Anti-ST2 antibodies and their applications

Abstract

An anti-ST2 antibody or a fragment thereof is provided. This antibody or fragment thereof specifically binds to human ST2, thereby inhibiting the combination of IL-33 and human ST2, blocking the IL-33 / ST2 intracellular signaling pathway, and inhibiting the promoting effect of different types of IL-33 on cell-derived IL5, IL6, and IL8 production. Compared to known anti-ST2 antibodies, this anti-ST2 antibody has higher biological activity and can be used to prevent, treat, or ameliorate diseases associated with ST2 expression or disorders of the IL-33 / ST2 pathway.

Description

[Technical Field]

[0001] This application claims the benefit of priority from Chinese Patent Application No. 202010072085.X, filed on January 21, 2020, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to the field of antibody drugs, in particular to antibodies against human ST2 and their use in preparing drugs. [Background technology]

[0003] Interleukin-33 (IL-33) is a cytokine related to IL-1 and IL-18 and is also known as NF-HEV or IL-1F11. ST2 (ST2L, IL-1RL1, T1, Fit-1, DER-4, IL-1R4, or ST2α) is a binding receptor for IL-33 and is a member of the Toll / IL-1 receptor family. It is expressed on the cell surface of a variety of immune cells, including lymphocytes, particularly helper T cells expressing IL-5 and IL-13, natural killer (NK) cells, and natural killer T (NKT) cells, as well as many so-called innate immune cells, such as mast cells, basophils, eosinophils, macrophages, and innate helper cells (also known as novel immune cells (neuocytes) (Non-Patent Document 1)).

[0004] ST2 can downregulate the reactivity of Toll-like receptors TLR2, TLR4, and TLR9, and can induce the release of type 2 cytokines through activation by its ligand IL-33 and association with the accessory protein IL-1RAcP. Related literature has proposed models of interactions between ST2, IL-33, and IL-1RAcP, as well as between IL-1R1 and IL-1RAcP (Non-Patent Document 2, Non-Patent Document 3).

[0005] IL-33 is present in its full-length form in the nuclei of epithelial and endothelial cells during homeostasis, but can be cleaved and released during cell necrosis, hence the name "alarmin." Examples of IL-33-induced cellular responses include the production of proinflammatory cytokines such as IL-5, IL-6, IL-13, TNF, IFN-γ, and GM-CSF, as well as chemokines such as CXCL8, CCL17, and CCL24. IL-33 has also been shown to enhance acute allergic responses by enhancing mast cell and basophil activation induced by IgE receptor signaling or other mast cell and basophil activating factors. IL-33 also enhances the recruitment, survival, and adhesive properties of ST2-expressing immune cells, and is therefore crucial for the initiation and maintenance of cellular inflammation in local tissues.

[0006] Dysregulation of the IL-33 / ST2 pathway has been shown to be associated with a variety of immune-mediated diseases, including asthma, rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, allergic rhinitis, nasal polyps, and systemic sclerosis. Therefore, therapeutic blocking of the IL-33 / ST2 pathway may help overcome hyperimmune responses. Inhibitors of this pathway primarily include IL33 antibodies (e.g., MEDI3506, ANB020, REGN3500, MT-2990, LY-3375880, PF-06817024) and ST2 antibodies (e.g., CNTO7160, AMG-282), which are currently being developed in phase 1 and 2 clinical trials for indications including allergic rhinitis, atopic dermatitis, chronic obstructive pulmonary disease, and asthma.

[0007] At present, the incidence of allergic inflammation and respiratory diseases is gradually increasing, and the medicines available on the market are mainly glucocorticoids and β2 receptor agonists.All antibodies reported to date can block the interaction between ST2 and its ligand, but the resulting biological activities are different.Differences in biological activity may lead to differences in the clinical efficacy and dosage of antibodies, therefore, there is still a need in the art for ST2 antibodies that provide high affinity, high stability, and high biological activity. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Neill, Wong et al., 2010 [Non-patent document 2] Lingel et al., Cell 17: 1398-1410, 2009 [Non-patent document 3] Wang et al, Nat Immunol, 11: 905-11, 2010 Summary of the Invention [Problem to be solved by the invention]

[0009] The technical problem to be solved by the present invention is to obtain novel high-affinity antibodies that bind to ST2 and are suitable for the treatment of diseases or any indications associated with the IL-33 / ST2 pathway by immunizing mice with human ST2 as an immunogen, obtaining murine antibodies through B cell panning, and further antibody engineering and humanization techniques.

[0010] In view of the above technical problems, an object of the present invention is to provide an antibody or a functional fragment thereof that specifically binds to ST2, and to provide uses thereof. [Means for solving the problem]

[0011] The technical solution of the present invention is as follows:

[0012] As used herein, a "fragment" of an antibody as described in the present invention includes various functional or active fragments of an antibody, such as an antigen-binding portion thereof, such as Fab, F(ab')2, or scFv.

[0013] In one aspect, the present invention provides an antibody or fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region and the light chain variable region are selected from the following combinations: (I-1) a heavy chain variable region as shown in SEQ ID NO: 1 and a light chain variable region as shown in SEQ ID NO: 29; (I-2) a heavy chain variable region as shown in SEQ ID NO: 2 and a light chain variable region as shown in SEQ ID NO: 30; (I-3) a heavy chain variable region as shown in SEQ ID NO: 2 and a light chain variable region as shown in SEQ ID NO: 31; (I-4) a heavy chain variable region as shown in SEQ ID NO: 3 and a light chain variable region as shown in SEQ ID NO: 30; (I-5) a heavy chain variable region as shown in SEQ ID NO: 3 and a light chain variable region as shown in SEQ ID NO: 31; (II-1) A heavy chain variable region as shown in SEQ ID NO: 4 and a light chain variable region as shown in SEQ ID NO: 32; (II-2) a heavy chain variable region as shown in SEQ ID NO: 5 and a light chain variable region as shown in SEQ ID NO: 33; (II-3) A heavy chain variable region as shown in SEQ ID NO: 5 and a light chain variable region as shown in SEQ ID NO: 34; (II-4) A heavy chain variable region as shown in SEQ ID NO: 5 and a light chain variable region as shown in SEQ ID NO: 35; (II-5) a heavy chain variable region as shown in SEQ ID NO: 6 and a light chain variable region as shown in SEQ ID NO: 33; (II-6) A heavy chain variable region as shown in SEQ ID NO: 6 and a light chain variable region as shown in SEQ ID NO: 34; (II-7) A heavy chain variable region as shown in SEQ ID NO: 6 and a light chain variable region as shown in SEQ ID NO: 35; (III-1) a heavy chain variable region as shown in SEQ ID NO: 7 and a light chain variable region as shown in SEQ ID NO: 36; (III-2) a heavy chain variable region as shown in SEQ ID NO: 8 and a light chain variable region as shown in SEQ ID NO: 37; (III-3) a heavy chain variable region as shown in SEQ ID NO: 8 and a light chain variable region as shown in SEQ ID NO: 38; (III-4) a heavy chain variable region as shown in SEQ ID NO: 9 and a light chain variable region as shown in SEQ ID NO: 37; (III-5) a heavy chain variable region as shown in SEQ ID NO: 9 and a light chain variable region as shown in SEQ ID NO: 38; (IV-1) a heavy chain variable region as shown in SEQ ID NO: 10 and a light chain variable region as shown in SEQ ID NO: 39; (IV-2) a heavy chain variable region as shown in SEQ ID NO: 11 and a light chain variable region as shown in SEQ ID NO: 40; (IV-3) a heavy chain variable region as shown in SEQ ID NO: 11 and a light chain variable region as shown in SEQ ID NO: 41; (IV-4) a heavy chain variable region as shown in SEQ ID NO: 12 and a light chain variable region as shown in SEQ ID NO: 40; (IV-5) a heavy chain variable region as shown in SEQ ID NO: 12 and a light chain variable region as shown in SEQ ID NO: 41; (IV-6) a heavy chain variable region as shown in SEQ ID NO: 13 and a light chain variable region as shown in SEQ ID NO: 40; (IV-7) a heavy chain variable region as shown in SEQ ID NO: 13 and a light chain variable region as shown in SEQ ID NO: 41; (IV-8) a heavy chain variable region as shown in SEQ ID NO: 14 and a light chain variable region as shown in SEQ ID NO: 40; (IV-9) a heavy chain variable region as shown in SEQ ID NO: 14 and a light chain variable region as shown in SEQ ID NO: 41; (V-1) a heavy chain variable region as shown in SEQ ID NO: 15 and a light chain variable region as shown in SEQ ID NO: 42; (V-2) a heavy chain variable region as shown in SEQ ID NO: 16 and a light chain variable region as shown in SEQ ID NO: 43; (V-3) a heavy chain variable region as shown in SEQ ID NO: 16 and a light chain variable region as shown in SEQ ID NO: 44; (V-4) a heavy chain variable region as shown in SEQ ID NO: 16 and a light chain variable region as shown in SEQ ID NO: 45; (V-5) a heavy chain variable region as shown in SEQ ID NO: 17 and a light chain variable region as shown in SEQ ID NO: 43; (V-6) a heavy chain variable region as shown in SEQ ID NO: 17 and a light chain variable region as shown in SEQ ID NO: 44; (V-7) a heavy chain variable region as shown in SEQ ID NO: 17 and a light chain variable region as shown in SEQ ID NO: 45; (V-8) a heavy chain variable region as shown in SEQ ID NO: 18 and a light chain variable region as shown in SEQ ID NO: 43; (V-9) a heavy chain variable region as shown in SEQ ID NO: 18 and a light chain variable region as shown in SEQ ID NO: 44; (V-10) a heavy chain variable region as shown in SEQ ID NO: 18 and a light chain variable region as shown in SEQ ID NO: 45; (V-11) a heavy chain variable region as shown in SEQ ID NO: 19 and a light chain variable region as shown in SEQ ID NO: 43; (V-12) a heavy chain variable region as shown in SEQ ID NO: 19 and a light chain variable region as shown in SEQ ID NO: 44; (V-13) a heavy chain variable region as shown in SEQ ID NO: 19 and a light chain variable region as shown in SEQ ID NO: 45; (VI-1) a heavy chain variable region as shown in SEQ ID NO: 20 and a light chain variable region as shown in SEQ ID NO: 46; (VI-2) a heavy chain variable region represented by SEQ ID NO: 21 and a light chain variable region represented by SEQ ID NO: 47; (VI-3) a heavy chain variable region as shown in SEQ ID NO: 21 and a light chain variable region as shown in SEQ ID NO: 48; (VI-4) a heavy chain variable region as shown in SEQ ID NO: 22 and a light chain variable region as shown in SEQ ID NO: 47; (VI-5) a heavy chain variable region as shown in SEQ ID NO: 22 and a light chain variable region as shown in SEQ ID NO: 48; (VI-6) a heavy chain variable region as shown in SEQ ID NO: 23 and a light chain variable region as shown in SEQ ID NO: 47; (VI-7) a heavy chain variable region as shown in SEQ ID NO: 23 and a light chain variable region as shown in SEQ ID NO: 48; (VI-8) a heavy chain variable region as shown in SEQ ID NO: 24 and a light chain variable region as shown in SEQ ID NO: 47; (VI-9) a heavy chain variable region as shown in SEQ ID NO: 24 and a light chain variable region as shown in SEQ ID NO: 48; (VII-1) a heavy chain variable region as shown in SEQ ID NO: 25 and a light chain variable region as shown in SEQ ID NO: 49; (VII-2) a heavy chain variable region as shown in SEQ ID NO: 26 and a light chain variable region as shown in SEQ ID NO: 52; (VII-3) a heavy chain variable region represented by SEQ ID NO: 26 and a light chain variable region represented by SEQ ID NO: 53; (VII-4) a heavy chain variable region as shown in SEQ ID NO: 26 and a light chain variable region as shown in SEQ ID NO: 50; (VII-5) a heavy chain variable region represented by SEQ ID NO: 26 and a light chain variable region represented by SEQ ID NO: 51; (VII-6) a heavy chain variable region represented by SEQ ID NO: 27 and a light chain variable region represented by SEQ ID NO: 52; (VII-7) A heavy chain variable region represented by SEQ ID NO: 27 and a light chain variable region represented by SEQ ID NO: 53; (VII-8) a heavy chain variable region represented by SEQ ID NO: 27 and a light chain variable region represented by SEQ ID NO: 50; (VII-9) a heavy chain variable region represented by SEQ ID NO: 27 and a light chain variable region represented by SEQ ID NO: 51; (VII-10) a heavy chain variable region as shown in SEQ ID NO: 28 and a light chain variable region as shown in SEQ ID NO: 52; (VII-11) a heavy chain variable region as shown in SEQ ID NO: 28 and a light chain variable region as shown in SEQ ID NO: 53; (VII-12) A heavy chain variable region as shown in SEQ ID NO: 28 and a light chain variable region as shown in SEQ ID NO: 50, and (VII-13) a heavy chain variable region as shown in SEQ ID NO: 28 and a light chain variable region as shown in SEQ ID NO: 51; The present invention provides an antibody or a fragment thereof, comprising a heavy chain CDR1 (H-CDR1), a heavy chain CDR2 (H-CDR2), a heavy chain CDR3 (H-CDR3), and a light chain CDR1 (L-CDR1), a light chain CDR2 (L-CDR2), and a light chain CDR3 (L-CDR3) from a heavy chain variable region and a light chain variable region selected from any one of the following:

