Anti-st2 antibody and pharmaceutical composition

Abstract

Provided are an anti-ST2 antibody and a pharmaceutical composition. Specifically provided is an anti-ST2 antibody or an antigen-binding fragment thereof. The anti-ST2 antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1 to HCDR3, and the light chain variable region comprises LCDR1 to LCDR3, wherein the amino acid sequence of HCDR1 is as shown in SEQ ID NO: 1, the amino acid sequence of HCDR2 is as shown in SEQ ID NO: 2, and the amino acid sequence of HCDR3 is as shown in SEQ ID NO: 3; and the amino acid sequence of LCDR1 is as shown in SEQ ID NO: 4, the amino acid sequence of LCDR2 is as shown in SEQ ID NO: 5, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO: 6. The antibody can bind to human ST2, block the binding of human IL-33 to ST2, and inhibit the activation of downstream signaling pathways of a human IL-33 / ST2 pathway.

Description

Anti-ST2 antibody and pharmaceutical composition

[0001] Cross-references to related applications

[0002] This application is based on and claims priority to CN application number 202510072939.7, 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-ST2 antibody and a pharmaceutical composition. Specifically, the pharmaceutical composition is a pharmaceutical composition comprising the anti-ST2 antibody. This invention also relates to the use of the anti-ST2 antibody or the pharmaceutical composition, such as for pharmaceutical applications. Background Technology

[0004] 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.

[0005] 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).

[0006] 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).

[0007] 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).

[0008] Asthma is currently considered a Th2 cell-mediated disease, with T lymphocytes, eosinophils, and mast cells playing important roles in its pathogenesis. Changes in the airway of asthma patients, such as hyperreactivity, inflammation, tissue remodeling, and damage to the airway epithelial cell layer, can all lead to the release of damage-associated pattern molecules (DAMPs), including IL-33. Prefontaine et al. found that IL-33 protein and mRNA were increased in airway smooth muscle cells of subjects with severe respiratory asthma (Préfontaine 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). Studies using ST2 or IL-33 deficient mice to investigate the role of IL-33 and ST2 in allergic airway inflammation revealed that 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 a crucial role in the development and progression 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).

[0009] There is still a need to develop new antibody drugs with high affinity for ST2 for the treatment of autoimmune diseases, so that they can have better therapeutic effects and lower toxic side effects. Summary of the Invention

[0010] Through in-depth research and creative labor, the inventors have obtained a specific antibody (named 3H11) that specifically binds to human ST2, and this antibody can effectively block the binding of human ST2 and IL-33. Furthermore, the inventors have creatively prepared humanized antibodies against human ST2 (named 3H11H4L7, 3H11H4L8, 3H11H40L8, 3H11H45L7, 3H11H45L8, 3H11H46L7, 3H11H46L8, etc.). The antibodies of this invention can effectively bind to human ST2, block the binding of human IL-33 to ST2, and inhibit the activation of downstream signaling pathways in the human IL-33 / ST2 pathway; they have the potential to be used in the prevention and treatment of diseases such as chronic obstructive pulmonary disease (COPD), asthma, rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, allergic rhinitis, sinusitis, nodular prurigo, chronic urticaria, nasal polyps, and systemic sclerosis. Thus, the following invention is provided:

[0011] One aspect of the present invention relates to an anti-ST2 antibody or an antigen-binding fragment thereof, said anti-ST2 antibody comprising a heavy chain variable region and a light chain variable region, said heavy chain variable region comprising HCDR1 to HCDR3, and said light chain variable region comprising LCDR1 to LCDR3, wherein:

[0012] HCDR1 contains the amino acid sequence shown in SEQ ID NO:1, HCDR2 contains the amino acid sequence shown in SEQ ID NO:2, and HCDR3 contains the amino acid sequence shown in SEQ ID NO:3.

[0013] LCDR1 contains the amino acid sequence shown in SEQ ID NO:4, LCDR2 contains the amino acid sequence shown in SEQ ID NO:5, and LCDR3 contains the amino acid sequence shown in SEQ ID NO:6.

[0014] In some embodiments of the present invention, the anti-ST2 antibody or its antigen-binding fragment, wherein,

[0015] The heavy chain variable region of the anti-ST2 antibody comprises an amino acid sequence selected from SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:34; and

[0016] The light chain variable region of the anti-ST2 antibody comprises an amino acid sequence selected from SEQ ID NO:10, SEQ ID NO:19, SEQ ID NO:21 and SEQ ID NO:35.

[0017] In some embodiments of the present invention, the anti-ST2 or its antigen-binding fragment, wherein,

[0018] (1) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:8, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:10;

[0019] (2) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:8, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:19;

[0020] (3) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:8, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:21;

[0021] (4) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:8, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:35;

[0022] (5) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:10;

[0023] (6) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:19;

[0024] (7) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:21;

[0025] (8) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:35;

[0026] (9) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:10;

[0027] (10) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:19;

[0028] (11) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:21;

[0029] (12) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:35;

[0030] (13) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:10;

[0031] (14) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:19;

[0032] (15) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:21;

[0033] (16) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:35;

[0034] (17) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:17, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:10;

[0035] (18) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:17, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:19;

[0036] (19) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:17, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:21;

[0037] (20) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:17, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:35;

[0038] (21) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:34, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:10;

[0039] (22) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:34, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:19;

[0040] (23) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:34, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:21; or

[0041] (24) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:34, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:35.

[0042] In some embodiments of the present invention, the anti-ST2 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.

[0043] In some embodiments of the present invention, the anti-ST2 antibody or its antigen-binding fragment, wherein,

[0044] The anti-ST2 antibody is an IgG1, IgG2, IgG3, or IgG4 subtype.

