted human IL-33 protein (IL33-Nhis, Akesobio) was added to the corresponding wells of the negative control, isotype control, and experimental antibody groups and incubated for 5 h. 50 μL of Luciferase Assay System (Promega, catalog number: E2620) was added to each well, and fluorescence values ​​were detected within 5 min using a multi-label microplate reader.

[0913] The experimental results are shown in Tables 27 and 28, and Figures 9A and 9B.

[0914] Table 28: L4RT04(LS) blocks the binding of IL-33 to ST2

[0915] The results showed that IL-33 binding to ST2 on the cell membrane surface of 293T-ST2-Luc cells can activate the reporter gene signaling pathway and upregulate luciferase expression. Under the same experimental conditions, 3H11H45L7(hG1WT), 3H11H46L7(hG1WT), TL4R01, L4RT03, TL4R04, L4RT04, L4RT04(LS) and the positive control antibody AMG282 can all effectively block the binding of IL-33 to ST2 and downregulate luciferase expression in a dose-dependent manner. Moreover, the blocking activity of 3H11H45L7(hG1WT), 3H11H46L7(hG1WT), and TL4R01 is stronger than that of AMG282.

[0916] Example 10: Reporter gene assay to detect the blocking of IL-4 / IL-13 binding to IL-4RA by anti-IL-4RA-anti-ST2 bispecific antibody.

[0917] 1. Reporter gene assay to detect the blocking of IL-13-IL-4RA binding by anti-IL-4RA bispecific antibody against ST2.

[0918] The following groups were set up: a blank control (293T-STAT6Luc cell group), a negative control (cell + IL-13 group), an isotype control, and an experimental antibody group. Each sample was replicated twice. The final concentrations of the experimental antibodies were 30 nM, 10 nM, 3.33 nM, 0.833 nM, 0.208 nM, 0.042 nM, 0.0083 nM, and 0.0017 nM (see Figure 10A) or 10 nM, 3.33 nM, 0.833 nM, 0.208 nM, 0.042 nM, 0.0083 nM, 0.00083 nM, and 0.000083 nM (see Figure 10B). The final concentration of the isotype control antibody hIgG4 was 30 nM (see Figure 10A) or 10 nM (see Figure 10B). The final concentration of human IL-13 protein was 100 pM.

[0919] 293T-STAT6Luc cells (constructed by Kangfang Biotechnology) were collected by routine digestion and resuspended in DMEM complete medium (DMEM + 10% FBS) at a ratio of 5*10. 4Cells / well were seeded into 96-well black plates (Costar, model: 3916). Dupilumab (Regeneron) antibodies (Regeneron, batch number: 7L615F) were diluted with DMEM complete medium and added to the corresponding wells, then incubated for 30 min. Human IL-13 protein (Sino Biological, Cat: 10369-HNAC-20) was diluted with DMEM complete medium and added to the corresponding wells of the negative control, isotype control, and experimental antibody groups, then incubated for 5 h. A Luciferase Assay System (Promega, catalog number: E2650) was added to each well, and fluorescence values ​​were detected within 5 min using a multi-label microplate reader.

[0920] The experimental results are shown in Tables 29 and 30, and Figures 10A and 10B.

[0921] Table 29: 14A10H49L25, L4RT03, L4RT04, TL4R04, and Dupilumab block the binding of IL-13 to IL-4RA.

[0922] Table 30: L4RT04 (LS) and Dupilumab (Regeneron) block the binding of IL-13 and IL-4RA

[0923] The results showed that the binding of IL-13 to IL-4RA on the cell membrane surface of 293T-STAT6Luc cells could activate the reporter gene signaling pathway and upregulate luciferase expression. Under the same experimental conditions, 14A10H49L25, L4RT03, L4RT04, TL4R04, L4RT04(LS) and the positive control antibody Dupilumab could all effectively block the binding of IL-13 to IL-4RA and downregulate luciferase expression in a dose-dependent manner.

[0924] 2. Reporter gene assay to detect the blocking of IL-4 binding to IL-4RA by anti-IL-4RA-anti-ST2 bispecific antibody.

[0925] The study included a blank control (293T-STAT6Luc cell group), a negative control (cell + IL-4 group), an isotype control, and an experimental antibody group, with two replicates for each sample. The final concentrations of the experimental antibodies were 30 nM, 10 nM, 3.33 nM, 0.833 nM, 0.208 nM, 0.042 nM, 0.0083 nM, and 0.0017 nM (see Figure 10C) or 30 nM, 10 nM, 3.33 nM, 0.833 nM, 0.208 nM, 0.042 nM, 0.0083 nM, and 0.00083 nM (see Figure 10D). The final concentration of the isotype control antibody hIgG4 was 30 nM, and the final concentration of human IL-4 protein was 100 pM.

[0926] 293T-STAT6Luc cells (constructed by Kangfang Biotechnology) were collected via routine digestion, centrifuged at 170xg for 5 min, and the supernatant was discarded. The cells were resuspended in DMEM complete medium (DMEM + 10% FBS), and the cell density was adjusted to 1*10⁻⁶ cells / mL. 4 Cells / well were seeded into 96-well black plates (Costar, model: 3916). Antibodies such as Dupilumab (Regeneron, batch number: 7L615F) were diluted with DMEM complete medium and added to the corresponding wells, then incubated for 30 min. Human IL-4 protein (Peprotech, catalog number: 200-04) was diluted with DMEM complete medium and added to the corresponding wells, then incubated for 5 hrs. 50 μL of Luciferase Assay System (Promega, catalog number: E2650) was added to each well, and fluorescence values ​​were detected within 5 min using a multi-label microplate reader.

[0927] The experimental results are shown in Tables 31 and 32, and Figures 10C and 10D.

[0928] Table 31: L4RT04, TL4R04, and Dupilumab block the binding of IL-4 to IL-4RA

[0929] Table 32: L4RT04(LS), 14A10H49L25, and Dupilumab block the binding of IL-4 to IL-4RA.

[0930] The results showed that the binding of IL-4 to IL-4RA on the cell membrane surface of 293T-STAT6Luc cells could activate the reporter gene signaling pathway and upregulate luciferase expression. Under the same experimental conditions, 14A10H49L25, L4RT04, TL4R04, L4RT04(LS) and the positive control antibody Dupilumab could all effectively block the binding of IL-4 to IL-4RA and downregulate luciferase expression in a dose-dependent manner.

[0931] Example 11: Anti-IL-4RA-anti-ST2 bispecific antibody inhibits IL-33-mediated IL-6 secretion

[0932] The study included a blank control (A549-ST2 cell group), a negative control (cells + IL-33 group), an isotype control, and an experimental antibody group, with two replicates for each sample. The final concentrations of the experimental antibodies were 1200 nM, 240 nM, 48 nM, 9.6 nM, 1.92 nM, 0.384 nM, 0.0384 nM, and 0.00384 nM. The final concentration of the isotype control antibody hIgG1 was 1200 nM, and the final concentration of human IL-33 protein was 2.5 ng / mL.