[0014] Based on the predetermined amino acid sequences of the light chain variable region and heavy chain variable region as described above, one skilled in the art can routinely determine the amino acid sequences of the heavy chain CDRs and light chain CDRs contained in these regions. For example, according to certain embodiments of the present invention, the CDRs in the variable region amino acid sequences are determined using the Kabat, IMGT, ABM, and Chothia numbering schemes. Light chain CDRs and heavy chain CDRs obtained by other methods known in the art, as well as combinations thereof, are also within the scope of the present invention.

[0015] Preferably, the heavy chain variable region and the light chain variable region comprise heavy chain CDRs and light chain CDRs shown in any one of the following combinations selected from the following: (I) H-CDR1 (GYSITSDYAWN), H-CDR2 (YIDYSGSTTYNPSLKS), H-CDR3 (TVIDSMDY) as shown in the order of SEQ ID NOs: 56, 63, 70, and L-CDR1 (RASKSVSTSGHSYMH), L-CDR2 (LASNLES), L-CDR3 (QHSREFPFT) as shown in the order of SEQ ID NOs: 85, 93, 97; (II-1) H-CDR1 (GYSITSDYAWD), H-CDR2 (YIRYSGDTYYNPSLKS), H-CDR3 (TMMDTMDY) as shown in SEQ ID NOs: 57, 64, 71, and L-CDR1 (RASKSVSTSGNSYMH), L-CDR2 (LASNLES), L-CDR3 (QHSREFPLT) as shown in SEQ ID NOs: 86, 93, 98, (II-2) H-CDR1 (GYSITSDYAWD), H-CDR2 (YIRYSGDTYYNPSLKS), H-CDR3 (TMMDTMDY) as shown in SEQ ID NOs: 57, 64, 71, and L-CDR1 (RASKSVSTSGNTYMH), L-CDR2 (LASNLES), L-CDR3 (QHSREFPLT) as shown in SEQ ID NOs: 87, 93, 98, (III) H-CDR1 (GFSLSTSGMGVG), H-CDR2 (HIWWDDVKQYNPALKS), H-CDR3 (IGGDYDYFDF) as shown in the order of SEQ ID NOs: 58, 65, 72, and L-CDR1 (RASESVEYSGTSLMQ), L-CDR2 (VASNVES), L-CDR3 (QQSRKVPWT) as shown in the order of SEQ ID NOs: 88, 94, 99; (IV-1) H-CDR1 (GYTFTDSEMY), H-CDR2 (AIDPETGDTAFNQKFKG), H-CDR3 (AFDNDNDDGFAY) as shown in the order of SEQ ID NOs: 59, 66, 73, and L-CDR1 (SASSSVNYMH), L-CDR2 (DTSKLAS), L-CDR3 (QQWSSNPLT) as shown in the order of SEQ ID NOs: 89, 95, 100; (IV-2) H-CDR1 (GYTFTDSEMY), H-CDR2 (AIDPETGDTAFNQKFKG), H-CDR3 (AFDNDNDEGFAY) as shown in the order of SEQ ID NOs: 59, 66, 74, and L-CDR1 (SASSSVNYMH), L-CDR2 (DTSKLAS), L-CDR3 (QQWSSNPLT) as shown in the order of SEQ ID NOs: 89, 95, 100; (IV-3) H-CDR1 (GYTFTDSEMY), H-CDR2 (AIDPETGDTAFNQKFKG), H-CDR3 (AFDNDNDDAFAY) as shown in the order of SEQ ID NOs: 59, 66, 75, and L-CDR1 (SASSSVNYMH), L-CDR2 (DTSKLAS), L-CDR3 (QQWSSNPLT) as shown in the order of SEQ ID NOs: 89, 95, 100; (V-1) H-CDR1 (GYTFTDYELH), H-CDR2 (TIDPETGDTVYNQKFKA), H-CDR3 (AFYNDYDDGFAY) as shown in the order of SEQ ID NOs: 60, 67, 76, and L-CDR1 (SVSSSVSYMH), L-CDR2 (DTSKLAS), L-CDR3 (QQWNSSPLT) as shown in the order of SEQ ID NOs: 90, 95, 101; (V-2) H-CDR1 (GYTFTDYELH), H-CDR2 (TIDPETGDTVYNQKFKA), H-CDR3 (AFYNDYDDGFAY) as shown in the order of SEQ ID NOs: 60, 67, 76, and L-CDR1 (SVSSSVSYMH), L-CDR2 (DTSKLAS), L-CDR3 (QQWNTSPLT) as shown in the order of SEQ ID NOs: 90, 95, 102, (V-3) H-CDR1 (GYTFTDYELH), H-CDR2 (TIDPETGDTVYNQKFKA), H-CDR3 (AFYNDYDEGFAY) as shown in the order of SEQ ID NOs: 60, 67, 77, and L-CDR1 (SVSSSVSYMH), L-CDR2 (DTSKLAS), L-CDR3 (QQWNSSPLT) as shown in the order of SEQ ID NOs: 90, 95, 101, (V-4) H-CDR1 (GYTFTDYELH), H-CDR2 (TIDPETGDTVYNQKFKA), H-CDR3 (AFYNDYDEGFAY) as shown in the order of SEQ ID NOs: 60, 67, 77, and L-CDR1 (SVSSSVSYMH), L-CDR2 (DTSKLAS), L-CDR3 (QQWNTSPLT) as shown in the order of SEQ ID NOs: 90, 95, 102, (V-5) H-CDR1 (GYTFTDYELH), H-CDR2 (TIDPETGDTVYNQKFKA), H-CDR3 (AFYNDYDDAFAY) as shown in the order of SEQ ID NOs: 60, 67, 78, and L-CDR1 (SVSSSVSYMH), L-CDR2 (DTSKLAS), L-CDR3 (QQWNSSPLT) as shown in the order of SEQ ID NOs: 90, 95, 101; (V-6) H-CDR1 (GYTFTDYELH), H-CDR2 (TIDPETGDTVYNQKFKA), H-CDR3 (AFYNDYDDAFAY) as shown in SEQ ID NOs: 60, 67, 78, and L-CDR1 (SVSSSVSYMH), L-CDR2 (DTSKLAS), L-CDR3 (QQWNTSPLT) as shown in SEQ ID NOs: 90, 95, 102, (VI-1) H-CDR1 (GYRFTDSEMH), H-CDR2 (TIDPETGGTVYNQKFKG), H-CDR3 (AFYNDFDDGFAY) as shown in the order of SEQ ID NOs: 61, 68, 79, and L-CDR1 (SASTSVSYMH), L-CDR2 (DTSKLAS), L-CDR3 (QQWSSNPLT) as shown in the order of SEQ ID NOs: 91, 95, 100; (VI-2) H-CDR1 (GYRFTDSEMH), H-CDR2 (TIDPETGGTVYNQKFKG), H-CDR3 (AFYNDFDEGFAY) as shown in the order of SEQ ID NOs: 61, 68, 80, and L-CDR1 (SASTSVSYMH), L-CDR2 (DTSKLAS), L-CDR3 (QQWSSNPLT) as shown in the order of SEQ ID NOs: 91, 95, 100, (VI-3) H-CDR1 (GYRFTDSEMH), H-CDR2 (TIDPETGGTVYNQKFKG), H-CDR3 (AFYNDFDDAFAY) as shown in the order of SEQ ID NOs: 61, 68, 81, and L-CDR1 (SASTSVSYMH), L-CDR2 (DTSKLAS), L-CDR3 (QQWSSNPLT) as shown in the order of SEQ ID NOs: 91, 95, 100; (VII-1) H-CDR1 (GYTFINYGMN), H-CDR2 (WINTYIGEPTYGDNFKG), H-CDR3 (EGDGFAY) as shown in the order of SEQ ID NOs: 62, 69, 82, and L-CDR1 (KSSQSLLYSGNQNNYLA), L-CDR2 (GASTRES), L-CDR3 (QNDHSYPYT) as shown in the order of SEQ ID NOs: 92, 96, 103, (VII-2) H-CDR1 (GYTFINYGMN), H-CDR2 (WINTYIGEPTYGDNFKG), H-CDR3 (EGEGFAY) as shown in the order of SEQ ID NOs: 62, 69, 83, and L-CDR1 (KSSQSLLYSGNQNNYLA), L-CDR2 (GASTRES), L-CDR3 (QNDHSYPYT) as shown in the order of SEQ ID NOs: 92, 96, 103, and (VII-3) H-CDR1 (GYTFINYGMN), H-CDR2 (WINTYIGEPTYGDNFKG), H-CDR3 (EGDAFAY) as shown in the order of SEQ ID NOs: 62, 69, 84, and L-CDR1 (KSSQSLLYSGNQNNYLA), L-CDR2 (GASTRES), L-CDR3 (QNDHSYPYT) as shown in the order of SEQ ID NOs: 92, 96, 103.