[0045] In some embodiments of the present invention, the anti-ST2 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;

[0046] Preferably, according to the EU numbering system, the human IgG1 heavy chain constant region contains the following mutations:

[0047] L234A and L235A,

[0048] L234A, L235A, and G237A, or

[0049] L234A, L235A, G237A, M428L, and N434S;

[0050] Preferably, the amino acid sequence of the constant region of the human IgG1 heavy chain is as shown in SEQ ID NO:31 or SEQ ID NO:32;

[0051] Preferably, the amino acid sequence of the constant region of the human Kappa chain is shown in SEQ ID NO:30.

[0052] In some embodiments of the present invention, the anti-ST2 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;

[0053] Preferably, according to the EU numbering system, the human IgG4 heavy chain constant region contains the following mutations: M252Y, S254T, and T256E;

[0054] Preferably, the amino acid sequence of the constant region of the human IgG4 heavy chain is as shown in SEQ ID NO:33 or SEQ ID NO:29;

[0055] Preferably, the amino acid sequence of the constant region of the human Kappa chain is shown in SEQ ID NO:30.

[0056] In some embodiments of the present invention, the anti-ST2 antibody or its antigen-binding fragment, wherein,

[0057] The antibody includes a non-CDR region, and the non-CDR region is derived from human antibodies, mouse antibodies, or rabbit antibodies.

[0058] In some embodiments of the present invention, the anti-ST2 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.

[0059] In some embodiments of the present invention, the anti-ST2 antibody or its antigen-binding fragment is selected from (1) to (7) below:

[0060] (1) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:11, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:19; 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.

[0061] (2) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:11, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:21; 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.

[0062] (3) 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; 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.

[0063] (4) 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:19; 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.

[0064] (5) 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; 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.

[0065] (6) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:17, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:19; 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; or

[0066] (7) 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; 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.

[0067] In some embodiments of the present invention, the anti-ST2 antibody or its antigen-binding fragment is selected from (1) to (7) below:

[0068] (1) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:11, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:19; 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.

[0069] (2) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:11, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:21; 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.

[0070] (3) 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; 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.

[0071] (4) 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:19; 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.

[0072] (5) 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; 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.

[0073] (6) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:17, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:19; 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; or

[0074] (7) 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; 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.

[0075] In some embodiments of the present invention, the anti-ST2 antibody or its antigen-binding fragment is selected from (1) to (7) below:

[0076] (1) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:11, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:19; the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:29, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:30.

[0077] (2) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:11, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:21; the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:29, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:30.

[0078] (3) 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; the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:29, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:30.

[0079] (4) 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:19; the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:29, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:30.

[0080] (5) 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; the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:29, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:30.

[0081] (6) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:17, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:19; the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:29, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:30; or

[0082] (7) 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; the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:29, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:30.

[0083] In some embodiments of the present invention, the anti-ST2 antibody or its antigen-binding fragment, wherein:

[0084] The anti-ST2 antibody or its antigen-binding fragment inhibits the binding of ST2 to IL-33 on the cell surface of EC. 50 The value is less than or equal to 1.8 nM, less than or equal to 1.4 nM, or less than or equal to 1.3 nM;

[0085] Preferably, the EC 50 This was determined using the reporter gene method.

[0086] In some embodiments of the present invention, the anti-ST2 antibody or its antigen-binding fragment, wherein, according to the EU numbering system, the heavy chain constant region of the anti-ST2 antibody contains the following mutation:

[0087] L234A and L235A; and / or

[0088] M252Y, S254T, and T256E.

[0089] 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.

[0090] In some embodiments of the present invention, the anti-ST2 antibody is a human IgG1 subtype;

[0091] According to the EU numbering system, the heavy chain constant region of the anti-ST2 antibody contains the following mutations:

[0092] L234A and L235A;

[0093] In some embodiments of the present invention, the anti-ST2 antibody is a human IgG4 subtype;

[0094] According to the EU numbering system, the heavy chain constant region of the antibody contains the following mutations:

[0095] M252Y, S254T, and T256E.

[0096] According to any one of the anti-ST2 antibodies or their antigen-binding fragments, any one of the antibody-drug conjugates or the pharmaceutical compositions of the present invention, they are used to treat or prevent diseases, wherein the diseases are selected from one or more of tumors, inflammatory diseases, allergic diseases and autoimmune diseases;

[0097] Preferably, the disease is mediated by the IL-33 / ST2 signaling pathway;

[0098] Preferably, the disease is selected from one or more of the following: asthma, rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, allergic rhinitis, sinusitis, nodular prurigo, chronic urticaria, nasal polyps, chronic obstructive pulmonary disease, ankylosing spondylitis, eosinophilic esophagitis, pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), systemic lupus erythematosus, cardiovascular disease, and systemic sclerosis.

[0099] Preferably, the asthma is allergic asthma;

[0100] Preferably, the allergic rhinitis is seasonal allergic rhinitis (SAR);

[0101] Preferably, the sinusitis is chronic sinusitis;

[0102] Preferably, the chronic obstructive pulmonary disease is moderate to severe chronic obstructive pulmonary disease;

[0103] Preferably, the atopic dermatitis is moderate to severe atopic dermatitis;

[0104] Preferably, the single dose based on the anti-ST2 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;

[0105] 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;

[0106] Preferably, the administration method is intravenous infusion, intravenous injection, intradermal injection, subcutaneous injection, intramuscular injection, or nasal administration.

[0107] Another aspect of the present invention relates to isolated nucleic acid molecules that encode the anti-ST2 antibody or its antigen-binding fragment as described in any one of the present invention.

[0108] Another aspect of the invention relates to a recombinant vector containing the isolated nucleic acid molecules of the invention.

[0109] Another aspect of the invention relates to a host cell containing the isolated nucleic acid molecules of the invention or the recombinant vector of the invention.

[0110] Another aspect of the present invention relates to antibody-drug conjugates 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-ST2 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.

[0111] In some embodiments of the present invention, the antibody-drug conjugate is wherein the antibody or its antigen-binding fragment is linked to a small molecule drug via a linker.