[0933] A549-ST2 cells (constructed by Kangfang Biotechnology) in the logarithmic growth phase were collected by routine digestion, resuspended in an appropriate amount of analytical medium (DMEM + 10% FBS), and then cultured at a ratio of 8*10. 4 Cells were seeded per well in 96-well plates and incubated overnight. Antibodies such as AMG282 were diluted with analytical medium and added to the corresponding wells, then incubated for 30 min in a cell culture incubator. Human IL-33 protein (IL33-Nhis, manufactured by Zhongshan Kangfang Biotechnology) was diluted with analytical medium and added to the corresponding wells of the negative control, isotype control, and experimental antibody groups, then incubated for 24 hrs in a cell culture incubator. The 96-well cell culture plates were centrifuged, and the cell supernatant was collected and detected using a human IL-6 kit (Dakeway, catalog number: 1110602).

[0934] The experimental results are shown in Figures 11A and 11B. The results showed that 2.5 ng / mL of IL-33 could induce A549-ST2 cells to secrete IL-6. Under the same experimental conditions, 3H11H46L7 (hG1WT), TL4R04, L4RT03, L4RT04, L4RT04 (LS), and AMG282 all significantly inhibited IL-6 secretion from IL-33-stimulated A549-ST2 cells in a concentration-dependent manner. Among these, at low antibody concentrations (0.0384 nM and below), TL4R04, L4RT03, L4RT04, and L4RT04 (LS) showed stronger inhibitory activity than the control antibody AMG282.

[0935] Example 12: Anti-IL-4RA-anti-ST2 bispecific antibody inhibits TF-1 cell proliferation

[0936] The experiment included a blank control (TF-1 cells only), a negative control (IL-4 group), an isotype control group (hIgG4), and an experimental antibody group, with two replicates for each treatment. The final concentrations of the experimental antibodies were 5 nM, 0.5 nM, and 0.05 nM (see Figure 12A) or 50 nM, 12.5 nM, 2.5 nM, 0.5 nM, 0.1 nM, 0.02 nM, 0.004 nM, and 0.0008 nM (see Figure 12B). The final concentration of the isotype control antibody hIgG4 was 5 nM or 50 nM, and the final concentration of IL-4 was 0.25 nM.

[0937] TF-1 cells in logarithmic growth phase were routinely collected (purchased from the American Type Culture Collection, catalog number: CRL-2003), resuspended in analytical medium (1640 + 10% FBS + 2.5 g / L glucose), and seeded into 96-well plates at 20,000 cells per well. Antibodies such as Dupilumab (Regeneron, batch number: 7L615F) were diluted with analytical medium and added to the corresponding wells, and pre-incubated at 37°C for 30 min. IL-4 (Peprotech, catalog number: 200-04) diluted with analytical medium was added, and the plates were incubated at 37°C in a 5% CO2 incubator. After 72 hours, OD values ​​were read at 450 nm and 600 nm using a microplate reader according to the instructions of the CCK-8 assay kit (Dojin, catalog number: CK04).

[0938] The experimental results are shown in Figures 12A and 12B. The results show that human IL-4 can promote the proliferation of TF-1 cells; compared with the isotype control antibody (hIgG4), 14A10H49L25, L4RT03, L4RT04, TL4R04, and L4RT04 (LS) can all specifically inhibit the proliferation of TF-1 cells induced by IL-4 in a dose-dependent manner.

[0939] Example 13: Anti-IL-4RA-anti-ST2 bispecific antibody inhibits the upregulation of CD23 expression in PBMC cells

[0940] The experiment included a blank control (group with only PBMC cells and secondary antibody), a negative control (group with IL-4 or IL-13 added to the blank control), an isotype control group (hIgG4), and an experimental antibody group. The final concentrations of the experimental antibodies were 25 nM, 2.5 nM, and 0.25 nM, respectively. The final concentration of the isotype control human IgG4 was 25 nM, the final concentration of human IL-4 was 100 pM, and the final concentration of human IL-13 was 100 ng / mL.

[0941] Using Ficoll-Paque TM Plus (GE, catalog number: 17-1440-02) isolated PBMCs from peripheral blood donated by healthy volunteers and cryopreserved them. PBMCs were resuscitated and cultured overnight at 37°C in a 5% FBS-containing RPMI 1640 complete medium. PBMCs were routinely collected and added at 500,000 cells per well to very low adsorption round-bottom 96-well plates (Corning, catalog number: 7007). According to the experimental design, experimental antibodies such as Dupilumab (Regeneron, batch number: 7L615F) or isotype control human IgG4 were added to the cells, and the plates were pre-incubated for 30 min. After 30 min, human IL-4 (peprotech, catalog number: 200-04) or human IL-13 (Sino Biological, catalog number: 10369-HNAC-20) was added, and the plates were incubated for 2 days. Two days later, PBMCs from each group were transferred to 96-well flow cytometry plates and washed once with 1% PBSA (i.e., PBS + 1% BSA). Mouse IgG Isotype Control (Thermofisher, catalog number: 10400C, diluted 20-fold with 1% PBSA) was added, and the plates were incubated on ice for 20 min. After washing once with an appropriate amount of 1% PBSA, FITC anti-human CD3 Antibody (Biolegend, catalog number: 344804, 1:100 dilution) and Brilliant Violet 421 were added. TM The secondary antibody mixture of anti-human CD19 Antibody (Biolegend, catalog number: 302234, 1:100 dilution) and PE anti-human CD23 Antibody (Biolegend, catalog number: 338508, 1:50 dilution) was incubated on ice in the dark for 30 min. After washing once with 1% PBSA, the sample was analyzed.

[0942] The test results are shown in Figures 13A and 13B.

[0943] The results showed that human IL-4 and human IL-13 could upregulate the expression level of CD23 on the surface of B cells in human PBMCs. Dupilumab, 14A10H49L25, L4RT04 and L4RT04 (LS) could specifically bind to human IL-4RA, thereby effectively blocking the upregulation of CD23 expression level on the surface of B cells by IL-4 and IL-13.

[0944] Example 14: Anti-IL-4RA-anti-ST2 bispecific antibody inhibits IL-4-stimulated CCL26 secretion

[0945] The experiment included a blank control (cells only), a negative control (cells + IL-4), an isotype control, and an experimental antibody group, with two replicates for each sample. The final concentrations of the experimental antibody were 100 nM, 10 nM, and 1 nM, the final concentration of the isotype control antibody hIgG1 was 100 nM, and the final concentration of human IL-4 was 5 ng / mL.