[0016] In particular, the antibodies or fragments thereof of the present invention comprise at least a heavy chain variable region and a light chain variable region, both of which comprise the above-mentioned CDRs and the framework regions (FRs) therebetween, and the domains in the heavy chain variable region and the light chain variable region are arranged as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Furthermore, for "at least 75% identity," optionally, up to 25% differences in amino acid sequence may exist in any framework region in the heavy chain variable region or light chain variable region, or in any domain or sequence in the antibodies or fragments thereof of the present invention other than the heavy chain variable region and light chain variable region. Differences may arise from amino acid deletions, additions, or substitutions at any position, and substitutions may be conservative or non-conservative.

[0017] Preferably, the heavy chain variable region comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 1 to 28, or an amino acid sequence having at least 75% identity to such an amino acid sequence, and / or the light chain variable region comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 29 to 53, or an amino acid sequence having at least 75% identity to such an amino acid sequence.

[0018] According to a particular embodiment of the invention, the antibody or fragment thereof of the invention is selected from the group consisting of: (I-1) a heavy chain variable region as shown in SEQ ID NO: 1 and a light chain variable region as shown in SEQ ID NO: 29; (I-2) a heavy chain variable region as shown in SEQ ID NO: 2 and a light chain variable region as shown in SEQ ID NO: 30; (I-3) a heavy chain variable region as shown in SEQ ID NO: 2 and a light chain variable region as shown in SEQ ID NO: 31; (I-4) a heavy chain variable region as shown in SEQ ID NO: 3 and a light chain variable region as shown in SEQ ID NO: 30; (I-5) a heavy chain variable region as shown in SEQ ID NO: 3 and a light chain variable region as shown in SEQ ID NO: 31; (II-1) A heavy chain variable region as shown in SEQ ID NO: 4 and a light chain variable region as shown in SEQ ID NO: 32; (II-2) a heavy chain variable region as shown in SEQ ID NO: 5 and a light chain variable region as shown in SEQ ID NO: 33; (II-3) A heavy chain variable region as shown in SEQ ID NO: 5 and a light chain variable region as shown in SEQ ID NO: 34; (II-4) A heavy chain variable region as shown in SEQ ID NO: 5 and a light chain variable region as shown in SEQ ID NO: 35; (II-5) a heavy chain variable region as shown in SEQ ID NO: 6 and a light chain variable region as shown in SEQ ID NO: 33; (II-6) A heavy chain variable region as shown in SEQ ID NO: 6 and a light chain variable region as shown in SEQ ID NO: 34; (II-7) A heavy chain variable region as shown in SEQ ID NO: 6 and a light chain variable region as shown in SEQ ID NO: 35; (III-1) a heavy chain variable region as shown in SEQ ID NO: 7 and a light chain variable region as shown in SEQ ID NO: 36; (III-2) a heavy chain variable region as shown in SEQ ID NO: 8 and a light chain variable region as shown in SEQ ID NO: 37; (III-3) a heavy chain variable region as shown in SEQ ID NO: 8 and a light chain variable region as shown in SEQ ID NO: 38; (III-4) a heavy chain variable region as shown in SEQ ID NO: 9 and a light chain variable region as shown in SEQ ID NO: 37; (III-5) a heavy chain variable region as shown in SEQ ID NO: 9 and a light chain variable region as shown in SEQ ID NO: 38; (IV-1) a heavy chain variable region as shown in SEQ ID NO: 10 and a light chain variable region as shown in SEQ ID NO: 39; (IV-2) a heavy chain variable region as shown in SEQ ID NO: 11 and a light chain variable region as shown in SEQ ID NO: 40; (IV-3) a heavy chain variable region as shown in SEQ ID NO: 11 and a light chain variable region as shown in SEQ ID NO: 41; (IV-4) a heavy chain variable region as shown in SEQ ID NO: 12 and a light chain variable region as shown in SEQ ID NO: 40; (IV-5) a heavy chain variable region as shown in SEQ ID NO: 12 and a light chain variable region as shown in SEQ ID NO: 41; (IV-6) a heavy chain variable region as shown in SEQ ID NO: 13 and a light chain variable region as shown in SEQ ID NO: 40; (IV-7) a heavy chain variable region as shown in SEQ ID NO: 13 and a light chain variable region as shown in SEQ ID NO: 41; (IV-8) a heavy chain variable region as shown in SEQ ID NO: 14 and a light chain variable region as shown in SEQ ID NO: 40; (IV-9) a heavy chain variable region as shown in SEQ ID NO: 14 and a light chain variable region as shown in SEQ ID NO: 41; (V-1) a heavy chain variable region as shown in SEQ ID NO: 15 and a light chain variable region as shown in SEQ ID NO: 42; (V-2) a heavy chain variable region as shown in SEQ ID NO: 16 and a light chain variable region as shown in SEQ ID NO: 43; (V-3) a heavy chain variable region as shown in SEQ ID NO: 16 and a light chain variable region as shown in SEQ ID NO: 44; (V-4) a heavy chain variable region as shown in SEQ ID NO: 16 and a light chain variable region as shown in SEQ ID NO: 45; (V-5) a heavy chain variable region as shown in SEQ ID NO: 17 and a light chain variable region as shown in SEQ ID NO: 43; (V-6) a heavy chain variable region as shown in SEQ ID NO: 17 and a light chain variable region as shown in SEQ ID NO: 44; (V-7) a heavy chain variable region as shown in SEQ ID NO: 17 and a light chain variable region as shown in SEQ ID NO: 45; (V-8) a heavy chain variable region as shown in SEQ ID NO: 18 and a light chain variable region as shown in SEQ ID NO: 43; (V-9) a heavy chain variable region as shown in SEQ ID NO: 18 and a light chain variable region as shown in SEQ ID NO: 44; (V-10) a heavy chain variable region as shown in SEQ ID NO: 18 and a light chain variable region as shown in SEQ ID NO: 45; (V-11) a heavy chain variable region as shown in SEQ ID NO: 19 and a light chain variable region as shown in SEQ ID NO: 43; (V-12) a heavy chain variable region as shown in SEQ ID NO: 19 and a light chain variable region as shown in SEQ ID NO: 44; (V-13) a heavy chain variable region as shown in SEQ ID NO: 19 and a light chain variable region as shown in SEQ ID NO: 45; (VI-1) a heavy chain variable region as shown in SEQ ID NO: 20 and a light chain variable region as shown in SEQ ID NO: 46; (VI-2) a heavy chain variable region represented by SEQ ID NO: 21 and a light chain variable region represented by SEQ ID NO: 47; (VI-3) a heavy chain variable region as shown in SEQ ID NO: 21 and a light chain variable region as shown in SEQ ID NO: 48; (VI-4) a heavy chain variable region as shown in SEQ ID NO: 22 and a light chain variable region as shown in SEQ ID NO: 47; (VI-5) a heavy chain variable region as shown in SEQ ID NO: 22 and a light chain variable region as shown in SEQ ID NO: 48; (VI-6) a heavy chain variable region as shown in SEQ ID NO: 23 and a light chain variable region as shown in SEQ ID NO: 47; (VI-7) a heavy chain variable region as shown in SEQ ID NO: 23 and a light chain variable region as shown in SEQ ID NO: 48; (VI-8) a heavy chain variable region as shown in SEQ ID NO: 24 and a light chain variable region as shown in SEQ ID NO: 47; (VI-9) a heavy chain variable region as shown in SEQ ID NO: 24 and a light chain variable region as shown in SEQ ID NO: 48; (VII-1) a heavy chain variable region as shown in SEQ ID NO: 25 and a light chain variable region as shown in SEQ ID NO: 49; (VII-2) a heavy chain variable region as shown in SEQ ID NO: 26 and a light chain variable region as shown in SEQ ID NO: 52; (VII-3) a heavy chain variable region represented by SEQ ID NO: 26 and a light chain variable region represented by SEQ ID NO: 53; (VII-4) a heavy chain variable region as shown in SEQ ID NO: 26 and a light chain variable region as shown in SEQ ID NO: 50; (VII-5) a heavy chain variable region represented by SEQ ID NO: 26 and a light chain variable region represented by SEQ ID NO: 51; (VII-6) a heavy chain variable region represented by SEQ ID NO: 27 and a light chain variable region represented by SEQ ID NO: 52; (VII-7) A heavy chain variable region represented by SEQ ID NO: 27 and a light chain variable region represented by SEQ ID NO: 53; (VII-8) a heavy chain variable region represented by SEQ ID NO: 27 and a light chain variable region represented by SEQ ID NO: 50; (VII-9) a heavy chain variable region represented by SEQ ID NO: 27 and a light chain variable region represented by SEQ ID NO: 51; (VII-10) a heavy chain variable region as shown in SEQ ID NO: 28 and a light chain variable region as shown in SEQ ID NO: 52; (VII-11) a heavy chain variable region as shown in SEQ ID NO: 28 and a light chain variable region as shown in SEQ ID NO: 53; (VII-12) A heavy chain variable region as shown in SEQ ID NO: 28 and a light chain variable region as shown in SEQ ID NO: 50, and (VII-13) a heavy chain variable region as shown in SEQ ID NO: 28 and a light chain variable region as shown in SEQ ID NO: 51; The present invention comprises a heavy chain variable region and a light chain variable region as set forth in any one of the following:

[0019] The antibodies or fragments thereof provided by the present invention bind to ST2, preferably mammalian ST2, more preferably primate ST2, even more preferably human ST2 or cynomolgus ST2, and particularly human ST2. Experiments have demonstrated that the antibodies provided by the present invention have the following activities: (1) Specific binding to human ST2, (2) Inhibition of IL-33 binding to human ST2; (3) Blocking the IL-33 / ST2 intracellular signaling pathway (4) inhibiting the stimulatory effects of different forms of IL-33 on cellular production of IL-5; (5) inhibiting the stimulatory effect of IL-33 on the cellular production of IL5, IL6, and IL8; and (6) Possession of a long half-life in vivo.

[0020] Generally, the antibodies or fragments thereof provided by the present invention may be of any type, such as monoclonal antibodies, single chain antibodies, diabodies, single domain antibodies, nanobodies, fully or partially humanized antibodies, or chimeric antibodies, or fragments thereof. Preferably, the antibodies are IgA, IgD, IgE, IgG, or IgM, more preferably IgG1, IgG2, or IgG4 antibodies.

[0021] Preferably, the fragment is a functionally active fragment of an antibody capable of specifically binding to ST2 or any portion thereof. More preferably, the fragment is a single-chain variable fragment (scFv), a bivalent single-chain variable fragment (BsFv), a disulfide-stabilized Fv fragment (dsFv), a (disulfide-stabilized Fv fragment)2 ((dsFv)2), an antigen-binding fragment (Fab), a Fab' fragment, a F(ab')2 fragment, or a variable fragment (Fv) of an antibody.

[0022] More preferably, the antibody or fragment thereof further comprises a human or murine constant region, preferably a human heavy chain constant region (CH) and / or a light chain constant region (CL) or a murine heavy chain constant region (CH) and / or a light chain constant region (CL). Preferably, the antibody or fragment thereof comprises a heavy chain and a light chain, more preferably, the antibody comprises two heavy chains and two light chains.

[0023] Preferably, the antibody or fragment thereof comprises a heavy chain constant region selected from the group consisting of IgG, IgA, IgM, IgD and IgE constant regions, and / or a κ-type or λ-type light chain constant region. According to certain embodiments of the invention, the antibody comprises a heavy chain constant region of the IgG1, IgG2 or IgG4 subtype, or the antibody comprises a light chain constant region of the κ subtype. More preferably, the heavy chain constant region comprises an amino acid sequence as set forth in SEQ ID NO: 54 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth in SEQ ID NO: 55 or an amino acid sequence having at least 75% identity to the amino acid sequence as set forth.

[0024] At least 75% identity in the context of the present invention can be any percent identity greater than or equal to 75%, such as at least 75%, at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99% identity.

[0025] According to a particular embodiment of the invention, the present invention particularly preferably relates to antibodies such as: an antibody designated "5888-116-H0L1" having a heavy chain variable region as set forth in SEQ ID NO: 11 and a light chain variable region as set forth in SEQ ID NO: 41; An antibody designated "5888-153-H0L1" having a heavy chain variable region as set forth in SEQ ID NO: 16 and a light chain variable region as set forth in SEQ ID NO: 44, and an antibody designated "5886-156-H1L0" having a heavy chain variable region as set forth in SEQ ID NO: 3 and a light chain variable region as set forth in SEQ ID NO: 30; each of the antibodies has a heavy chain constant region as set forth in SEQ ID NO: 54 and a light chain constant region as set forth in SEQ ID NO: 55. The antibodies are monoclonal antibodies, each comprising two heavy chains and two light chains.

[0026] Based on the antibodies or fragments thereof provided by the present invention, the present invention also provides conjugates or fusion proteins comprising the antibodies of the present invention or fragments thereof. The conjugates or fusion proteins may contain other moieties, such as cell surface receptors, small molecule compounds such as amino acids and carbohydrates, small molecule polymers, or any other moieties that modify the antibodies of the present invention, or further active proteins or polypeptides, chemically or physically bound to the antibodies of the present invention or fragments thereof.

[0027] In another aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a heavy chain CDR, a light chain CDR, a heavy chain variable region, a light chain variable region, a heavy chain or a light chain comprised in any antibody or fragment thereof according to the present invention.

[0028] In yet another aspect, the present invention provides a vector comprising the nucleic acid molecule of the present invention. The vector may be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, a phage vector, etc.

[0029] The vectors or nucleic acid molecules of the invention may be used to transform or transfect or in any way enter host cells, such as for antibody storage or expression. Thus, in a further aspect, the invention provides a host cell comprising or transformed or transfected with a nucleic acid molecule and / or vector in accordance with the invention. The host cell may be a prokaryotic or eukaryotic cell, for example a bacterial or insect, fungal, plant or animal cell.

[0030] In accordance with the disclosure of the present invention, the antibodies or fragments thereof provided by the present invention, and, where appropriate, conjugates or fusion proteins, nucleic acid molecules, vectors, and / or host cells can be obtained using any conventional techniques known in the art. The antibodies or fragments thereof, conjugates or fusion proteins, nucleic acid molecules, vectors, and / or host cells can be contained in compositions, more particularly pharmaceutical compositions, e.g., pharmaceuticals, to be used for various purposes as actually required.

[0031] Thus, in a still further aspect, the present invention also provides a composition comprising an antibody or fragment thereof, a conjugate or fusion protein, a nucleic acid molecule, a vector, and / or a host cell according to the invention. Preferably, the composition is a pharmaceutical composition, optionally comprising a pharmaceutically acceptable excipient.

[0032] The present invention also provides the following related uses of the above-mentioned subject matter, which are based on antibodies or fragments thereof capable of specifically binding to ST2 or any part thereof.

[0033] In a further aspect, the present invention provides use of the antibody or fragment thereof, conjugate or fusion protein, nucleic acid molecule, vector, host cell and / or composition in the manufacture of a medicament for preventing, treating or ameliorating a disease, preferably the disease is associated with ST2 expression or dysregulation of the IL-33 / ST2 pathway. Preferably, the disease is an inflammatory disease or an autoimmune disease, more preferably the disease is heart failure, allergic rhinitis, nasal polyps, atopic dermatitis, chronic obstructive pulmonary disease, asthma, pulmonary fibrosis, sepsis, inflammatory bowel disease, systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, Wegener's granulomatosis, or chemotherapy-associated diarrhea.

[0034] Furthermore, the present invention provides a method for preventing, treating, or ameliorating a disease, comprising administering to a subject in need thereof the antibody or fragment thereof, conjugate or fusion protein, nucleic acid molecule, vector, host cell, and / or composition of the present invention, and optionally, an additional agent or means. Preferably, the disease is associated with dysregulation of ST2 expression or the IL-33 / ST2 pathway. Preferably, the disease is an inflammatory disease or an autoimmune disease, more preferably, the disease is heart failure, allergic rhinitis, nasal polyps, atopic dermatitis, chronic obstructive pulmonary disease, asthma, pulmonary fibrosis, sepsis, inflammatory bowel disease, systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, Wegener's granulomatosis, or chemotherapy-associated diarrhea. Optional additional agent or means refers to any other hormonal or immunomodulatory agent or means that may be administered in combination with the antibody or fragment thereof, conjugate or fusion protein, nucleic acid molecule, vector, host cell and / or composition of the present invention, such as glucocorticoids, mepolizumab, dupilumab, tezepelumab, etc. Co-administration of the two may be in any manner, including simultaneous, sequential or spaced apart. The subject is a mammal, preferably a primate, more preferably a human or cynomolgus monkey; preferably, the subject is a human.

[0035] Thus, the present invention also provides pharmaceutical combinations of the antibody or fragment thereof, conjugate or fusion protein, nucleic acid molecule, vector, host cell and / or composition of the present invention, and optionally a further agent. The optional further agent refers to any other hormonal or immunomodulatory agent or means, such as glucocorticoids, mepolizumab, dupilumab, tezepelumab, etc., that may be administered in combination with the antibody or fragment thereof, conjugate or fusion protein, nucleic acid molecule, vector, host cell and / or composition of the present invention.

[0036] The present invention provides methods for detecting or diagnosing a disease, the methods comprising contacting a sample from a subject with an antibody or fragment thereof, a conjugate or fusion protein, a nucleic acid molecule, a vector, a host cell, and / or a composition. Preferably, the disease is associated with ST2 expression or dysregulation of the IL-33 / ST2 pathway. Preferably, the disease is an inflammatory disease or an autoimmune disease, more preferably, the disease is heart failure, allergic rhinitis, nasal polyps, atopic dermatitis, chronic obstructive pulmonary disease, asthma, pulmonary fibrosis, sepsis, inflammatory bowel disease, systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, Wegener's granulomatosis, or chemotherapy-associated diarrhea. The subject is a mammal, preferably a primate, more preferably a human or a cynomolgus monkey, and preferably, the subject is a human.

[0037] In yet another aspect, the present invention provides kits comprising the antibodies or fragments thereof, conjugates or fusion proteins, nucleic acid molecules, vectors, host cells and / or compositions of the invention, which can be used for detection or diagnosis, e.g., in methods for detecting or diagnosing a disease as described above.