[0112] Another aspect of the invention relates to a pharmaceutical composition comprising an effective amount of any one of the anti-ST2 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. The excipients may be pharmaceutically acceptable carriers and / or excipients. Preferably, the pharmaceutical composition is an injection, a nasal spray, or a nasal drop.

[0113] Another aspect of the present invention relates to a packaged product comprising the anti-ST2 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;

[0114] Preferably, the packaging container is a syringe, injection pen, nasal spray, or dropper bottle;

[0115] Preferably, the packaged product further includes a product instruction manual.

[0116] Another aspect of the invention relates to a combination product comprising a first product and a second product in individually packaged form, wherein,

[0117] The first product comprises an anti-ST2 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;

[0118] The second product contains one or more glucocorticoids;

[0119] Preferably, the glucocorticoid is selected from one or more of prednisone, methylprednisolone, betamethasone, beclomethasone dipropionate, prednisolone, hydrocortisone, dexamethasone, and triamcinolone;

[0120] Preferably, the first product and the second product further comprise one or more pharmaceutically acceptable excipients.

[0121] Preferably, the combined product further includes a product instruction manual.

[0122] Another aspect of the present invention relates to the use of any anti-ST2 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 tumors, inflammatory diseases, allergic diseases, and autoimmune diseases;

[0123] Preferably, the disease is mediated by the IL-33 / ST2 signaling pathway;

[0124] Preferably, the disease is selected from one or more of the following: asthma, rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, allergic rhinitis, sinusitis, nodular prurigo, chronic urticaria, nasal polyps, chronic obstructive pulmonary disease, ankylosing spondylitis, eosinophilic esophagitis, pulmonary fibrosis, chronic obstructive pulmonary disease, systemic lupus erythematosus, cardiovascular disease, and systemic sclerosis.

[0125] Preferably, the asthma is allergic asthma;

[0126] Preferably, the allergic rhinitis is seasonal allergic rhinitis;

[0127] Preferably, the sinusitis is chronic sinusitis;

[0128] Preferably, the chronic obstructive pulmonary disease is moderate to severe chronic obstructive pulmonary disease;

[0129] Preferably, the atopic dermatitis is moderate to severe atopic dermatitis;

[0130] Preferably, the single dose based on the anti-ST2 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;

[0131] 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;

[0132] Preferably, the administration method is intravenous infusion, intravenous injection, intradermal injection, subcutaneous injection, intramuscular injection, or nasal administration.

[0133] 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-ST2 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 tumors, inflammatory diseases, allergic diseases and autoimmune diseases;

[0134] Preferably, the disease is mediated by the IL-33 / ST2 signaling pathway;

[0135] Preferably, the disease is selected from one or more of the following: asthma, rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, allergic rhinitis, sinusitis, nodular prurigo, chronic urticaria, nasal polyps, chronic obstructive pulmonary disease, ankylosing spondylitis, eosinophilic esophagitis, pulmonary fibrosis, chronic obstructive pulmonary disease, systemic lupus erythematosus, cardiovascular disease, and systemic sclerosis.

[0136] Preferably, the asthma is allergic asthma;

[0137] Preferably, the allergic rhinitis is seasonal allergic rhinitis;

[0138] Preferably, the sinusitis is chronic sinusitis;

[0139] Preferably, the chronic obstructive pulmonary disease is moderate to severe chronic obstructive pulmonary disease;

[0140] Preferably, the atopic dermatitis is moderate to severe atopic dermatitis.

[0141] In some embodiments of the present invention, the method wherein...

[0142] The single-dose dose, calculated based on the anti-ST2 antibody or its antigen-binding fragment, is 0.1-100 mg per kilogram of body weight, preferably 5-50 mg or 5-15 mg per kilogram of body weight;

[0143] 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;

[0144] Preferably, the administration method is intravenous infusion, intravenous injection, intradermal injection, subcutaneous injection, intramuscular injection, or nasal administration.

[0145] The variable regions of the light and heavy chains determine antigen binding; each chain's variable region contains three hypervariable regions called complementarity-determining regions (CDRs) (the CDRs of the heavy chain (H) include HCDR1, HCDR2, and HCDR3, and the CDRs of the light chain (L) include LCDR1, LCDR2, and LCDR3; these were named by Kabat et al., see Bethesda Md, Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 1991; 1-3:91-3242).

[0146] Preferably, the CDR can also be defined by the IMGT numbering system. See Ehrenmann, Francois, Quentin Kaas, and Marie-Paule Lefranc. IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF. Nucleic acids research 2009; 38(suppl_1):D301-D307.

[0147] The assignment of amino acids to various 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.

[0148] Using techniques well known to those skilled in the art, such as analyzing the amino acid sequence of the CDR region of a monoclonal antibody sequence according to the IMGT definition.

[0149] 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 herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0150] As used in this article, the term EC 50 The half-maximal concentration (50% of maximal effect) refers to the concentration that produces a 50% maximal effect.

[0151] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of a "light" (L) chain and a "heavy" (H) chain). Antibody light chains can be classified as κ and λ light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within both light and heavy chains, variable and constant regions are linked by "J" regions of approximately 12 or more amino acids, and heavy chains also contain "D" regions of approximately 3 or more amino acids. Each heavy chain is further divided by a heavy chain variable region (V... H ) and heavy chain constant region (C H The heavy chain constant region consists of three structural domains (C). H1 C H2 and C H3It consists of ) light chains. Each light chain is composed of a light chain variable region (V L ) and light chain constant region (C L It consists of a light chain constant region composed of a structural domain C. L Composition. The constant region of an antibody mediates the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. V H and V L The region can be further subdivided into highly variable regions (called complementary determinant regions (CDRs)), interspersed with more conservative regions called framework regions (FRs). Each V H and V L It consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. The variable region (V) of each heavy chain / light chain pair... H and V L These amino acids form antigen-binding sites. The allocation of amino acids to each region or domain follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda Md (1987 and 1991)), or the definitions in the IMGT numbering system, see Ehrenmann, Francois, Quentin Kaas, and Marie-Paule Lefranc. "IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF." Nucleic acids research 2009; 38(suppl_1):D301-D307. The term "antibody" is not limited to any specific method of antibody production. For example, these include, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0152] 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—that is, a group of identical antibody molecules except for the possibility of spontaneous natural mutations. Monoclonal antibodies exhibit high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies that typically recognize different epitopes on an antigen. Monoclonal antibodies can usually be obtained using the hybridoma technique first reported by Kohler et al. G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity[J]. Nature, 1975; 256(5517):495), but it can also be obtained using recombinant DNA technology (see USPatent4,816,567).