[0946] HUVEC cells (Allcells, catalog number: H-001F-C) were routinely collected and resuspended in endothelial cell complete medium (Allcells, catalog number: H-004). 20,000 cells / well were seeded into 96-well plates and incubated at 37°C with 5% CO2 for 2 hours until cell adhesion. Antibodies such as Dupilumab (Regeneron, batch number: 7L615F) were diluted with endothelial cell complete medium and added to the corresponding wells, then incubated for 30 minutes. Human IL-4 (Peprotech, catalog number: 200-04) diluted with endothelial cell complete medium was added, and the plates were incubated for 3 days. The cell supernatant was collected, and CCL26 levels were measured according to the Human CCL26 / Eotaxin-3 Quantikine ELISA Kit (R&D, catalog number: DCC260B) instructions.

[0947] The experimental results are shown in Figure 14. IL-4 can stimulate HUVEC cells to secrete CCL26. Under the same experimental conditions, L4RT04, L4RT04(LS), 14A10H49L25, and Dupilumab can all significantly inhibit IL-4-induced CCL26 secretion from HUVEC cells in a concentration-dependent manner. Furthermore, the inhibitory activity of L4RT04 and L4RT04(LS) is stronger than that of 14A10H49L25 and Dupilumab.

[0948] Example 15: Anti-IL-4RA-anti-ST2 bispecific antibody inhibits IL-6 secretion induced by IL-4 / IL-33 co-stimulation.

[0949] The experiment included a blank control (cells only), an IL-4 control (cells + IL-4), an IL-33 control (cells + IL-33), a negative control (cells + IL-4 + IL-33), an isotype control (a negative control supplemented with hIgG1DM), and an experimental antibody group. Each sample was replicated in two places. The final concentrations of the experimental antibodies were 0.08 nM, 0.4 nM, 2 nM, 20 nM, 200 nM, and 1000 nM. In the Dupilumab + AMG282 group, the final concentrations of each antibody were 0.08 nM, 0.4 nM, 2 nM, 20 nM, 200 nM, and 1000 nM. The final concentration of the isotype control antibody hIgG1DM was 1000 nM. The final concentration of human IL-4 or human IL-33 was 10 ng / mL.

[0950] HUVEC cells (Allcells, catalog number: H-001F-C) were collected via routine digestion and resuspended in endothelial cell complete medium (Allcells, catalog number: H-004). Cells were seeded at a rate of 10,000 cells / well in 96-well plates and incubated at 37°C with 5% CO2 for 2 hours until cell adhesion. Experimental antibodies such as Dupilumab and AMG282 were diluted with endothelial cell complete medium and added to the corresponding wells, then incubated for 30 minutes. Human IL-4 (Peprotech, catalog number: 200-04) and human IL-33 (manufactured by Zhongshan Kangfang Biotechnology) diluted with endothelial cell complete medium were added, and the cells were incubated for 2 days. Cell supernatant was collected, and IL-6 levels were determined according to the instructions of the human IL-6 kit (Dakewei, catalog number: 1110602).

[0951] The experimental results are shown in Figure 15. The results showed that IL-4 combined with IL-33 stimulated HUVEC cells to secrete large amounts of IL-6. Under the same experimental conditions, L4RT04(LS), L4RT04, Dupilumab, AMG282, 14A10H49L25, 3H11H46L7, and the combination of 14A10H49L25+3H11H46L7 and Dupilumab+AMG282 all inhibited the secretion of IL-6 by HUVEC cells induced by the co-stimulation of IL-4 and IL-33 in a concentration-dependent manner. L4RT04(LS) and L4RT04 showed superior inhibitory activity compared to Dupilumab, AMG282, and the monoclonal antibodies 14A10H49L25 and 3H11H46L7. Furthermore, under low concentration conditions, L4RT04(LS) and L4RT04 showed superior inhibitory activity compared to the combination of Dupilumab+AMG282.

[0952] Example 16: Anti-IL-4RA-anti-ST2 bispecific antibody inhibits IL-6 secretion induced by IL-13 / IL-33 co-stimulation.

[0953] The experiment included a blank control (cells only), an IL-13 control (cells + IL-13), an IL-33 control (cells + IL-33), a negative control (cells + IL-13 + IL-33), an isotype control (a negative control supplemented with hIgG1DM), and an experimental antibody group. Each sample was replicated. The final antibody concentrations were 0.08 nM, 0.4 nM, 2 nM, 20 nM, 200 nM, and 1000 nM. In the Dupilumab + AMG282 group, the final antibody concentrations were also 0.08 nM, 0.4 nM, 2 nM, 20 nM, 200 nM, and 1000 nM. The final concentration of the isotype control antibody hIgG1DM was 1000 nM. The final concentration of human IL-13 or human IL-33 was 10 ng / mL.

[0954] HUVEC cells (Allcells, catalog number: H-001F-C) were collected via routine digestion and resuspended in endothelial cell complete medium (Allcells, catalog number: H-004). Cells were seeded at a rate of 10,000 cells / well in 96-well plates and incubated at 37°C with 5% CO2 for 2 hours until cell adhesion. Experimental antibodies such as Dupilumab (Regeneron, batch number: 7L615F) and AMG282 were diluted with endothelial cell complete medium and added to the corresponding wells, then incubated for 30 minutes. Human IL-13 (sinobiological, catalog number: 10369-HNAC-20) and human IL-33 (manufactured by Zhongshan Kangfang Biotechnology) diluted with endothelial cell complete medium were added and incubated for 2 days. Cell supernatant was collected, and IL-6 levels were determined according to the instructions of the human IL-6 kit (Dakewei, catalog number: 1110602).

[0955] The experimental results are shown in Figure 16. The results show that IL-13 combined with IL-33 can stimulate HUVEC cells to secrete large amounts of IL-6. Under the same experimental conditions, L4RT04(LS), L4RT04, Dupilumab, AMG282, 14A10H49L25, 3H11H46L7, and the combination of 14A10H49L25+3H11H46L7 and Dupilumab+AMG282 can all inhibit the secretion of IL-6 by HUVEC cells induced by the co-stimulation of IL-13 and IL-33 in a concentration-dependent manner. L4RT04(LS) exhibits superior inhibitory activity compared to Dupilumab, AMG282, 14A10H49L25, and 3H11H46L7 monoclonal antibodies. Furthermore, at low concentrations, L4RT04(LS) and L4RT04 show superior inhibitory activity compared to the combination of 14A10H49L25 and 3H11H46L7, as well as the combination of Dupilumab and AMG282.

[0956] Example 17: Anti-IL-4RA-anti-ST2 bispecific antibody inhibits IL-6 secretion induced by IL-4 / IL-13 / IL-33 co-stimulation.