[0038] In the present invention, mice were immunized with human ST2, culture supernatants were obtained through B cell panning, and positive clones were obtained through ELISA and further functional assay screening. The murine antibody was then humanized through antibody engineering to obtain a humanized antibody. Through activity screening experiments involving in vitro ligand binding by the antibody, antibody inhibition of in vitro effector cell activation by the ligand, and antibody inhibition of the ligand's stimulation of effector cell production of IL5, IL6, IL8, etc., antibody affinity determination, and animal drug metabolism experiments, the antibody of the present invention has been demonstrated to have higher biological activity than currently available anti-ST2 antibodies.

[0039] Embodiments of the present invention are described in detail below with reference to the accompanying figures. [Brief explanation of the drawings]

[0040] [Figure 1] 1 shows the inhibition rates of B cell clones derived from different mouse numbers in the KU812-NF-κB reporter gene assay. [Figure 2] 1 shows the inhibition rates of B cell clones derived from different mouse numbers in the KU812-NF-κB reporter gene assay. [Figure 3] 1 shows the inhibition rates of B cell clones derived from different mouse numbers in the KU812-NF-κB reporter gene assay. [Figure 4] 1 shows the inhibition rates of B cell clones derived from different mouse numbers in the KU812-NF-κB reporter gene assay. [Figure 5] 1 shows the inhibition rates of B cell clones derived from different mouse numbers in the KU812-NF-κB reporter gene assay. [Figure 6A] FIG. 1 shows the inhibitory activity of murine antibodies against IL-33-induced promotion of KU812-IL5 production. [Figure 6B] This is a continuation of Figure 6A. [Figure 7] FIG. 1 shows binding of murine antibodies to cynomolgus monkey ST2. [Figure 8] FIG. 1 shows binding of murine antibodies to mouse ST2. [Figure 9A] FIG. 1 shows blocking assay screening of humanized antibodies for binding of IL33 to human ST2. [Figure 9B] This is a continuation of Figure 9A. [Figure 9C] This is a continuation of Figure 9B. [Figure 10A] FIG. 1 shows the inhibitory activity of humanized antibodies against IL-33-induced promotion of KU812-IL5 production. [Figure 10B] This is a continuation of Figure 10A. [Figure 11]FIG. 1 shows the inhibitory activity of humanized antibodies against the promotion of KU812-IL5 production by oxidized IL-33. [Figure 12] FIG. 1 shows the inhibitory activity of humanized antibodies against the promotion of KU812-IL5 production by reduced IL-33. [Figure 13] FIG. 1 shows the inhibitory activity of humanized antibodies against IL33-induced promotion of HUVEC-IL6 production. [Figure 14] FIG. 1 shows the inhibitory activity of humanized antibodies against IL33-induced promotion of HMC-1-IL8 production. [Figure 15A] Figure 1 shows the PK curves of humanized antibodies in mice: Panel 15-1: 5888-116-H0L1, Panel 15-2: 5888-153-H0L1, Panel 15-3: CNTO7160, Panel 15-4: 5886-156-H1L0. [Figure 15B] This is a continuation of Figure 15A. DETAILED DESCRIPTION OF THE INVENTION

[0041] The present invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are merely illustrative of the invention and do not limit the scope of the invention in any way.

[0042] All experimental methods in the following examples are conventional unless otherwise specified. All materials and reagents used in the following examples are commercially available products unless otherwise specified.

[0043] Human ST2: NP_003847.2 (Met1~Phe328) Human ST2-his: Human ST2 fused with a 6-histidine tag at the C-terminus Human ST2-fc: Human ST2 fused to a human IgG1 Fc tag at the C-terminus Human IL33: NP_254274.1 (Ser112-Thr270) Human IL33-his: Human IL33 fused to a 6-histidine tag at the C-terminus Oxidized human IL33-his: Human IL-33-his was diluted to 300 μg / ml in IMDM, incubated at 37°C for 18 hours, and then purified using an S75 16:600 Superdex column (GE Healthcare). Reduced human IL33-his: Human IL33-his with Cys208Ser and Cys259Ser Control antibody CNTO7160: having a heavy chain as set forth in SEQ ID NO: 104 and a light chain as set forth in SEQ ID NO: 105 Cynomolgus monkey ST2: XP_005575214.1 (Met1~Cys331) Cynomolgus ST2-fc: Cynomolgus ST2 fused to a human IgG1 Fc tag at the C-terminus Mouse ST2: NP_001020773.1 (Met1 to Arg332) Mouse ST2-fc: Mouse ST2 fused to a human IgG1 Fc tag at the C-terminus [Example]

[0044] Example 1: Murine antibody screening 1.1 Animal immunity Human ST2-his was expressed using CHO-K1 cells, and six BALB / c mice were then immunized with Freund's adjuvant according to a conventional immunization program. Two batches of mice, each containing three mice, were immunized, with a two-week interval between batches. Each batch of mice was immunized four times and tested by ELISA using human ST2-his. Any mouse with a serum titer greater than 1:100,000 was considered final immunization, and spleens were harvested three to four days later.

[0045] 1.2 B cell panning and culture Two days before the formal experiment, feeder cells were plated in four 10 cm plates (Corning, Cat. No. 430167) using culture medium, and one day before the formal experiment, they were treated with 25 μg / mL MMC for 6 hours, and then plated into a 96-well plate (Corning, Cat. No. 3599) at 10,000 cells / well and 100 μL / well. Furthermore, the antigen human ST2-his was coated into a 6-well plate overnight at 4°C.

[0046] The spleens collected from immunized mice were mashed, filtered, and centrifuged, and red blood cell lysate was added to remove red blood cells. Treatment with red blood cell lysate was repeated several times until no visible red blood cells were present, and DC cells were then removed from the spleen cells. Spleen cells from one spleen were uniformly plated in a 6-well plate coated with the above antigen for panning. After antigen panning, B cells were harvested with trypsin, counted, and plated in a 96-well plate coated with the above feeder cells. The cells were cultured at 37°C and 5% CO2 for 10 to 14 days, and the B cell culture supernatant from wells in which clear clones were formed was collected for the following screening.

[0047] 1.3 Screening of supernatants containing murine antibodies (1) ELISA screening of murine antibodies that bind to human ST2-his The immunogen human ST2-his, as described above, was diluted to 1 μg / mL in coating buffer and added to ELISA plates at 50 μL / well and coated overnight at 4°C. The next day, the coated plates were removed and washed three times with PBST, followed by incubation at room temperature for 1 hour using blocking buffer. Subsequently, the plates were washed three times with PBST again, and B cell culture supernatant was added to the ELISA plates and incubated at room temperature for 1 hour. The plates were washed three times with PBST, and then goat anti-mouse secondary antibody (1:10,000) was added to the plates at 50 μL / well, followed by incubation at room temperature for 1 hour. The 96-well plates were washed three times with PBST, and TMB was added at 50 μL / well for 10 minutes of color development in the dark. The reaction was then stopped by adding 2 M sulfuric acid, and the OD values ​​were read at 450 nm using a microplate reader.

[0048] The results showed that the B cell clones derived from two mice, numbered 5883 and 5884, had a positive rate of over 97% in ELISA for screening murine antibodies binding to human ST2-his, where an OD value 10 times higher than that of the negative control (blank medium) was used as the criterion for positivity.

[0049] 1.4 KU812-NF-κB reporter gene assay screening 1 x 10 in logarithmic growth phase 6 KU812 cells were harvested, washed by centrifugation once, and resuspended in 20 μL of buffer R from the Neon Transfection System 10 μL Kit. 1 μg of pGL4.32[luc2P / NF-κB-RE / Hygro] vector was added to the cells, followed by transfection by a single electric shock at 1000 V for 50 ms. After transfection, pressure screening was performed using hygromycin B, and the final cell line, KU812 / NF-κB-1#, was obtained.

[0050] Human IL33-his was diluted to 1 μg / mL in culture medium and mixed with each B cell culture supernatant at a 1:1 ratio to obtain test samples. Additionally, a negative control sample (diluted human IL33-his mixed with blank medium at a 1:1 ratio) and a positive control sample (diluted human IL33-his mixed with 1 μg / mL CNTO7160 at a 1:1 ratio) were prepared. The samples were added to a 384-well plate at 20 μL / well, respectively. KU812 / NF-κB-1# cells in the logarithmic growth phase were harvested and added to the 384-well plate at 20,000 / well and 20 μL / well, respectively, and incubated overnight (16-24 hours) at 37°C, 5% CO2. Then, 40 μL / well of Bright-glo reagent was added into the 384-well plate, the plate was shaken for 3 minutes, and detected by a microplate reader to read the RLU value.

[0051] The inhibition rate of each clone was calculated relative to the negative and positive control values, and the results are shown in Figures 1 to 5.

[0052] Example 2: Antibody Engineering 2.1 Antibody sequencing in B cells mRNA was extracted from B cell clones using the PureLink™ RNA Mini Kit according to the instructions in the kit, subpackaged, and stored at -80°C. The extracted mRNA was used as a template and reverse transcribed into cDNA using the PrimeScript™ II First Strand cDNA Synthesis Kit according to the instructions in the kit, which was then subpackaged and stored at -80°C.

[0053] Using Ex Taq enzyme and the heavy chain VH and light chain VL amplification primers shown in Table 1-1 and Table 1-2, the VH and VL sequences were amplified using the above cDNA as a template and then ligated into the pMD18T vector for sequencing.

[0054] [Table 1-1]

[0055] [Table 1-2A] [Table 1-2B]

[0056] 2.2 Recombinant expression and screening of murine antibodies (1) Recombinant expression of murine antibodies Light and heavy chains from the same clone (e.g., those shown in Figures 1-5) were co-transfected into CHO-K1 cells in pairs. 24 hours after transfection, 10 μg / mL MSX was added for pressure screening. After recovering cell density and viability, the cells were inoculated for fed-batch expression. Upon completion of expression, the supernatant was centrifuged and purified by protein A. After determining the antibody concentration by the BCA method, the resulting antibody was used for quantitative screening.