[0153] 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 in the framework 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 1986; 321:522-525; Reichmann et al., Nature 1988; 332:323-329; Presta, Curr. Op. Struct. Biol., 1992; 2:593-596; and Clark M. Antibody humanization: a case of the 'Emperor's new clothes'? [J]. Immunol. Today, 2000; 21(8):397-402.

[0154] 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.

[0155] As used herein, 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 a light chain is located at the amino terminus of the polypeptide. Light chains include κ chains and λ chains.

[0156] As used herein, the term "heavy chain" includes the full-length heavy chain and its segments having 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.

[0157] As used herein, the term “antigen-binding fragment” of an antibody refers to a polypeptide containing a fragment of the full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen; it is also referred to as the “antigen-binding moiety.” See also Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of the intact antibody. In some cases, antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabody antibodies, and polypeptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] As used in this invention, the term "Fv region" includes variable regions from both the heavy and light chains, but lacks constant regions.

[0163] 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)).

[0164] As used in this invention, the term “dAb” segment (Ward et al., Nature 341:544-546 (1989)) consists of a VH domain.

[0165] 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" entity, 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 one. 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 substance's activity.

[0166] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0167] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, GS cells, BHK cells, HEK 293 cells, or human cells.

[0168] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its target antigen. In some embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific to an antigen) means that the antibody binds to an antigen at a concentration of less than about 10. -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.

[0169] As used in this article, the term "K" D "" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. Typically, antibodies have an equilibrium dissociation constant of less than approximately 10. -5 M, for example, less than approximately 10 -6M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or a smaller dissociation equilibrium constant (K) D K binds to an antigen (e.g., ST2 protein). K can be measured using methods known to those skilled in the art. D For example, measurements can be taken using a Fortebio molecular interaction analyzer.

[0170] As used herein, 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.

[0171] 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, and is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), including but not limited to: pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride. Pharmaceutical compositions may generally contain, but are not limited to, antibodies, buffers, protectants, surfactants, etc.

[0172] 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.

[0173] As used herein, the term "effective amount" means an amount sufficient to achieve or at least partially achieve the desired effect. For example, an effective amount for disease prevention means an amount sufficient to prevent, stop, or delay the onset of disease; an effective amount for disease treatment means an amount sufficient to cure or at least partially stop the disease and its complications in patients who already have the disease.

[0174] 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:8 may refer to SEQ ID NO:8 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:8, or can achieve the effects of this invention.

[0175] Beneficial effects of the invention

[0176] This invention achieves one or more of the following technical effects:

[0177] (1) The anti-ST2 antibody or its antigen-binding fragment of the present invention can bind to ST2 specifically and well.

[0178] (2) The anti-ST2 antibody or its antigen-binding fragment of the present invention can effectively block the binding of human ST2 and IL-33, inhibit the activation of downstream signaling pathways of human IL-33 / ST2 pathway, and reduce the secretion of IL6 and / or IL-8 by cells mediated by IL-33 / ST2 (e.g., A549-ST2 cells, HUVEC cells).

[0179] (3) The anti-ST2 antibody or its antigen-binding fragment of the present invention can effectively inhibit the secretion of IFN-γ by NK cells co-induced by IL12, IL-15 and IL-33.

[0180] (4) The anti-ST2 antibody or its antigen-binding fragment of the present invention has shown excellent efficacy in animal models such as mouse models of rhinitis and mouse models of asthma.

[0181] (5) The anti-ST2 antibody or its antigen-binding fragment of the present invention can effectively treat or prevent the diseases described in the present invention, such as asthma, rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, allergic rhinitis, sinusitis, nodular prurigo, chronic urticaria, nasal polyps, chronic obstructive pulmonary disease, ankylosing spondylitis, eosinophilic esophagitis, pulmonary fibrosis, chronic obstructive pulmonary disease, systemic lupus erythematosus, cardiovascular disease or systemic sclerosis, etc. Attached Figure Description

[0182] Figure 1: Detection results of the affinity constants of 3H11(CH) and ST2-His. The antibody concentrations added to each pair of curves from top to bottom in the figure are 100 nM, 33.3 nM, 11.1 nM, 3.70 nM, and 1.24 nM, respectively.

[0183] Figure 2: Detection results of the affinity constant between 3H11H4L7 and ST2-His. The antibody concentrations added to each pair of curves from top to bottom in the figure are 100 nM, 33.3 nM, 11.1 nM, 3.70 nM, and 1.24 nM, respectively.

[0184] Figure 3: Detection results of the affinity constant between 3H11H4L8 and ST2-His. The antibody concentrations added to each pair of curves from top to bottom in the figure are 100 nM, 33.3 nM, 11.1 nM, 3.70 nM, and 1.24 nM, respectively.

[0185] Figure 4: Detection results of the affinity constant between 3H11H40L8 and ST2-His. The antibody concentrations added to each pair of curves from top to bottom in the figure are 100 nM, 33.3 nM, 11.1 nM, 3.70 nM, and 1.24 nM, respectively.

[0186] Figure 5: Detection results of the affinity constant between 3H11H45L7 and ST2-His. The antibody concentrations added to each pair of curves from top to bottom in the figure are 100 nM, 33.3 nM, 11.1 nM, 3.70 nM, and 1.24 nM, respectively.