[0957] The experiment included a blank control (cells only), an IL-4 control (cells + IL-4), an IL-13 control (cells + IL-13), an IL-33 control (cells + IL-33), an IL-4 + IL-13 control (cells + IL-4 + IL-13), a negative control (cells + IL-4 + IL-13 + IL-33), an isotype control (a negative control plus hIgG4 or hIgG1™), and an experimental antibody group. Each sample was replicated twice. The final concentrations of the experimental antibodies were 0.08 nM, 0.4 nM, 2 nM, 20 nM, 200 nM, and 1000 nM. In the Dupilumab+AMG282 group, the final concentrations of each antibody were 0.08 nM, 0.4 nM, 2 nM, 20 nM, 200 nM, and 1000 nM. The final concentrations of the isotype control antibodies hIgG4 or hIgG1TM were 1000 nM, and the final concentrations of human IL-4, human IL-13, or human IL-33 were 1, 5, and 0.25 ng / mL, respectively.

[0958] HUVEC cells (Allcells, catalog number: H-001F-C) were routinely digested and collected. After resuspending the cells in endothelial cell complete medium (Allcells, catalog number: H-004), they were seeded at a rate of 10,000 cells / well in 96-well plates and cultured at 37°C with 5% CO2 for 2 hours until cell adhesion. Experimental antibodies such as Dupilumab (Regeneron, batch number: 7L615F) and AMG282 were diluted with endothelial cell complete medium and added to the corresponding wells, then incubated in a cell culture incubator for 30 minutes. Human IL-13 (sinobiological, catalog number: 10369-HNAC-20), human IL-33 (manufactured by Zhongshan Kangfang Biotechnology), and human IL-4 (peprotech, catalog number: 200-04) diluted with endothelial cell complete medium were added, and the plates were incubated for 24 hours. Collect cell supernatant and detect IL-6 content according to the instructions of the human IL-6 kit (Dakow, catalog number: 1110602).

[0959] The experimental results are shown in Figure 17. The results indicate that the combination of IL-4, IL-13, and IL-33 stimulates HUVEC cells to secrete large amounts of IL-6. Under the same experimental conditions, the combination of L4RT04(LS) and Dupilumab+AMG282 both inhibited the secretion of IL-6 by HUVEC cells induced by the co-stimulation of IL-4, IL-13, and IL-33. The inhibitory activity of L4RT04(LS) was superior to that of Dupilumab+AMG282.

[0960] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and all such changes are within the scope of protection of this invention. The full scope of this invention is given by the appended claims and any equivalents thereof.

Claims

1. An anti-IL-4RA antibody or its antigen-binding fragment, wherein the anti-IL-4RA antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising complementarity-determining regions HCDR1 to HCDR3, and the light chain variable region comprising complementarity-determining regions LCDR1 to LCDR3, wherein: HCDR1 contains the amino acid sequence shown in SEQ ID NO:5, HCDR2 contains the amino acid sequence shown in SEQ ID NO:96, and HCDR3 contains the amino acid sequence shown in SEQ ID NO:7; and LCDR1 contains the amino acid sequence shown in SEQ ID NO:8, LCDR2 contains the amino acid sequence shown in SEQ ID NO:9, and LCDR3 contains the amino acid sequence shown in SEQ ID NO:

10.

2. The anti-IL-4RA or its antigen-binding fragment according to claim 1, wherein, HCDR2 contains the amino acid sequence shown in SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:6; or The heavy chain variable region comprises an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:50 and SEQ ID NO:88, and the light chain variable region comprises an amino acid sequence selected from SEQ ID NO:4, SEQ ID NO:21, SEQ ID NO:52 and SEQ ID NO:

90.

3. The anti-IL-4RA or its antigen-binding fragment according to any one of claims 1 to 2, wherein: (1) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:2, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:4; (2) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:13, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:4; (3) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:15, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:4; (4) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:17, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:4; (5) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:19, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:4; (6) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:50, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:4; (7) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:88, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:4; (8) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:2, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:21; (9) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:13, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:21; (10) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:15, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:21; (11) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:17, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:21; (12) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:19, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:21; (13) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:50, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:21; (14) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:88, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:21; (15) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:2, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:52; (16) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:13, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:52; (17) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:15, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:52; (18) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:17, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:52; (19) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:19, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:52; (20) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:50, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:52; (21) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:88, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:52; (22) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:2, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:90; (23) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:13, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:90; (24) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:15, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:90; (25) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:17, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:90; (26) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:19, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:90; (27) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:50, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:90; or (28) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:88, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:

90.

4. The anti-IL-4RA antibody or its antigen-binding fragment according to any one of claims 1 to 3, comprising a heavy chain constant region and a light chain constant region, wherein, The heavy chain constant region is the human IgG heavy chain constant region, and the light chain constant region is the human Kappa chain constant region or the human Lambda chain constant region.

5. The anti-IL-4RA antibody or its antigen-binding fragment according to any one of claims 1 to 4, wherein, The heavy chain constant region is the human IgG1 heavy chain constant region, and the light chain constant region is the human Kappa chain constant region; Preferably, according to the EU numbering system, the human IgG1 heavy chain constant region contains the following mutations: L234A and L235A, L234A, L235A, and G237A, or L234A, L235A, G237A, M428L, and N434S; Preferably, the amino acid sequence of the constant region of the human IgG1 heavy chain is as shown in any of SEQ ID NOs:66-69; Preferably, the amino acid sequence of the constant region of the human Kappa chain is shown in SEQ ID NO:

72.

6. The anti-IL-4RA antibody or its antigen-binding fragment according to any one of claims 1 to 4, wherein, The heavy chain constant region is the human IgG4 heavy chain constant region, and the light chain constant region is the human Kappa chain constant region; Preferably, according to the EU numbering system, the human IgG4 heavy chain constant region contains the following mutations: M252Y, S254T, and T256E; Preferably, the amino acid sequence of the constant region of the human IgG4 heavy chain is as shown in SEQ ID NO:70 or SEQ ID NO:71; Preferably, the amino acid sequence of the constant region of the human Kappa chain is shown in SEQ ID NO:72; Optionally, the amino acid sequence of the heavy chain of the anti-IL-4RA antibody is shown in SEQ ID NO:11, and the amino acid sequence of the light chain is shown in SEQ ID NO:

12.

7. The anti-IL-4RA antibody or its antigen-binding fragment according to any one of claims 1 to 6, wherein, The antibody includes a non-CDR region, and the non-CDR region is derived from human antibodies, mouse antibodies, or rabbit antibodies.

8. The anti-IL-4RA antibody or its antigen-binding fragment according to any one of claims 1 to 7, wherein, The anti-IL-4RA antibody or its antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fv, single-chain antibody (scFv), scFv-Fc, humanized antibody or chimeric antibody.

9. The anti-IL-4RA antibody or its antigen-binding fragment according to any one of claims 1 to 8, wherein, The anti-IL-4RA antibody is selected from (1) to (4) below: (1) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:13, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:21; its heavy chain constant region is the human IgG4 heavy chain constant region, and its light chain constant region is the human Kappa chain constant region. (2) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:15, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:21; its heavy chain constant region is the human IgG4 heavy chain constant region, and its light chain constant region is the human Kappa chain constant region. (3) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:17, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:21; its heavy chain constant region is the human IgG4 heavy chain constant region, and its light chain constant region is the human Kappa chain constant region. (4) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:19, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:21; its heavy chain constant region is the human IgG4 heavy chain constant region, and its light chain constant region is the human Kappa chain constant region.

