Anti-ST2 antibody and its application

Abstract

An anti-ST2 antibody or a fragment thereof is provided. The antibody or fragment thereof can specifically bind to human ST2, inhibit the combination of IL-33 and human ST2, block the IL-33 / ST2 intracellular signaling pathway, and inhibit the stimulating effects of various forms of intracellular IL-33 that induce the production of IL5, IL6, and IL8. The anti-ST2 antibody exhibits higher biological activity compared to conventional anti-ST2 antibodies and can be used for the prevention, treatment, or alleviation of diseases associated with ST2 expression or IL-33 / ST2 pathway disorders.

Description

Technology Field

[0001] Technology field

[0002] The present invention relates to the field of antibody drugs, particularly to antibodies against human ST2 and their use in the manufacture of medicines.

[0003] This application claims priority to Chinese patent application No. CN202010072085.X filed on January 21, 2020, the entirety of which is incorporated herein by reference. Background Technology

[0004] Background of the Invention

[0005] Interleukin 33 (IL-33) is a cytokine related to IL-1 and IL-18, also known as NF-HEV or IL-1F11. ST2 (ST2L, IL-1RL1, T1, Fit-1, DER-4, IL-1R4, or ST2 alpha) is a binding receptor for IL-33 and a member of the Toll / IL-1 receptor family; it is expressed on the cell surface of various immune cells, including lymphocytes, particularly helper T cells, natural killer (NK) cells, and natural killer-T (NKT) cells expressing IL-5 and IL-13, and on the cell surface of many so-called innate immune cells such as mast cells, basophils, eosinophils, macrophages, and innate helper cells (also called new immune cells (nuocytes) (Neill, Wong et al, 2010)).

[0006] ST2 downregulates the responsiveness of Toll-like receptors TLR2, TLR4, and TLR9 and can induce the release of type 2 cytokines through activation by the ligand IL-33 and binding to the helper protein IL-1RAcP. Related literature has proposed interaction models between ST2, IL-33, and IL-1RAcP, as well as between IL-1R1 and IL-1RAcP (Lingel et al, Cell 17: 1398-1410, 2009; Wang et al, Nat Immunol , 11: 905-11, 2010).

[0007] IL-33 was described as "alamine" because, while it exists in its full-length form in the nuclei of epithelial and endothelial cells during homeostasis, it can be cleaved and released during cell necrosis. Examples of cellular responses induced by IL-33 include the production of inflammatory 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 reactions by strengthening mast cell and basophil activation induced by IgE receptor signaling or other mast cell and basophil activators. IL-33 also improves the recruitment, survival, and adhesion characteristics of ST2-expressing immune cells and is therefore crucial for the excitation and maintenance of cellular inflammation in local tissues.

[0008] Dysregulation of the IL-33 / ST2 pathway has been shown to be associated with various immune-mediated diseases, including asthma, rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, allergic rhinitis, nasal polyps, and systemic sclerosis. Therefore, therapeutic blockade of the IL-33 / ST2 pathway may help overcome hyperimmune responses. Inhibitors of this pathway mainly include IL33 antibodies (e.g., MEDI3506, ANB020, REGN3500, MT-2990, LY-3375880, PF-06817024) and ST2 antibodies (e.g., CNTO7160, AMG-282), and are currently being developed in Phase 1 and 2 clinical trials for indications such as allergic rhinitis, atopic dermatitis, chronic obstructive pulmonary disease, and asthma.

[0009] Currently, the incidence of allergic inflammation and respiratory diseases is gradually increasing, and glucocorticoids and β2 receptor agonists remain the dominant drugs available on the market. While all antibodies reported to date can block the interaction between ST2 and its ligand, they differ in their biological activity. Since differences in biological activity can lead to variations in the clinical efficacy and dosage of antibodies, there is still a demand in the industry for ST2 antibodies that provide high affinity, high stability, and high biological activity.

[0010] Summary of the Invention

[0011] The technical problem that the present invention aims to solve is to immunize mice using human ST2 as an immunogen, obtain murine antibodies through B cell panning, and further utilize antibody engineering and humanization technologies to obtain novel high-affinity antibodies that bind to ST2 and are suitable for treating diseases or indications associated with the IL-33 / ST2 pathway.

[0012] Regarding the above technical problem, the object of the present invention is to provide an antibody or a functional fragment thereof that specifically binds to ST2 and its uses.

[0013] The technical solution of the present invention is as follows.

[0014] As described in the present invention, the “fragment” of the antibody as described in the present invention comprises various functional or active fragments of the antibody, such as its antigen-binding portion, such as Fab, F(ab')2 or scFv.