[0187] Figure 6: Detection results of the affinity constant between 3H11H45L8 and ST2-His. The antibody concentrations added to each pair of curves from top to bottom in the figure are 100 nM, 33.3 nM, 11.1 nM, 3.70 nM, and 1.24 nM, respectively.

[0188] Figure 7: Detection results of the affinity constant between 3H11H46L7 and ST2-His. The antibody concentrations added to each pair of curves from top to bottom in the figure are 100 nM, 33.3 nM, 11.1 nM, 3.70 nM, and 1.24 nM, respectively.

[0189] Figure 8: Detection results of the affinity constant between 3H11H46L8 and ST2-His. The antibody concentrations added to each pair of curves from top to bottom in the figure are 100 nM, 33.3 nM, 11.1 nM, 3.70 nM, and 1.24 nM, respectively.

[0190] Figure 9: Detection results of the affinity constants of AMG282 and ST2-His. The antibody concentrations added to each pair of curves from top to bottom in the figure are 100 nM, 33.3 nM, 11.1 nM, 3.70 nM, and 1.24 nM, respectively.

[0191] Figure 10: Binding activity of anti-ST2 antibody to 293T-NFκB-Luc-ST2 cell surface antigen.

[0192] Figure 11: Reporter gene assay analysis shows that anti-ST2 antibody inhibits the binding of ST2 and IL-33 on the surface of 293T-NFκB-Luc-ST2 cells.

[0193] Figure 12: Inhibition of IL6 secretion by A549-ST2 cells by anti-ST2 antibody.

[0194] The sequences involved in this invention are shown in Table A below. All CDRs are numbered according to the IMGT numbering system.

[0195] Table A Detailed Implementation

[0196] The embodiments of the present invention will be described in detail below with reference to examples. 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. Where specific techniques or conditions are not specified in the examples, they should be performed according to the techniques or conditions described in the literature in the art (e.g., refer to J. Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 3rd edition, Science Press, translated by Huang Peitang et al.) or according to the product instructions. Reagents or instruments whose manufacturers are not specified are conventional products that can be purchased on the market. For example, 293T cells and A549 cells can be purchased from ATCC.

[0197] In the experimental examples of the present invention, the isotype control antibodies used, namely hIgG1 and hIgG4, are 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 as the heavy chain constant region and Ig kappa chain C region, ACCESSION: P01834 as the light chain constant region. The heavy chain constant region of hIgG4 uses Ig gamma-4 chain C region, ACCESSION: P01861.1 as the heavy chain constant region and introduces the S228P mutation to improve stability. Cregion, ACCESSION: P01834 is the light chain constant region; hIgG1 and hIgG4 were both prepared in the laboratory of Zhongshan Kangfang Biopharmaceutical Co., Ltd.

[0198] In the following embodiments of the present invention, the sequence of the positive control antibody AMG282 used is shown in SEQ ID NOs:23-24.

[0199] The 293T-NFκB-Luc-ST2 cell line was constructed by Zhongshan Kangfang Biopharmaceutical Co., Ltd. The 293T-NFκB-Luc-ST2 cell line was obtained from HEK293T cells via viral infection. 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 pNF-kB-Luc2P-hygro (where the vector pCDH-Hygro was modified based on pCDH-CMV-MCS-EF1-Puro (purchased from Youbao Biotechnology, catalog number: VT1480)) and pCDH-CMV-IL1RL1-FL (where IL1RL1 is ST2, and its amino acid sequence is as shown in SEQ ID). NO:26; the vector pCDH-CMV-Puro was purchased from UBO Biotechnology, product number: VT1480).

[0200] 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: 26; the vector pCDH-CMV-Puro was purchased from Youbao Biotechnology, product number: VT1480).

[0201] Preparation Example 1: Preparation of Anti-ST2 Antibody

[0202] 1. Preparation of hybridoma cell lines

[0203] The antigen used to prepare the anti-ST2 antibody was human ST2-ECD-His (sequence shown in SEQ ID NO:25). Spleen cells from immunized mice were fused with mouse myeloma cells to create hybridoma cells. Using human ST2-ECD-His as the antigen, the 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.

[0204] 2. Preparation of anti-ST2 antibody 3H11

[0205] The cell lines prepared above were cultured in CD (Chemical Defined Medium) (CD medium containing 1% penicillin and streptomycin, cultured in a 5% CO2, 37°C cell culture incubator). After 7 days, the cell culture supernatant was collected, centrifuged at high speed, filtered through a microporous membrane under vacuum, and purified using a HiTrap protein A HP column to obtain antibody 3H11.

[0206] Preparation Example 2: Sequence Analysis of Anti-ST2 Antibody 3H11

[0207] mRNA was extracted from the hybridoma cell line obtained in Preparation Example 1 according to the method of the Total RNA Extraction Kit for Cultured Bacteria (Tiangen, catalog number DP430).

[0208] According to Invitrogen III. First-Strand Synthesis System for RT-PCR Kit Instructions: Synthesize cDNA and perform PCR amplification.

[0209] The PCR amplification products were directly cloned using TA. For specific procedures, please refer to the instructions of the pEASY-T1Cloning Kit (Transgen CT101).

[0210] The TA clone product was directly sequenced, and the sequencing results are as follows:

[0211] The nucleic acid sequence of the heavy chain variable region is shown in SEQ ID NO:7, and the amino acid sequence it encodes is shown in SEQ ID NO:8.

[0212] The nucleic acid sequence of the light chain variable region is shown in SEQ ID NO:9, and the amino acid sequence it encodes is shown in SEQ ID NO:10.

[0213] According to the IMGT numbering system:

[0214] The sequence of heavy chain CDR1 (HCDR1) is shown in SEQ ID NO:1, the sequence of heavy chain CDR2 (HCDR2) is shown in SEQ ID NO:2, and the sequence of heavy chain CDR3 (HCDR3) is shown in SEQ ID NO:3.