10. The anti-IL-4RA antibody or its antigen-binding fragment according to any one of claims 1 to 9, Its EC binding to the IL4-RA antigen on the surface of 293T-IL4Rα cells 50 Less than or equal to the ECG binding of the antibody Dupilumab to the IL4-RA antigen on the surface of 293T-IL4Rα cells 50 Preferably, the measurement is performed by fluorescence-activated cell sorting. Its activity in blocking the upregulation of CD23 expression levels on the surface of B cells by IL-4 is greater than or equal to that of the antibody Dupilumab; Its activity in blocking the upregulation of CD23 expression on the surface of B cells by IL-13 is greater than or equal to that of the antibody Dupilumab; Its activity in blocking the upregulation of CD23 expression levels on the surface of monocytes by IL-4 is greater than or equal to that of the antibody Dupilumab; and / or Its activity in blocking the upregulation of CD23 expression on the surface of monocytes by IL-13 is greater than or equal to that of the antibody Dupilumab.

11. An isolated nucleic acid molecule encoding the anti-IL-4RA antibody or its antigen-binding fragment as described in any one of claims 1 to 10.

12. A recombinant vector comprising the isolated nucleic acid molecule of claim 11.

13. A host cell comprising the isolated nucleic acid molecule of claim 11, or the recombinant vector of claim 12.

14. Antibody-drug conjugates, comprising an antibody or its antigen-binding fragment and a small molecule drug, wherein, The antibody or its antigen-binding fragment is the anti-IL-4RA antibody or its antigen-binding fragment as described in any one of claims 1 to 10; preferably, the small molecule drug is a small molecule cytotoxic drug; more preferably, the small molecule drug is an antitumor chemotherapy drug.

15. A pharmaceutical composition comprising an effective amount of the anti-IL-4RA antibody or its antigen-binding fragment as claimed in any one of claims 1 to 10 or the antibody-drug conjugate as claimed in claim 14; optionally, the pharmaceutical composition further comprising one or more pharmaceutically acceptable excipients; Preferably, the pharmaceutical composition is an injection, a nasal spray, or a nasal drop.

16. A packaged product comprising an anti-IL-4RA antibody or an antigen-binding fragment thereof as claimed in any one of claims 1 to 10, an antibody-drug conjugate as claimed in claim 14, or a pharmaceutical composition as claimed in claim 15, and a packaging container; Preferably, the packaging container is a syringe, injection pen, nasal spray, or dropper bottle; Preferably, the packaged product further includes a product instruction manual.

17. A combination product comprising a first product and a second product in individually packaged form, wherein, The first product comprises an anti-IL-4RA antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 10, an antibody-drug conjugate as described in claim 14, or a pharmaceutical composition as described in claim 15; The second product contains one or more glucocorticoids; Preferably, the glucocorticoid is selected from one or more of prednisone, methylprednisolone, betamethasone, beclomethasone dipropionate, prednisolone, hydrocortisone, dexamethasone, and triamcinolone; Preferably, the first product and the second product further comprise one or more pharmaceutically acceptable excipients. Preferably, the combined product also includes a product instruction manual.

18. Use of the anti-IL-4RA antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, the antibody-drug conjugate according to claim 14, or the pharmaceutical composition according to claim 15 in the preparation of a medicament for treating or preventing a disease, wherein the disease is one or more selected from allergic diseases, autoimmune diseases, inflammatory diseases, and tumors; Preferably, the disease is mediated by IL-4 and / or IL-13; Preferably, the disease is selected from one or more of the following: asthma, allergic rhinitis, sinusitis, nodular prurigo, chronic urticaria, nasal polyps, chronic obstructive pulmonary disease, pulmonary fibrosis, food allergy, rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, ankylosing spondylitis, eosinophilic esophagitis, pulmonary fibrosis, systemic lupus erythematosus, cardiovascular disease, and systemic sclerosis. Preferably, the asthma is allergic asthma; Preferably, the allergic rhinitis is seasonal allergic rhinitis; Preferably, the sinusitis is chronic sinusitis; Preferably, the chronic obstructive pulmonary disease is moderate to severe chronic obstructive pulmonary disease; Preferably, the atopic dermatitis is moderate to severe atopic dermatitis; Preferably, the single dose, calculated based on the anti-IL-4RA antibody or its antigen-binding fragment, is 0.1-100 mg per kilogram of body weight, more preferably 5-50 mg or 5-15 mg per kilogram of body weight; Preferably, each patient is given a single dose of 0-1000 mg, 0-900 mg, 0-800 mg, 0-700 mg, 0-600 mg, 0-500 mg, 0-400 mg, 0-300 mg, 0-200 mg, 0-100 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or 1000 mg, calculated according to the anti-IL-4RA antibody or its antigen-binding fragment. Preferably, the medication is administered once every 3 days, every 4 days, every 5 days, every 6 days, every 10 days, every week, every 2 weeks, or every 3 weeks; Preferably, the administration method is intravenous infusion, intravenous injection, intradermal injection, subcutaneous injection, intramuscular injection, or nasal administration.

19. The anti-IL-4RA antibody or its antigen-binding fragment according to any one of claims 1 to 10, the antibody-drug conjugate according to claim 14, or the pharmaceutical composition according to claim 15, for the treatment or prevention of a disease, wherein the disease is one or more selected from allergic diseases, autoimmune diseases, inflammatory diseases, and tumors; Preferably, the disease is mediated by IL-4 and / or IL-13; Preferably, the disease is selected from one or more of the following: asthma, allergic rhinitis, sinusitis, nodular prurigo, chronic urticaria, nasal polyps, chronic obstructive pulmonary disease, pulmonary fibrosis, food allergy, rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, ankylosing spondylitis, eosinophilic esophagitis, pulmonary fibrosis, systemic lupus erythematosus, cardiovascular disease, and systemic sclerosis. Preferably, the asthma is allergic asthma; Preferably, the allergic rhinitis is seasonal allergic rhinitis; Preferably, the sinusitis is chronic sinusitis; Preferably, the chronic obstructive pulmonary disease is moderate to severe chronic obstructive pulmonary disease; Preferably, the atopic dermatitis is moderate to severe atopic dermatitis; Preferably, the single dose, calculated based on the anti-IL-4RA antibody or its antigen-binding fragment, is 0.1-100 mg per kilogram of body weight, more preferably 5-50 mg or 5-15 mg per kilogram of body weight; Preferably, each patient is given a single dose of 0-1000 mg, 0-900 mg, 0-800 mg, 0-700 mg, 0-600 mg, 0-500 mg, 0-400 mg, 0-300 mg, 0-200 mg, 0-100 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or 1000 mg, calculated according to the anti-IL-4RA antibody or its antigen-binding fragment. Preferably, the medication is administered once every 3 days, every 4 days, every 5 days, every 6 days, every 10 days, every week, every 2 weeks, or every 3 weeks; Preferably, the administration method is intravenous infusion, intravenous injection, intradermal injection, subcutaneous injection, intramuscular injection, or nasal administration.