[0057] Murine antibodies were named after the cell clone number from which their light and heavy chains (H+L) were derived. For example, murine antibody "5883-105H+L" refers to a murine antibody derived from cell clone numbered 5883-105, whose heavy chain is 5883-105H and whose light chain is 5883-105L.

[0058] (2) Screening of murine antibodies for binding activity to human ST2 Human ST2-his was diluted to 1 μg / mL in coating buffer and added to the plate at 50 μL / well for overnight coating at 4°C. The next day, the coated plate was removed, washed three times with PBST, and then incubated at room temperature for 1 hour using blocking buffer. The plate was then washed three times with PBST again. Starting at 100 ng / mL, each murine antibody was diluted three-fold to obtain a total of eight serial dilutions, which were then added to the 96-well plate at 50 μL / well. The plate was incubated at room temperature for 1 hour, washed three times with PBST, and then goat anti-mouse secondary antibody (1:10,000) was added to the 96-well plate at 50 μL / well, followed by incubation at room temperature for 1 hour. The 96-well plate was washed three times with PBST, and TMB was added at 50 μL / well for 10 minutes of development in the dark. The reaction was then stopped by adding 2M sulfuric acid, and the OD value was read at 450 nm using a microplate reader. The results are shown in Table 2.

[0059] [Table 2A] [Table 2B] [Table 2C]

[0060] (3) Blocking assay screening of murine antibodies against IL33 binding to human ST2 Human ST2-fc was diluted to 10 μg / mL in coating buffer and added to a 96-well plate at 50 μL / well, followed by overnight coating at 4°C in a refrigerator. The coated plate was washed three times with PBST, and then 100 μL / well of blocking buffer was added to the plate, followed by 1 hour of incubation at room temperature. The plate was then washed three times with PBST. Human IL33-his was diluted to 200 ng / mL in diluent solution, and each murine antibody was diluted to 200 μg / mL in diluent solution and then diluted three-fold starting from 200 μg / mL to obtain a total of eight serial dilutions. The diluted human IL33-his and each diluted murine antibody were mixed at a 1:1 ratio, and the mixture was then added to a 96-well plate at 50 μL / well, followed by 1 hour of incubation at room temperature. The plate was washed three times with PBST, and then His-tagged secondary antibody (1:2500) was added at 50 μL / well. The plate was incubated at room temperature for 1 hour. The 96-well plate was washed three times again with PBST, and TMB was added at 50 μL / well. The color was developed in the dark for 10 minutes. The reaction was then stopped by adding 100 μL / well of 2 M sulfuric acid, and the OD values ​​at 450 nm and 650 nm were read using a microplate reader. The results are shown in Table 3.

[0061] [Table 3A] [Table 3B] [Table 3C]

[0062] (4) Screening of murine antibodies against IL33-induced activation of the KU812-NF-κB reporter gene. Human IL33-his was diluted to 1 μg / mL in culture medium. The murine antibody and control antibody CNTO7160 were each diluted to 50 μg / mL in culture medium and then diluted 3-fold to obtain a total of 12 serial dilutions. The antibody was mixed with diluted human IL33-his at a 1:1 ratio to obtain test samples. In addition, a negative control sample (diluted human IL33-his mixed with blank medium at a 1:1 ratio) and a positive control sample (blank medium) were prepared. 20 μL of each sample was added to a 384-well plate.

[0063] Logarithmically growing KU812 / NF-κB-1# cells were centrifuged, transferred to fresh medium, and added to the 384-well plate at 20,000 / well and 20 μL / well. The cells were incubated overnight (16-24 hours) at 37°C and 5% CO2. Bright-glo developer was then added to the 384-well plate at 40 μL / well. The plate was shaken for 3 minutes and then detected using a microplate reader, and the RLU values ​​were read. The results are shown in Table 4.

[0064] [Table 4A] [Table 4B]

[0065] (5) Affinity screening of murine antibodies Based on the combined results of the quantitative screening of the above murine antibodies, 19 murine antibodies were selected for affinity determination and in vitro pharmacological studies. Experiments on the interaction between anti-human ST2 antibodies and human ST2-his were performed using a Biacore X100 in HBS-EP (1x) buffer (pH 7.4) at 25°C.

[0066] Each anti-human ST2 antibody was diluted to 10 nM and captured onto the surface of a Protein A chip (60-second capture time). After antibody capture, human ST2-his solutions (2-fold gradient dilutions from 11.8 nM to 0.7375 nM, total of five concentrations) were injected. Association was monitored for 4 minutes, and dissociation for 10 minutes. The sensor surface was regenerated by injecting a glycine solution at pH 2.0. The data generated for the kinetic and affinity assays were analyzed using BIAevaluation software. A simple 1:1 binding model was used to analyze the kinetic data, and the results are shown in Table 5.

[0067] [Table 5]

[0068] (6) In vitro pharmacological studies of murine antibodies Human IL33-his was diluted to 80 ng / mL in culture medium. Each murine antibody was diluted to 40 μg / mL in culture medium and then diluted 4-fold to obtain a total of eight serial dilutions. The diluted antibody was mixed with diluted human IL33-his at a 1:1 ratio, and the resulting mixture was added to a 96-well plate at 50 μL / well. Logarithmically growing KU812 cells were centrifuged and added to a 96-well plate at 100,000 / well and 50 μL / well, followed by incubation for 48 hours.

[0069] One day prior to the assay, 50 μL of the capture antibody included in the human IL-5 DuoSet ELISA kit was coated overnight at 4°C in an ELISA plate at 50 μL / well, using a 2 μg / mL working solution prepared by diluting the 240 μg / mL stock solution 120-fold with PBS, according to the instructions. The plate was then blocked for 1 hour and washed three times. The 120 ng / mL standard was diluted 400-fold to 300 pg / mL, and then diluted 2-fold to obtain a total of seven serial dilutions. 50 μL each of the cell culture supernatant and one of the diluted standards was pipetted into the ELISA plate and incubated for 2 hours. The plate was washed three times, and 50 μL / well of a 125 ng / mL working solution prepared by diluting the detection antibody (from a 7.5 μg / mL stock solution) 60-fold was added to the plate at 50 μL / well, followed by incubation for 2 hours. The plate was washed three times again, and a 125 ng / mL working solution obtained by diluting SA-HRP 40-fold was added to the plate at 50 μL / well, followed by incubation for 20-30 minutes. Substrate solution was added to the plate at 50 μL / well, and color development was allowed to occur in the dark for 5-10 minutes. The reaction was then stopped by adding 100 μL / well of 2 M sulfuric acid, and the OD values ​​at 450 nm and 650 nm were read using a microplate reader. The results are shown in Table 6 and Figures 6A and 6B.

[0070] [Table 6]

[0071] (7) Cross-reactivity of mouse ST2 and cynomolgus monkey ST2 with murine antibodies Cynomolgus monkey ST2-fc was diluted to 1 μg / mL in coating buffer and added to a 96-well plate at 50 μL / well for overnight coating at 4°C. The next day, the coated plate was removed, washed three times with PBST, and then incubated at room temperature for 1 hour with blocking buffer. The plate was then washed three times with PBST. Starting at 1000 ng / mL, each murine antibody was diluted three-fold to obtain a total of eight serial dilutions, which were then added to a 96-well plate at 50 μL / well. The plate was incubated at room temperature for 1 hour, washed three times with PBST, and then goat anti-mouse secondary antibody (1:10,000) was added to the 96-well plate at 50 μL / well, followed by incubation at room temperature for 1 hour. The 96-well plate was washed three times with PBST, and TMB was added at 50 μL / well for 10 minutes of development in the dark. The reaction was then stopped by adding 2 M sulfuric acid, and the OD value at 450 nm was read using a microplate reader. The results are shown in Table 7 and Figure 7. The murine antibody was found to be able to bind to cynomolgus monkey ST2, and the binding tendency was consistent with that of human ST2.

[0072] [Table 7]

[0073] Experiments on the interaction between anti-human ST2 antibodies and cynomolgus ST2-fc were performed at 25°C in HBS-EP (1x) buffer (pH 7.4) using a Biacore X100. Each antibody was immobilized on the surface of a CM5 chip using an amino coupling kit. After antibody immobilization, cynomolgus ST2-fc solution (2-fold gradient dilution from a starting concentration of 8 nM to obtain a total of six concentrations; if the signal of some samples was too low, the starting concentration could be increased) was injected. Association was monitored for 2 minutes and dissociation for 10 minutes, and the sensor surface was regenerated by injecting a glycine solution at pH 1.5. The kinetic data were analyzed using a simple 1:1 binding model, and the results are shown in Table 8.

[0074] [Table 8]

[0075] Mouse ST2-fc was diluted to 1 μg / mL in coating buffer and added to a 96-well plate at 50 μL / well and coated overnight at 4°C. The next day, the coated plate was removed, washed three times with PBST, and then incubated at room temperature for 1 hour using blocking buffer. The plate was then washed three times with PBST again. Starting at 1000 ng / mL, each murine antibody was diluted three-fold to obtain a total of eight serial dilutions, which were then added to a 96-well plate at 50 μL / well. The plate was incubated at room temperature for 1 hour, washed three times with PBST, and then goat anti-mouse secondary antibody (1:10,000) was added to the 96-well plate at 50 μL / well, followed by incubation at room temperature for 1 hour. The 96-well plate was washed three times with PBST, and TMB was added at 50 μL / well and allowed to develop in the dark for 10 minutes. Then, the reaction was stopped by adding 2 M sulfuric acid, and the OD value at 450 nm was read using a microplate reader. The results are shown in Figure 8. It can be seen that the murine antibody can bind weakly to mouse ST2, similar to the control antibody CNTO7160.

[0076] 2.3 Humanization Seven murine antibodies were selected for humanization design: 5886-156H+L, 5887-41H+L, 5887-537H3H+L1, 5888-116H1+L1, 5888-153H1+L2, 5888-357H+L, and 5888-379H1+L2.