[0215] The sequence of light chain CDR1 (LCDR1) is shown in SEQ ID NO:4, the sequence of light chain CDR2 (LCDR2) is shown in SEQ ID NO:5, and the sequence of light chain CDR3 (LCDR3) is shown in SEQ ID NO:6.

[0216] Preparation Example 3: Design and Preparation of Anti-ST2 Chimeric Antibody 3H11(CH)

[0217] Based on the variable region sequence of antibody 3H11 obtained in Preparation Example 2, a chimeric antibody was designed. The constant region of the heavy chain of this chimeric antibody adopted the human Ig gamma-1 chain C region; the constant region of the light chain was the human Ig kappa chain C region. Following the EU numbering system, a point mutation (L234A) was introduced at position 234 of the heavy chain constant region, and another point mutation (L235A) was introduced at position 235, resulting in the mutated chimeric antibody 3H11(CH). The amino acid sequence of the 3H11(CH) heavy chain is shown in SEQ ID NO:27, and the amino acid sequence of the 3H11(CH) light chain is shown in SEQ ID NO:28.

[0218] 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.

[0219] Preparation Example 4: Design and preparation of humanized antibodies and mutants against ST2

[0220] 1. Design of light and heavy chains of humanized antibodies against human ST2: 3H11H4L7, 3H11H4L8, 3H11H40L8, 3H11H45L7, 3H11H45L8, 3H11H46L7, and 3H11H46L8

[0221] Based on the sequence of hybridoma antibody 3H11 obtained in Preparation Example 2, 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 1 below.

[0222] Table 1

[0223] The heavy chain constant regions of the antibodies all adopted the human Ig gamma-4 chain C region; the light chain constant regions were human Ig kappa chain C regions; 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 sequences of their heavy chain constant regions are shown in SEQ ID NO:29, and the amino acid sequences of their light chain constant regions are shown in SEQ ID NO:30.

[0224] 2. Preparation of humanized antibodies 3H11H4L7, 3H11H4L8, 3H11H40L8, 3H11H45L7, 3H11H45L8, 3H11H46L7, and 3H11H46L8

[0225] 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.

[0226] pUC57simple-3H11H4, pUC57simple-3H11L8;

[0227] pUC57simple-3H11H40, pUC57simple-3H11L8;

[0228] pUC57simple-3H11H45, pUC57simple-3H11L7;

[0229] pUC57simple-3H11H45, pUC57simple-3H11L8;

[0230] pUC57simple-3H11H46, pUC57simple-3H11L7; and

[0231] pUC57simple-3H11H46, pUC57simple-3H11L8.

[0232] 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 containing the corresponding light and heavy chain recombinant plasmids were designed (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).

[0233] HEK293 cells were co-transfected with pcDNA3.1-3H11H46 / pcDNA3.1-3H11L7 and pcDNA3.1-3H11H46 / pcDNA3.1-3H11L8, 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.

[0234] Preparation Example 5: Design and preparation of humanized antibodies 3H11H45L7 (hG1WT) and 3H11H46L7 (hG1WT)

[0235] 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:31), and the light chain constant region was replaced with the human Ig kappa chain C region (SEQ ID NO:30), thereby obtaining humanized antibodies 3H11H45L7(hG1WT) and 3H11H46L7(hG1WT). The preparation method of antibodies 3H11H45L7(hG1WT) and 3H11H46L7(hG1WT) is the same as in Preparation Example 4 above.

[0236] Example 1: Determination of kinetic parameters of binding between anti-ST2 humanized antibody and antigen ST2-His using a Fortebio molecular interaction analyzer

[0237] The sample dilution buffer was PBS, 0.02% Tween-20, 0.1% BSA, pH 7.4.

[0238] ST2-His samples at a concentration of 3 μg / ml were used to immobilize ST2-His onto the NTA sensor at a height of 0.5 nm. The sensor was equilibrated in buffer for 60 s. The immobilized ST2-His then bound to a humanized antibody at a concentration of 3.13–50 nM (two-fold dilution) for 90 s. The antibody dissociated in buffer for 480 s. The sensor was regenerated using 10 mM glycine at pH 1.7 for 5 s, repeated 5 times. The sensor was then equilibrated in 10 mM nickel sulfate buffer for 60 s. 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 and analysis were performed using Fortebio Data Acquisition 12.0 and Fortebio Data Analysis 12.0 software, respectively.

[0239] The results of the detection of the affinity constant between the anti-ST2 humanized antibody and the antigen ST2-His are shown in Table 2 and Figures 1 to 9.

[0240] Table 2: Detection results of affinity constants between anti-ST2 humanized antibody and antigen ST2-His.

[0241] K D K is the affinity constant. D =kdis / kon.

[0242] The results showed that the humanized antibodies 3H11H4L7, 3H11H4L8, 3H11H40L8, 3H11H45L7, 3H11H45L8, 3H11H46L7, 3H11H46L8, 3H11H38L7 and 3H11(CH) could all effectively bind to the antigen ST2-His.

[0243] Example 2: FACS detection of the binding activity of anti-ST2 antibody to 293T-NFκB-Luc-ST2 cell surface antigen

[0244] 293T-NFκB-Luc-ST2 cells were routinely collected, centrifuged at 170×g for 5 min, and the supernatant was discarded. Cells were washed twice with PBS, and cell counts and viability were measured. 300,000 cells / sample were added to 96-well plates, with an appropriate amount of 1% PBSA (i.e., PBS + 1% BSA) added to each well. The plates were centrifuged at 500×g for 5 min, and the supernatant was discarded. Antibodies were diluted with 1% PBSA to 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, and isotyped to 900 nM. 100 μL of the diluted antibody was added to each well, and the cell clumps were resuspended. The plates were incubated on ice for 40 min. 150 μL of 1% PBSA was added to each well, and the plates were centrifuged at 500×g for 5 min, the supernatant was discarded, and the washing process was repeated twice. Dilute Mouse Anti-Human IgG Fc-Alexa with 1% PBSA After preparing 647 (500-fold dilution, working concentration: 10 μg / mL) (Southern Biotech, catalog number: 9040-31), add 100 μL of the diluted antibody to the corresponding sample in each well and resuspend the cell clumps. Incubate on ice in the dark for 30 min. Add 150 μL of 1% PBSA to each well, centrifuge at 500×g for 5 min, discard the supernatant, and repeat the washing process twice. Add 200 μL of 1% PBSA to each well, resuspend the cell clumps, transfer to sample tubes, and perform analysis.