20. A method of treating or preventing a disease, comprising administering to a subject in need an effective amount of the anti-IL-4RA antibody or its antigen-binding fragment as described in any one of claims 1 to 10, the antibody-drug conjugate as described in claim 14, or the pharmaceutical composition as described in claim 15, wherein the disease is one or more selected from allergic diseases, autoimmune diseases, inflammatory diseases, and tumors; Preferably, the disease is mediated by IL-4 and / or IL-13; Preferably, the disease is selected from one or more of the following: asthma, allergic rhinitis, sinusitis, nodular prurigo, chronic urticaria, nasal polyps, chronic obstructive pulmonary disease, pulmonary fibrosis, food allergy, rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, ankylosing spondylitis, eosinophilic esophagitis, pulmonary fibrosis, systemic lupus erythematosus, cardiovascular disease, and systemic sclerosis. Preferably, the asthma is allergic asthma; Preferably, the allergic rhinitis is seasonal allergic rhinitis; Preferably, the sinusitis is chronic sinusitis; Preferably, the chronic obstructive pulmonary disease is moderate to severe chronic obstructive pulmonary disease; Preferably, the atopic dermatitis is moderate to severe atopic dermatitis; Preferably, the single dose, calculated based on the anti-IL-4RA antibody or its antigen-binding fragment, is 0.1-100 mg per kilogram of body weight, more preferably 5-50 mg or 5-15 mg per kilogram of body weight; Preferably, each patient is given a single dose of 0-1000 mg, 0-900 mg, 0-800 mg, 0-700 mg, 0-600 mg, 0-500 mg, 0-400 mg, 0-300 mg, 0-200 mg, 0-100 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or 1000 mg, calculated according to the anti-IL-4RA antibody or its antigen-binding fragment. Preferably, the medication is administered once every 3 days, every 4 days, every 5 days, every 6 days, every 10 days, every week, every 2 weeks, or every 3 weeks; Preferably, the administration method is intravenous infusion, intravenous injection, intradermal injection, subcutaneous injection, intramuscular injection, or nasal administration.

21. Bispecific antibodies, including: Targeting the first protein functional region of IL-4RA, and Targeting a second protein functional region that is different from the target of IL-4RA; in: The first protein functional region comprises the anti-IL-4RA antibody or its antigen-binding fragment as described in any one of claims 1 to 10.

22. The bispecific antibody according to claim 21, wherein, The target that differs from IL-4RA is ST2.

23. The bispecific antibody according to any one of claims 21 to 22, wherein, The second protein functional region contains an anti-ST2 antibody or its antigen-binding fragment.

24. The bispecific antibody according to claim 23, wherein, The anti-ST2 antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region includes complementarity-determining regions HCDR1 to HCDR3, and the light chain variable region includes complementarity-determining regions LCDR1 to LCDR3, wherein: HCDR1 contains the amino acid sequence shown in SEQ ID NO:29, HCDR2 contains the amino acid sequence shown in SEQ ID NO:30, and HCDR3 contains the amino acid sequence shown in SEQ ID NO:31; and LCDR1 contains the amino acid sequence shown in SEQ ID NO:32, LCDR2 contains the amino acid sequence shown in SEQ ID NO:33, and LCDR3 contains the amino acid sequence shown in SEQ ID NO:

34.

25. The bispecific antibody according to any one of claims 23 to 24, wherein, The heavy chain variable region of the anti-ST2 antibody comprises an amino acid sequence selected from SEQ ID NO:26, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:49, and SEQ ID NO:92; and The light chain variable region of the anti-ST2 antibody comprises an amino acid sequence selected from SEQ ID NO:28, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:51 and SEQ ID NO:

93.

26. The bispecific antibody according to any one of claims 23 to 25, wherein, The anti-ST2 antibody mentioned above: (1) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:26, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:28; (2) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:37, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:28; (3) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:39, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:28; (4) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:41, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:28; (5) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:43, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:28; (6) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:49, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:28; (7) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:92, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:28; (8) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:26, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:45; (9) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:37, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:45; (10) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:39, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:45; (11) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:41, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:45; (12) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:43, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:45; (13) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:49, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:45; (14) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:92, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:45; (15) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:26, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:47; (16) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:37, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:47; (17) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:39, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:47; (18) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:41, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:47; (19) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:43, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:47; (20) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:49, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:47; (21) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:92, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:47; (22) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:26, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:51; (23) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:37, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:51; (24) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:39, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:51; (25) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:41, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:51; (26) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:43, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:51; (27) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:49, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:51; (28) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:92, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:51; (29) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:26, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:93; (30) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:37, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:93; (31) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:39, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:93; (32) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:41, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:93; (33) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:43, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:93; (34) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:49, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:93; or (35) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:92, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:

93.

27. The bispecific antibody according to any one of claims 21 to 26, wherein, The first protein functional region is directly connected to the second protein functional region or connected through a connector; Preferably, the linker is selected from the amino acid sequences of SEQ ID NOs:53, 54 and 76-78; Preferably, the first protein functional region and the second protein functional region are independently one, two, or more.

28. The bispecific antibody according to any one of claims 21 to 27, wherein, The first protein functional region is an immunoglobulin against IL-4RA, and the second protein functional region is a single-chain antibody against ST2; Preferably, the anti-ST2 single-chain antibody comprises two molecules, which are respectively linked to the N-terminus or C-terminus of the two heavy chains of the anti-IL-4RA immunoglobulin. Preferably, the bispecific antibody is a bispecific antibody in the form of IgG-scFv; Preferably, the bispecific antibody is a tetravalent bispecific antibody in the form of IgG-scFv.

29. The bispecific antibody according to any one of claims 21 to 28, wherein, The bispecific antibody includes: Targeting the first protein functional region of IL-4RA, and Targeting the second protein functional region of ST2; The first protein has one functional region, and the second protein has two functional regions; Wherein, the first protein functional region is an immunoglobulin against IL-4RA, and the second protein functional region is a single-chain antibody against ST2; The amino acid sequence of the heavy chain variable region of the anti-IL-4RA immunoglobulin is shown in SEQ ID NO:17, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:

21. The amino acid sequence of the heavy chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:49, and the amino acid sequence of the light chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:51; or, the amino acid sequence of the heavy chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:43, and the amino acid sequence of the light chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:

45. The anti-ST2 single-chain antibody is respectively linked to the C-terminus of the two heavy chains of the anti-IL-4RA immunoglobulin; The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the anti-ST2 single-chain antibody and the light chain variable region of the anti-ST2 single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different; Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO:53 and SEQ ID NO:

54.