[0077] The heavy and light chain variable region sequences of seven murine antibodies were compared with human germline sequences by blast searches in the IMGT database. Duplicate genes and those containing unpaired cysteines were removed from the human germline genes. The human germline gene with the best-matched framework and CDR regions was selected from the remaining ones, and the framework region within it was used as the human acceptor framework. FR-4 was selected based on sequence similarity to the IGHJ / IGJK germline genes. Tables 9-15 show the humanized sequences of seven murine antibodies, 5886-156H+L, 5887-41H+L, 5887-537H3H+L1, 5888-116H1+L1, 5888-153H1+L2, 5888-357H+L, and 5888-379H1+L2, respectively, where HZ0 represents the CDR-grafted version, HZ1 has backmutation(s) introduced, and HZ2 and further versions have mutation(s) directed at PTM sites present in the sequence. Specific sequences after humanization are shown in Tables 9-15, with the corresponding CDRs (as defined by the modified Chothia / AbM) underlined.

[0078] [Table 9]

[0079] [Table 10]

[0080] [Table 11]

[0081] [Table 12A] [Table 12B]

[0082] [Table 13A] [Table 13B]

[0083] [Table 14A] [Table 14B]

[0084] [Table 15A] [Table 15B]

[0085] 2.4 Recombinant Expression and Screening of Humanized Antibodies (1) Recombinant expression of humanized antibodies Humanized light and heavy chains derived from the same murine antibody were constructed using the heavy chain constant region as shown in SEQ ID NO: 54 and the light chain constant region as shown in SEQ ID NO: 55, and were co-transfected pairwise into CHO-K1 cells. 24 hours after transfection, 10 μg / mL MSX was added for pressure screening. After recovering cell density and viability, the cells were inoculated for fed-batch expression. Upon completion of expression, the supernatant was centrifuged and purified by protein A. After determining the antibody concentration by the BCA method, the resulting antibody was used for quantitative screening.

[0086] Humanized antibodies and the paired light chain variable regions and heavy chain variable regions contained in these antibodies are shown in Tables 16 to 22. Antibodies with names ending in "ix" are corresponding chimeric antibodies.

[0087] [Table 16]

[0088] [Table 17]

[0089] [Table 18]

[0090] [Table 19]

[0091] [Table 20]

[0092] [Table 21]

[0093] [Table 22]

[0094] (2) Blocking assay screening of humanized antibodies against IL33 binding to human ST2 Detection was performed according to the experimental method described in Section 2.2 above, "(3) Blocking assay screening of murine antibodies for binding of IL33 to human ST2." The results are shown in Figures 9A, B, and C and Table 23. In Figures 9A, B, and C, the negative control was a mixture of blank medium and diluted human IL33-his in a 1:1 ratio, and the positive control was blank medium.

[0095] [Table 23]

[0096] (3) Inhibitory activity of humanized antibodies against IL-33-induced promotion of KU812-IL5 production Human IL33-his was diluted to 80 ng / mL in culture medium. Each humanized antibody was diluted to 640 μg / mL in culture medium and then diluted 4-fold to obtain a total of 11 serial dilutions. The diluted antibody was mixed with diluted human IL33-his at a 1:1 ratio, and the resulting mixture was added to a 96-well plate at 50 μL / well. The subsequent experimental method was the same as that described in Section 2.2 above, "(6) In vitro pharmacological study of murine antibodies." The results are shown in Figures 10A and 10B and Table 24. In Figures 10A and 10B, the negative control was a mixture of blank medium and diluted human IL33-his at a 1:1 ratio, and the positive control was blank medium.

[0097] [Table 24]

[0098] (4) Screening of humanized antibodies against IL33-induced activation of the KU812-NF-κB reporter gene Detection was performed according to the experimental method described in Section 2.2 above, "(4) Screening of murine antibodies against IL33-induced activation of the KU812-NF-κB reporter gene." The results are shown in Table 25.

[0099] [Table 25]

[0100] (5) Inhibitory activity of humanized antibodies against oxidized IL-33-induced promotion of KU812-IL5 production Oxidized human IL33-his was diluted to 200 ng / mL in culture medium. Each humanized antibody was diluted to 640 μg / mL in culture medium and then diluted 4-fold to obtain a total of 11 serial dilutions. The diluted antibody was mixed with diluted human IL33-his in a 1:1 ratio, and the resulting mixture was added to a 96-well plate at 50 μL / well. The subsequent experimental method was the same as that described in Section 2.2 above, "(6) In vitro pharmacological study of murine antibodies." The results are shown in Figure 11 and Table 26. In Figure 11, the negative control was a mixture of blank medium and diluted human IL33-his in a 1:1 ratio, and the positive control was blank medium.

[0101] [Table 26]

[0102] (6) Inhibitory activity of humanized antibodies against the promotion of KU812-IL5 production by reduced IL-33 Reduced human IL33-his was diluted to 6 ng / mL in culture medium. Each humanized antibody was diluted to 640 μg / mL in culture medium and then diluted 4-fold to obtain a total of 11 serial dilutions. The diluted antibody was mixed with diluted human IL33-his at a 1:1 ratio, and the resulting mixture was added to a 96-well plate at 50 μL / well. The subsequent experimental method was the same as that described in Section 2.2 above, "(6) In vitro pharmacological study of murine antibodies." The results are shown in Figure 12 and Table 27. In Figure 12, the negative control was a mixture of blank medium and diluted human IL33-his at a 1:1 ratio, and the positive control was blank medium.

[0103] [Table 27]

[0104] (7) Inhibitory activity of humanized antibodies against IL33-induced promotion of HUVEC-IL6 production HUVEC cells were incubated in a 96-well plate at 10,000 / well and 100 μL / well at 37°C, 5% CO2 for 18-24 hours. Human IL33-his was diluted to 10 ng / mL in culture medium. Each humanized antibody was diluted to 400 μg / mL in culture medium and then diluted 4-fold to obtain a total of 11 serial dilutions. The diluted antibody was mixed with diluted human IL33-his at a 1:1 ratio, and the resulting mixture was added to the 96-well plate at 50 μL / well and then incubated at 37°C, 5% CO2 for 18-24 hours.

[0105] One day prior to the assay, 50 μL of the capture antibody included in the human IL-6 DuoSet ELISA Kit was coated into an ELISA plate overnight at 4°C, using 50 μL of the 2 μg / mL working solution obtained by diluting the 240 μg / mL stock solution 120-fold with PBS, according to the instructions. The plate was then blocked for 1 hour using blocking buffer and then washed three times. The 180 ng / mL standard was diluted 300-fold to 600 pg / mL, and then diluted 2-fold to obtain a total of seven serial dilutions. 50 μL each of the cell culture supernatant and one of the diluted standards was pipetted into the ELISA plate and incubated for 2 hours. The plate was washed three times, and 50 μL of the 50 ng / mL working solution obtained by diluting the detection antibody (from a 3 μg / mL stock solution) 60-fold was added to the plate at 50 μL / well, followed by incubation for 2 hours. The plate was washed three times again, and a 125 ng / mL working solution obtained by diluting SA-HRP 40-fold was added to the plate at 50 μL / well, followed by incubation for 20 to 30 minutes. Substrate solution was added to the plate at 50 μL / well, and color development was allowed to occur in the dark for 5 to 10 minutes. The reaction was then stopped by adding 100 μL / well of 2 M sulfuric acid, and the OD values ​​at 450 nm and 650 nm were read using a microplate reader. The results are shown in Table 28 and Figure 13. In Figure 13, the negative control was a 1:1 mixture of blank medium and diluted human IL33-his, and the positive control was blank medium.

[0106] [Table 28]

[0107] (8) Inhibitory activity of humanized antibodies against IL33-induced promotion of HMC-1 IL8 production Human IL33-his was diluted to a final concentration of 1000 ng / mL. Starting at 640 μg / mL, each humanized antibody was diluted 4-fold to obtain a total of 11 serial dilutions. The diluted antibody was mixed with diluted human IL33-his at a 1:1 ratio, and the resulting mixture was added to a 96-well plate at 50 μL / well. Logarithmic growth phase HMC-1 cells were harvested and added to a 96-well plate at 50,000 / well and 50 μL / well, and incubated at 37°C, 5% CO2 for 18 to 24 hours.

[0108] One day before, 50 μL of the capture antibody included in the human IL-8 DuoSet ELISA Kit was diluted 120-fold with PBS as a working solution and coated overnight at 4°C in an ELISA plate. The plate was then blocked for 1 hour using blocking buffer and washed three times. The standard was diluted 40-fold to 2000 pg / mL, then diluted 2-fold to obtain a total of seven serial dilutions. 50 μL each of the cell culture supernatant and one of the diluted standards was pipetted into the ELISA plate and incubated for 2 hours. The plate was washed three times, and 50 μL / well of the working solution, obtained by diluting the detection antibody 60-fold, was added to the plate and incubated for 2 hours. The plate was washed three times again, and 50 μL / well of the working solution, obtained by diluting SA-HRP 40-fold to 125 ng / mL, was added to the plate and incubated for 20 to 30 minutes. The substrate solution was added to the plate at 50 μL / well, and the color was allowed to develop in the dark for 5 to 10 minutes. The reaction was then stopped by adding 100 μL / well of 2 M sulfuric acid, and the OD values ​​at 450 nm and 650 nm were read using a microplate reader. The results are shown in Table 29 and Figure 14. In Figure 14, the negative control was a 1:1 mixture of blank medium and diluted human IL33-his, and the positive control was blank medium.

[0109] [Table 29]

[0110] (9) Determination of affinity of humanized antibodies An experiment on the interaction between anti-human ST2 antibody and human ST2-his was carried out using Biacore X100.

[0111] (9-1) Affinity experiment of dissociation kinetics of human ST2 from humanized antibody at pH 7.4: The experiment was carried out in HBS-EP (1×) buffer (pH 7.4) at 25°C. Each anti-human ST2 antibody was diluted to 2 μg / mL and captured on the surface of a Protein A chip with a 60-second capture time. After antibody capture, a human ST2-his solution (diluted in a two-fold gradient from a starting concentration of 20 nM, resulting in a total of six concentrations) was injected. Association was monitored for 180 seconds, dissociation was monitored for 700 seconds, and the sensor surface was regenerated by injecting a glycine solution at pH 2.0. Kinetic data were analyzed using a simple 1:1 binding model.