[0245] The experimental results are shown in Table 3 and Figure 10.

[0246] Table 3: Binding activity of anti-ST2 antibody to 293T-NFκB-Luc-ST2 cell surface antigen

[0247] The results showed that 3H11(CH), 3H11H45L7(hG1WT), 3H11H46L7(hG1WT), AMG282 and 293T-NFκB-Luc-ST2 cells bound to ECMO 50The concentrations were 0.3421 nM, 0.2910 nM, 0.2992 nM, and 0.4156 nM, respectively. Experimental results showed that the binding activity of 3H11(CH), 3H11H45L7(hG1WT), and 3H11H46L7(hG1WT) to the 293T-NFκB-Luc-ST2 cell surface antigen ST2 was stronger than that of the positive control antibody AMG282.

[0248] Example 3: Reporter gene assay to analyze the inhibition of ST2 binding to IL-33 on the surface of 293T-NFκB-Luc-ST2 cells by anti-ST2 antibody.

[0249] 293T-NFκB-Luc-ST2 cells were collected, centrifuged at 170×g for 5 min, and resuspended in DMEM + 10% FBS to dilute the cells. Cell counts and viability were determined. Then, 50 μL (containing 10,000 cells) of cell suspension was added to each well of a black 96-well plate (Costar, model: 3916) and placed in a cell culture incubator. Antibodies such as AMG282 were diluted with Opti-MEM (Gibco, catalog number: 31985088) to working concentrations of 300 nM, 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, 1.23 nM, 0.41 nM, and 0.041 nM. hIgG1 and hIgG4 were diluted with Opti-MEM to a working concentration of 100 nM. 25 μL of the diluted antibodies were added to each well of the 96-well plate. Antibody was prepared in duplicate. Positive control TNF-α (sinobiological, catalog number: GMP.10602-HNAE-50) was diluted to a working concentration of 10 ng / mL, with 50 μL added to each well. A blank control (293T-NFκB-Luc-ST2 cell group), a negative control (cells + IL-33 group), and isotype controls (hlgG1 and hlgG4) were also included, and incubated for 30 min. IL-33 (IL-33-Nhis bio, Kangfang Biotechnology) was diluted to a working concentration of 1 pM using opti-MEM, and 25 μL of IL-33 was added to each well of the corresponding 96-well plate. The plates were incubated for 5 hours. 50 μL of Bright-Glo was added to each well. TM Luciferase Assay Reagent (promega, catalog number: E2620), Envision reading fluorescence value (RLU).

[0250] The experimental results are shown in Table 4 and Figure 11.

[0251] Table 4: Anti-ST2 antibody inhibits the binding of ST2 and IL-33 on the surface of 293T-NFκB-Luc-ST2 cells.

[0252] The results showed that IL-33 could activate the 293T-NFκB-Luc-ST2 cell signaling pathway and promote luciferase expression. 3H11(CH), 3H11H4L7, 3H11H4L8, 3H11H40L8, 3H11H45L7, 3H11H45L8, 3H11H46L7, 3H11H46L8, and the positive control antibody AMG282 could all inhibit the binding of ST2 to IL-33 on the cell surface, blocking luciferase expression and decreasing RLU values. Among them, the inhibitory abilities of 3H11(CH), 3H11H4L7, 3H11H4L8, 3H11H45L7, 3H11H45L8, 3H11H46L7, and 3H11H46L8 were superior to those of the positive control antibody AMG282.

[0253] Example 4: ELISA detection of the bioactivity of anti-ST2 antibody in the IL-33-stimulated A549-ST2 IL-6 secretion system.

[0254] A549-ST2 cells were routinely collected (complete culture medium: DMEM + 10% FBS) and seeded at 8000 cells / 100 μL / well in 96-well plates (Corning, model: 3599) and cultured overnight. Antibodies were diluted to the appropriate concentrations (working concentrations: 0.00384 nM, 0.0384 nM, 0.384 nM, 1.92 nM, 9.6 nM, 48 nM, 240 nM, and 1200 nM) and added at 50 μL / well to the corresponding cell wells. Isotype controls (hlgG1 and hlgG4), negative controls (IL-33-2.5 ng / mL), and blank control groups were also included, with two replicates per well. Pre-incubation was performed at 37°C for 30 min. IL-33 (IL-33-Nhis, working concentration 2.5 ng / mL) was added at 50 μL / well to the corresponding cell wells, and incubation was performed at 37°C for 24 h. Cell supernatant was collected by centrifugation at 4°C and detected using an IL-6 kit (Human IL-6 ELISA Kit, Daco, catalog number: 1110602).

[0255] The experimental results are shown in Table 5 and Figure 12.

[0256] Table 5: Bioactivity of anti-ST2 antibodies in the IL-33-stimulated A549-ST2 IL-6 secretion system

[0257] The results showed that 3H11(CH), 3H11H45L7, 3H11H46L7 and the positive control antibody AMG282 could effectively inhibit IL-6 secretion induced by IL-33 stimulation of A549-ST2 cells, and the inhibitory ability of 3H11(CH) and 3H11H45L7 was stronger than that of AMG282.