30. The bispecific antibody according to any one of claims 21 to 29, wherein, The bispecific antibody includes: Targeting the first protein functional region of IL-4RA, and Targeting the second protein functional region of ST2; The first protein has one functional region, and the second protein has two functional regions; Wherein, the first protein functional region is an immunoglobulin against IL-4RA, and the second protein functional region is a single-chain antibody against ST2; The amino acid sequence of the heavy chain of the anti-IL-4RA immunoglobulin is shown in SEQ ID NO:85 or SEQ ID NO:86, and the amino acid sequence of its light chain is shown in SEQ ID NO:

64. The amino acid sequence of the heavy chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:49, and the amino acid sequence of the light chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:51; or, the amino acid sequence of the heavy chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:43, and the amino acid sequence of the light chain variable region of the anti-ST2 single-chain antibody is shown in SEQ ID NO:

45. The anti-ST2 single-chain antibody is respectively linked to the C-terminus of the two heavy chains of the anti-IL-4RA immunoglobulin; The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the anti-ST2 single-chain antibody and the light chain variable region of the anti-ST2 single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different; Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO:53 and SEQ ID NO:

54.

31. The bispecific antibody according to any one of claims 21 to 27, wherein, The first protein functional region is a single-chain antibody against IL-4RA, and the second protein functional region is an immunoglobulin against ST2; Preferably, the anti-IL-4RA single-chain antibody comprises two molecules, which are respectively linked to the N-terminus or C-terminus of the two heavy chains of the anti-ST2 immunoglobulin. Preferably, the bispecific antibody is a bispecific antibody in the form of IgG-scFv; Preferably, the bispecific antibody is a tetravalent bispecific antibody in the form of IgG-scFv; Preferably, the heavy chain constant region of the anti-ST2 immunoglobulin is the human IgG heavy chain constant region, and the light chain constant region is the human Kappa chain constant region or the human Lambda chain constant region.

32. The bispecific antibody according to claim 31, wherein, The heavy chain constant region of anti-ST2 immunoglobulin is the human IgG1 heavy chain constant region, and the light chain constant region is the human Kappa chain constant region. Preferably, according to the EU numbering system, the human IgG1 heavy chain constant region contains the following mutations: L234A and L235A, L234A, L235A, and G237A, or L234A, L235A, G237A, M428L, and N434S; Preferably, the amino acid sequence of the constant region of the human IgG1 heavy chain is as shown in any of SEQ ID NOs:66-69; Preferably, the amino acid sequence of the constant region of the human Kappa chain is shown in SEQ ID NO:

72.

33. The bispecific antibody according to claim 31, wherein, The heavy chain constant region of anti-ST2 immunoglobulin is the human IgG4 heavy chain constant region, and the light chain constant region is the human Kappa chain constant region. Preferably, according to the EU numbering system, the human IgG4 heavy chain constant region contains the following mutations: M252Y, S254T, and T256E; Preferably, the amino acid sequence of the constant region of the human IgG4 heavy chain is as shown in SEQ ID NO:70 or SEQ ID NO:71; Preferably, the amino acid sequence of the constant region of the human Kappa chain is shown in SEQ ID NO:

72.

34. The bispecific antibody according to any one of claims 21 to 27 and 31 to 33, wherein, The bispecific antibody includes: Targeting the first protein functional region of IL-4RA, and Targeting the second protein functional region of ST2; The first protein has two functional regions, and the second protein has one functional region; Wherein, the first protein functional region is a single-chain antibody against IL-4RA, and the second protein functional region is an immunoglobulin against ST2; The amino acid sequence of the heavy chain variable region of the anti-IL-4RA single-chain antibody is shown in SEQ ID NO:17, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:21; or, the amino acid sequence of the heavy chain variable region of the anti-IL-4RA single-chain antibody is shown in SEQ ID NO:50, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:

52. The amino acid sequence of the heavy chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:43, and the amino acid sequence of the light chain variable region of the anti-ST2 immunoglobulin is shown in SEQ ID NO:

45. The anti-IL-4RA single-chain antibody is respectively linked to the C-terminus of the two heavy chains of the anti-ST2 immunoglobulin; The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the anti-IL-4RA single-chain antibody and the light chain variable region of the anti-IL-4RA single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different; Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO:53 and SEQ ID NO:

54.

35. The bispecific antibody according to any one of claims 21 to 27 and 31 to 34, wherein, The bispecific antibody includes: Targeting the first protein functional region of IL-4RA, and Targeting the second protein functional region of ST2; The first protein has two functional regions, and the second protein has one functional region; Wherein, the first protein functional region is a single-chain antibody against IL-4RA, and the second protein functional region is an immunoglobulin against ST2; The amino acid sequence of the heavy chain variable region of the anti-IL-4RA single-chain antibody is shown in SEQ ID NO:17, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:21; or, the amino acid sequence of the heavy chain variable region of the anti-IL-4RA single-chain antibody is shown in SEQ ID NO:50, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:

52. The amino acid sequence of the heavy chain of the anti-ST2 immunoglobulin is shown in SEQ ID NO:87, and the amino acid sequence of the light chain of the anti-ST2 immunoglobulin is shown in SEQ ID NO:

65. The anti-IL-4RA single-chain antibody is respectively linked to the C-terminus of the two heavy chains of the anti-ST2 immunoglobulin; The first protein functional region and the second protein functional region are connected by a first linker; and the heavy chain variable region of the anti-IL-4RA single-chain antibody and the light chain variable region of the anti-IL-4RA single-chain antibody are connected by a second linker; the first linker and the second linker may be the same or different; Preferably, the amino acid sequences of the first linker and the second linker are independently selected from SEQ ID NO:53 and SEQ ID NO:

54.

36. The bispecific antibody according to any one of claims 21 to 35, It is a tetramer formed by two identical first peptide chains and two identical second peptide chains, wherein, The first peptide chain comprises an amino acid sequence selected from SEQ ID NOs:55-63, and the second peptide chain comprises an amino acid sequence shown in SEQ ID NO:64 or SEQ ID NO:65; Preferably, the first peptide chain and the second peptide chain, and / or the two first peptide chains are independently connected by one or more (e.g., 2, 3, 4 or 5) disulfide bonds.

37. An isolated nucleic acid molecule encoding the bispecific antibody as described in any one of claims 21 to 36.

38. A recombinant vector comprising the isolated nucleic acid molecule of claim 37.

39. A host cell comprising the isolated nucleic acid molecule of claim 37, or the recombinant vector of claim 38.

40. Antibody-drug conjugates, including bispecific antibodies and small molecule drugs, wherein, The bispecific antibody is the bispecific antibody according to any one of claims 21 to 36; preferably, the small molecule drug is a small molecule cytotoxic drug; more preferably, the small molecule drug is a tumor chemotherapy drug.