[0112] (9-2) Affinity experiments of dissociation kinetics of human ST2 from humanized antibody at pH 5.5: The experiment was carried out according to the experimental method as described in (9-1), except that the dissociation was carried out in HBS-EP (1x) buffer (pH 5.5) and the dissociation was monitored for 600 seconds.

[0113] The results are shown in Table 30.

[0114] [Table 30]

[0115] Example 3: Pharmacokinetics of antibodies in mice After an acclimation period, 20 mice were randomly divided into 4 groups of 5 mice each. Dosing information is shown in Table 31.

[0116] [Table 31]

[0117] Mice were administered according to group assignment, and the administration time was recorded. Blood samples were taken from each group of mice before administration (0 hours), and 4, 8, 24, 72, 120, 168, 240, 288, and 336 hours after administration. Serum was collected and stored at -60 to -80°C.

[0118] Blood drug concentrations for PK studies in mice were determined as follows. (1) Coating. Human ST2-his was diluted to 1 μg / mL in PBS (pH 7.2-7.4) and added to a 96-well ELISA plate at 50 μL / well, which was then sealed with film. The plate was simmered at 2-8°C for 15-20 hours and then washed three times with PBST (containing 0.05% (v / v) Tween-20 (pH 7.2-7.4)). (2) Blocking and drying. The plate was blocked with 200 μL / well of N502 at room temperature for 1 to 2 hours. After absorbing the blocking solution, the plate was dried in an incubator at 25°C for more than 1 hour, and then used immediately or sealed with film and stored at 2 to 8°C. (3) Preparation of standards used for plotting the standard curve and quality control. Antibody samples were diluted to 1000 ng / mL using mouse plasma (EDTA-K), followed by two-fold gradient dilutions to 15 ng / mL (seven concentrations, including 1000 ng / mL). Additionally, the same antibody samples used to plot the standard curve were quantitatively diluted to concentrations within the concentration range for quantitative analysis and used as quality controls (QCs). Specifically, the antibody samples were diluted to 1000 ng / mL, 100 ng / mL, and 20 ng / mL, respectively. (4) Preparation of samples to be detected: Samples at different blood sampling time points were obtained and diluted to concentrations within the concentration range used to plot the standard curve. (5) Dilution of standards, quality controls, and samples. Standards, quality controls, and samples were diluted 10-fold (e.g., 10 μL diluted with 90 μL) in 0.1% casein (pH 6.2, obtained by diluting casein stock solution with PBS (pH 6.2)) and added to the dried plate at 50 μL / well, creating two replicates per sample. The plate was sealed with film and incubated at room temperature for 2 hours, then washed three times with PBST (0.05% (v / v) Tween-20 (pH 7.2-7.4)). (6) Incubation with secondary antibody. Goat anti-human IgG Fc-HRP was diluted 50,000-fold with 10% goat serum and added to the plate at 50 μL / well. The plate was then sealed and incubated at room temperature for 1 hour. The plate was then washed three times with PBST (0.05% (v / v) Tween-20 (pH 7.2-7.4)). (7) Color development: TMB warmed to room temperature was added to the plate at 50 μL / well, and color development was carried out in the dark for 20 minutes. (8) Stopping the reaction and reading the OD value. 2M sulfuric acid was added to the plate at 100 μL / well, and then gently tapped to mix the sulfuric acid evenly. The OD reading at 450 nm was used as a reference at 650 nm to calculate the concentration of each tested sample.

[0119] The experimental results showed that the three antibodies had half-lives longer than 100 hours, with the order being 5886-156-H1L0 > 5888-153-H0L1 > 5888-116-H0L1. Of the three antibodies, antibody 5886-156H1L0 had a half-life of 10 days, comparable to that of CNTO7160. The results are shown in Figures 15A and 15B and Table 32.

[0120] [Table 32]

[0121] The above description of the embodiments of the present invention is not intended to limit the present invention, and those skilled in the art can make various changes and modifications to the present invention without departing from the spirit of the present invention, which should be included in the scope of the appended claims.

Claims

1. An antibody or antigen-binding fragment thereof that binds to ST2, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL); The heavy chain variable region and the light chain variable region are the following combination: (1) H-CDR1, H-CDR2, H-CDR3 shown in SEQ ID NOs: 56, 63, 70, and L-CDR1, L-CDR2, L-CDR3 shown in SEQ ID NOs: 85, 93, 97, (2) H-CDR1, H-CDR2, H-CDR3 shown in SEQ ID NOs: 60, 67, 76, and L-CDR1, L-CDR2, L-CDR3 shown in SEQ ID NOs: 90, 95, 101, and (3) H-CDR1, H-CDR2, H-CDR3 shown in SEQ ID NOs: 62, 69, 84, and L-CDR1, L-CDR2, L-CDR3 shown in SEQ ID NOs: 92, 96, 103, An antibody or antigen-binding fragment thereof comprising a heavy chain CDR and a light chain CDR selected from any one of the following:

2. The antibody or antigen-binding fragment thereof is a combination of: (1) the heavy chain variable region shown in SEQ ID NO: 1 and the light chain variable region shown in SEQ ID NO: 29; (2) the heavy chain variable region shown in SEQ ID NO: 3 and the light chain variable region shown in SEQ ID NO: 30; (3) the heavy chain variable region shown in SEQ ID NO: 3 and the light chain variable region shown in SEQ ID NO: 31; (4) The heavy chain variable region shown in SEQ ID NO: 15 and the light chain variable region shown in SEQ ID NO: 42; (5) The heavy chain variable region shown in SEQ ID NO: 16 and the light chain variable region shown in SEQ ID NO: 43; (6) The heavy chain variable region shown in SEQ ID NO: 16 and the light chain variable region shown in SEQ ID NO: 44; (7) The heavy chain variable region shown in SEQ ID NO: 17 and the light chain variable region shown in SEQ ID NO: 44; (8) The heavy chain variable region represented by SEQ ID NO: 28 and the light chain variable region represented by SEQ ID NO: 53, and (9) The heavy chain variable region shown in SEQ ID NO: 28 and the light chain variable region shown in SEQ ID NO: 50; The antibody or antigen-binding fragment thereof according to claim 1, comprising a heavy chain variable region and a light chain variable region selected from any one of the following:

3. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof binds to mammalian ST2.

4. An antibody or antigen-binding fragment thereof described in claim 1 or 2, wherein the antibody or antigen-binding fragment thereof binds to primate ST2.

5. An antibody or antigen-binding fragment thereof described in claim 1 or 2, wherein the antibody or antigen-binding fragment thereof binds to human ST2 or cynomolgus monkey ST2.

6. The antibody or antigen-binding fragment thereof of claim 1 or 2, wherein the antibody is a monoclonal antibody, a single-chain antibody, a diabody, a fully or partially humanized antibody, or a chimeric antibody.

7. An antibody or its antigen-binding fragment described in claim 1 or 2, wherein the antibody is IgA, IgD, IgE, IgG or IgM.

8. An antibody or its antigen-binding fragment described in claim 1 or 2, wherein the antibody is an IgG1, IgG2 or IgG4 antibody.

9. An antibody or its antigen-binding fragment described in claim 1 or 2, wherein the antigen-binding fragment is a functionally active fragment of the antibody that can specifically bind to ST2.

10. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antigen-binding fragment is an scFv, BsFv, dsFv, (dsFv) 2 , Fab, Fab', F(ab') 2 , or Fv of the antibody.

11. The antibody or antigen-binding fragment thereof described in claim 1 or 2, wherein the antibody further comprises a heavy chain constant region that is of the IgG1, IgG2, or IgG4 subtype, or the antibody comprises a light chain constant region that is of the κ subtype.

12. The antibody or antigen-binding fragment thereof described in claim 11, wherein the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 54 or an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO: 54, and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 55 or an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO:

55.

13. A conjugate or fusion protein comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 12.

14. A nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 12.

15. A vector comprising the nucleic acid molecule of claim 14.

16. A host cell comprising or transformed or transfected with a nucleic acid molecule according to claim 14 and / or a vector according to claim 15.

17. A composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 12, the conjugate or fusion protein of claim 13, the nucleic acid molecule of claim 14, the vector of claim 15, and / or the host cell of claim 16.

18. The composition of claim 17, which is a pharmaceutical composition and optionally comprises a pharmaceutically acceptable excipient.

19. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, a conjugate or fusion protein according to claim 13, a nucleic acid molecule according to claim 14, a vector according to claim 15, a host cell according to claim 16, and / or a composition according to claim 17 or 18 in the manufacture of a medicament for preventing, treating, or ameliorating a disease associated with ST2 expression or dysregulation of the IL-33 / ST2 pathway.

20. The use of claim 19, wherein the disease is an inflammatory disease or an autoimmune disease.

21. The use according to claim 19 or 20, wherein the disease is heart failure, allergic rhinitis, nasal polyps, atopic dermatitis, chronic obstructive pulmonary disease, asthma, pulmonary fibrosis, sepsis, inflammatory bowel disease, systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, Wegener's granulomatosis, or chemotherapy-induced diarrhea.

22. 19. A pharmaceutical combination comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, a conjugate or fusion protein according to claim 13, a nucleic acid molecule according to claim 14, a vector according to claim 15, a host cell according to claim 16, and / or a composition according to claim 17 or 18, and optionally a further agent.

23. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, a conjugate or fusion protein according to claim 13, a nucleic acid molecule according to claim 14, a vector according to claim 15, a host cell according to claim 16, and / or a composition according to claim 17 or 18 in the manufacture of an agent for detecting or diagnosing a disease associated with ST2 expression or dysregulation of the IL-33 / ST2 pathway.

24. The use of claim 23, wherein the disease is an inflammatory disease or an autoimmune disease.

25. The use according to claim 23 or 24, wherein the disease is heart failure, allergic rhinitis, nasal polyps, atopic dermatitis, chronic obstructive pulmonary disease, asthma, pulmonary fibrosis, sepsis, inflammatory bowel disease, systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, Wegener's granulomatosis, or chemotherapy-induced diarrhea.

26. 19. A kit comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 12, the conjugate or fusion protein of claim 13, the nucleic acid molecule of claim 14, the vector of claim 15, the host cell of claim 16, and / or the composition of claim 17 or 18.

Images