[0258] 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-ST2 antibody or its antigen-binding fragment, wherein the anti-ST2 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1 to HCDR3, and the light chain variable region comprising LCDR1 to LCDR3, wherein: HCDR1 contains the amino acid sequence shown in SEQ ID NO:1, HCDR2 contains the amino acid sequence shown in SEQ ID NO:2, and HCDR3 contains the amino acid sequence shown in SEQ ID NO:

3. LCDR1 contains the amino acid sequence shown in SEQ ID NO:4, LCDR2 contains the amino acid sequence shown in SEQ ID NO:5, and LCDR3 contains the amino acid sequence shown in SEQ ID NO:

6.

2. The anti-ST2 antibody or its antigen-binding fragment according to claim 1, wherein, The heavy chain variable region of the anti-ST2 antibody comprises an amino acid sequence selected from SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, and SEQ ID NO:34; and The light chain variable region of the anti-ST2 antibody comprises an amino acid sequence selected from SEQ ID NO:10, SEQ ID NO:19, SEQ ID NO:21 and SEQ ID NO:

35.

3. The anti-ST2 or its antigen-binding fragment according to any one of claims 1 to 2, wherein, (1) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:8, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:10; (2) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:8, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:19; (3) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:8, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:21; (4) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:8, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:35; (5) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:10; (6) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:19; (7) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:21; (8) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:11, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:35; (9) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:10; (10) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:19; (11) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:21; (12) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:13, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:35; (13) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:10; (14) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:19; (15) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:21; (16) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:15, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:35; (17) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:17, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:10; (18) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:17, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:19; (19) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:17, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:21; (20) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:17, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:35; (21) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:34, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:10; (22) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:34, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:19; (23) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:34, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:21; or (24) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:34, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

35.

4. The anti-ST2 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-ST2 antibody or its antigen-binding fragment according to any one of claims 1 to 4, wherein, The anti-ST2 antibody is an IgG1, IgG2, IgG3, or IgG4 subtype.

6. The anti-ST2 antibody or its antigen-binding fragment according to any one of claims 1 to 5, 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 SEQ ID NO:31 or SEQ ID NO:32; Preferably, the amino acid sequence of the constant region of the human Kappa chain is shown in SEQ ID NO:

30.

7. The anti-ST2 antibody or its antigen-binding fragment according to any one of claims 1 to 5, 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:33 or SEQ ID NO:29; Preferably, the amino acid sequence of the constant region of the human Kappa chain is shown in SEQ ID NO:

30.

8. The anti-ST2 antibody or its antigen-binding fragment according to any one of claims 1 to 7, wherein, The antibody includes a non-CDR region, and the non-CDR region is derived from human antibodies, mouse antibodies, or rabbit antibodies.

9. The anti-ST2 antibody or its antigen-binding fragment according to any one of claims 1 to 8, wherein, The anti-ST2 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.

10. The anti-ST2 antibody or its antigen-binding fragment according to any one of claims 1 to 9, wherein, The anti-ST2 antibody is selected from (1) to (21) below: (1) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:11, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:19; the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:29, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:

30. (2) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:11, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:21; the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:29, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:

30. (3) 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; the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:29, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:

30. (4) 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:19; the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:29, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:

30. (5) 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; the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:29, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:

30. (6) 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:19; the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:29, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:

30. (7) 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; the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO:29, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:

30. (8) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:11, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:19; 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. (9) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:11, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:21; 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. (10) 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; 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. (11) 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:19; 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. (12) 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; 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. (13) 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:19; 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. (14) 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; 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. (15) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:11, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:19; 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. (16) The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO:11, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO:21; 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. (17) 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; 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. (18) 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:19; 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. (19) 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; 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. (20) The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:17, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:19; 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; or (21) 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; 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.

11. An isolated nucleic acid molecule encoding the anti-ST2 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-ST2 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 anti-tumor chemotherapy drug; Preferably, the antibody or its antigen-binding fragment is linked to a small molecule drug via a linker.

15. A pharmaceutical composition comprising an effective amount of the anti-ST2 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-ST2 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-ST2 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 further includes a product instruction manual.

18. Use of the anti-ST2 antibody or its antigen-binding fragment as claimed in any one of claims 1 to 10, the antibody-drug conjugate as claimed in claim 14, or the pharmaceutical composition as claimed in claim 15 in the preparation of a medicament for treating or preventing a disease, wherein the disease is one or more selected from tumors, inflammatory diseases, allergic diseases, and autoimmune diseases; Preferably, the disease is mediated by the IL-33 / ST2 signaling pathway; Preferably, the disease is selected from one or more of the following: asthma, rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, allergic rhinitis, sinusitis, nodular prurigo, chronic urticaria, nasal polyps, chronic obstructive pulmonary disease, ankylosing spondylitis, eosinophilic esophagitis, pulmonary fibrosis, chronic obstructive pulmonary disease, 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 based on the anti-ST2 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, 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-ST2 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 tumors, inflammatory diseases, allergic diseases, and autoimmune diseases; Preferably, the disease is mediated by the IL-33 / ST2 signaling pathway; Preferably, the disease is selected from one or more of the following: asthma, rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, allergic rhinitis, sinusitis, nodular prurigo, chronic urticaria, nasal polyps, chronic obstructive pulmonary disease, ankylosing spondylitis, eosinophilic esophagitis, pulmonary fibrosis, chronic obstructive pulmonary disease, 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 based on the anti-ST2 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, 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-ST2 antibody or its antigen-binding fragment as claimed in any one of claims 1 to 10, the antibody-drug conjugate as claimed in claim 14, or the pharmaceutical composition as claimed in claim 15, wherein the disease is one or more selected from tumors, inflammatory diseases, allergic diseases, and autoimmune diseases; Preferably, the disease is mediated by the IL-33 / ST2 signaling pathway; Preferably, the disease is selected from one or more of the following: asthma, rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, allergic rhinitis, sinusitis, nodular prurigo, chronic urticaria, nasal polyps, chronic obstructive pulmonary disease, ankylosing spondylitis, eosinophilic esophagitis, pulmonary fibrosis, chronic obstructive pulmonary disease, 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 based on the anti-ST2 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, 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.

Images