41. A pharmaceutical composition comprising an effective amount of the bispecific antibody of any one of claims 21 to 36 or the antibody-drug conjugate of claim 40; optionally, the pharmaceutical composition further comprising one or more pharmaceutically acceptable excipients; Preferably, the pharmaceutical composition is an injection, a nasal spray, or a nasal drop.

42. A packaged product comprising a bispecific antibody as claimed in any one of claims 21 to 36, an antibody-drug conjugate as claimed in claim 40, or a pharmaceutical composition as claimed in claim 41, and a packaging container; Preferably, the packaging container is a syringe, injection pen, nasal spray, or dropper bottle; Preferably, the packaged product further includes a product instruction manual.

43. A combination product comprising a first product and a second product in individually packaged form, wherein, The first product comprises a bispecific antibody as described in any one of claims 21 to 36, an antibody-drug conjugate as described in claim 40, or a pharmaceutical composition as described in claim 41; The second product contains one or more glucocorticoids; Preferably, the glucocorticoid is selected from one or more of prednisone, methylprednisolone, betamethasone, beclomethasone dipropionate, prednisolone, hydrocortisone, dexamethasone, and triamcinolone; Preferably, the first product and the second product further comprise one or more pharmaceutically acceptable excipients. Preferably, the combined product also includes a product instruction manual.

44. Use of the bispecific antibody of any one of claims 21 to 36, the antibody-drug conjugate of claim 40, or the pharmaceutical composition of claim 41 in the preparation of a medicament for treating or preventing a disease, wherein the disease is one or more selected from allergic diseases, autoimmune diseases, inflammatory diseases, and tumors; Preferably, the disease is selected from one or more of the following: asthma, allergic rhinitis, sinusitis, nodular prurigo, chronic urticaria, nasal polyps, chronic obstructive pulmonary disease, pulmonary fibrosis, food allergy, rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, ankylosing spondylitis, eosinophilic esophagitis, pulmonary fibrosis, systemic lupus erythematosus, cardiovascular disease, and systemic sclerosis. Preferably, the asthma is allergic asthma; Preferably, the allergic rhinitis is seasonal allergic rhinitis; Preferably, the sinusitis is chronic sinusitis; Preferably, the chronic obstructive pulmonary disease is moderate to severe chronic obstructive pulmonary disease; Preferably, the atopic dermatitis is moderate to severe atopic dermatitis; Preferably, the single-dose dose based on the bispecific antibody is 0.1-100 mg per kilogram of body weight, more preferably 5-50 mg or 5-15 mg per kilogram of body weight; Preferably, each patient is given a single dose of 0-1000 mg, 0-900 mg, 0-800 mg, 0-700 mg, 0-600 mg, 0-500 mg, 0-400 mg, 0-300 mg, 0-200 mg, 0-100 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or 1000 mg, calculated according to the bispecific antibody. Preferably, the medication is administered once every 3 days, every 4 days, every 5 days, every 6 days, every 10 days, every week, every 2 weeks, or every 3 weeks; Preferably, the administration method is intravenous infusion, intravenous injection, intradermal injection, subcutaneous injection, intramuscular injection, or nasal administration.

45. The bispecific antibody according to any one of claims 21 to 36, the antibody-drug conjugate according to claim 40, or the pharmaceutical composition according to claim 41, for the treatment or prevention of a disease, wherein the disease is one or more selected from allergic diseases, autoimmune diseases, inflammatory diseases, and tumors; Preferably, the disease is selected from one or more of the following: asthma, allergic rhinitis, sinusitis, nodular prurigo, chronic urticaria, nasal polyps, chronic obstructive pulmonary disease, pulmonary fibrosis, food allergy, rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, ankylosing spondylitis, eosinophilic esophagitis, pulmonary fibrosis, systemic lupus erythematosus, cardiovascular disease, and systemic sclerosis. Preferably, the asthma is allergic asthma; Preferably, the allergic rhinitis is seasonal allergic rhinitis; Preferably, the sinusitis is chronic sinusitis; Preferably, the chronic obstructive pulmonary disease is moderate to severe chronic obstructive pulmonary disease; Preferably, the atopic dermatitis is moderate to severe atopic dermatitis; Preferably, the single-dose dose based on the bispecific antibody is 0.1-100 mg per kilogram of body weight, more preferably 5-50 mg or 5-15 mg per kilogram of body weight; Preferably, each patient is given a single dose of 0-1000 mg, 0-900 mg, 0-800 mg, 0-700 mg, 0-600 mg, 0-500 mg, 0-400 mg, 0-300 mg, 0-200 mg, 0-100 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or 1000 mg, calculated according to the bispecific antibody. Preferably, the medication is administered once every 3 days, every 4 days, every 5 days, every 6 days, every 10 days, every week, every 2 weeks, or every 3 weeks; Preferably, the administration method is intravenous infusion, intravenous injection, intradermal injection, subcutaneous injection, intramuscular injection, or nasal administration.

46. ​​A method of treating or preventing a disease, comprising the step of administering to a subject in need an effective amount of the bispecific antibody of any one of claims 21 to 36, the antibody-drug conjugate of claim 40, or the pharmaceutical composition of claim 41, wherein the disease is one or more selected from allergic diseases, autoimmune diseases, inflammatory diseases, and tumors; Preferably, the disease is selected from one or more of the following: asthma, allergic rhinitis, sinusitis, nodular prurigo, chronic urticaria, nasal polyps, chronic obstructive pulmonary disease, pulmonary fibrosis, food allergy, rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, ankylosing spondylitis, eosinophilic esophagitis, pulmonary fibrosis, systemic lupus erythematosus, cardiovascular disease, and systemic sclerosis. Preferably, the asthma is allergic asthma; Preferably, the allergic rhinitis is seasonal allergic rhinitis; Preferably, the sinusitis is chronic sinusitis; Preferably, the chronic obstructive pulmonary disease is moderate to severe chronic obstructive pulmonary disease; Preferably, the atopic dermatitis is moderate to severe atopic dermatitis; Preferably, the single-dose dose based on the bispecific antibody is 0.1-100 mg per kilogram of body weight, more preferably 5-50 mg or 5-15 mg per kilogram of body weight; Preferably, each patient is given a single dose of 0-1000 mg, 0-900 mg, 0-800 mg, 0-700 mg, 0-600 mg, 0-500 mg, 0-400 mg, 0-300 mg, 0-200 mg, 0-100 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or 1000 mg, calculated according to the bispecific antibody. Preferably, the medication is administered once every 3 days, every 4 days, every 5 days, every 6 days, every 10 days, every week, every 2 weeks, or every 3 weeks; Preferably, the administration method is intravenous infusion, intravenous injection, intradermal injection, subcutaneous injection, intramuscular injection, or nasal administration.

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