[0015] In one aspect, the present invention provides an antibody or a 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 comprise a heavy chain CDR1 (H-CDR1), CDR2 (H-CDR2), CDR3 (H-CDR3) and a light chain CDR1 (L-CDR1), CDR2 (L-CDR2), CDR3 (L-CDR3) from a heavy chain variable region and a light chain variable region appearing in any one of the combinations selected from the following:

[0016] (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;

[0017] (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;

[0018] (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;

[0019] (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;

[0020] (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;

[0021] (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;

[0022] (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;

[0023] (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;

[0024] (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;

[0025] (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;

[0026] (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;

[0027] (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;

[0028] (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;

[0029] (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;

[0030] (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;

[0031] (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;

[0032] (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;

[0033] (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;

[0034] (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;

[0035] (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;

[0036] (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;

[0037] (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;

[0038] (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;

[0039] (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;

[0040] (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;

[0041] (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;

[0042] (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;

[0043] (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;

[0044] (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;

[0045] (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;

[0046] (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;

[0047] (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;

[0048] (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;

[0049] (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;

[0050] (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;

[0051] (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;

[0052] (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;

[0053] (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;

[0054] (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;

[0055] (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;

[0056] (VI-2): heavy chain variable region represented by SEQ ID NO. 21 and light chain variable region represented by SEQ ID NO. 47;

[0057] (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;

[0058] (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;

[0059] (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;

[0060] (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;

[0061] (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;

[0062] (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;

[0063] (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;

[0064] (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;

[0065] (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;

[0066] (VII-3): heavy chain variable region represented by sequence number 26 and light chain variable region represented by sequence number 53;

[0067] (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;

[0068] (VII-5): heavy chain variable region represented by sequence number 26 and light chain variable region represented by sequence number 51;

[0069] (VII-6): heavy chain variable region represented by SEQ ID NO. 27 and light chain variable region represented by SEQ ID NO. 52;

[0070] (VII-7): heavy chain variable region represented by sequence number 27 and light chain variable region represented by sequence number 53;

[0071] (VII-8): heavy chain variable region represented by sequence number 27 and light chain variable region represented by sequence number 50;

[0072] (VII-9): heavy chain variable region represented by SEQ ID NO. 27 and light chain variable region represented by SEQ ID NO. 51;

[0073] (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;

[0074] (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;

[0075] (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

[0076] (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;

[0077] Based on the given amino acid sequences of the light and heavy chain variable regions as described above, those skilled in the art can routinely determine the amino acid sequences of the heavy and light chain CDRs included therein. For example, according to a specific embodiment of the present invention, the CDRs of the variable region amino acid sequences are determined using the Kabat, IMGT, ABM, and Chotia numbering schemes. Light and heavy chain CDRs obtained by other methods known in the art and combinations thereof are also included within the scope of the present invention.

[0078] Preferably, the heavy chain variable region and the light chain variable region contain a heavy chain CDR and a light chain CDR represented by any one of the following combinations:

[0079] (I) H-CDR1 (GYSITSDYAWN), H-CDR2 (YIDYSGSTTYNPSLKS), H-CDR3 (TVIDSMDY) as shown in sequence Nos. 56, 63, and 70; and L-CDR1 (RASKSVSTSGHSYMH), L-CDR2 (LASNLES), L-CDR3 (QHSREFPFT) as shown in sequence Nos. 85, 93, and 97;

[0080] (II-1): H-CDR1 (GYSITSDYAWD), H-CDR2 (YIRYSGDTYYNPSLKS), H-CDR3 (TMMDTMDY) as shown in sequence nos. 57, 64, and 71 in sequence; and L-CDR1 (RASKSVSTSGNSYMH), L-CDR2 (LASNLES), L-CDR3 (QHSREFPLT) as shown in sequence nos. 86, 93, and 98 in sequence nos. 86, 93, and 98 in sequence;

[0081] (II-2): H-CDR1 (GYSITSDYAWD), H-CDR2 (YIRYSGDTYYNPSLKS), H-CDR3 (TMMDTMDY) as shown in sequence nos. 57, 64, and 71 in sequence; and L-CDR1 (RASKSVSTSGNTYMH), L-CDR2 (LASNLES), L-CDR3 (QHSREFPLT) as shown in sequence nos. 87, 93, and 98 in sequence nos. 87, 93, and 98 in sequence;

[0082] (III): H-CDR1 (GFSLSTSGMGVG), H-CDR2 (HIWWDDVKQYNPALKS), H-CDR3 (IGGDYDYFDF) as shown in sequence Nos. 58, 65, and 72; and L-CDR1 (RASESVEYSGTSLMQ), L-CDR2 (VASNVES), L-CDR3 (QQSRKVPWT) as shown in sequence Nos. 88, 94, and 99;

[0083] (IV-1): H-CDR1 (GYTFTDSEMY), H-CDR2 (AIDPETGDTAFNQKFKG), H-CDR3 (AFDNDNDDGFAY) as shown in sequence nos. 59, 66, and 73; and L-CDR1 (SASSSVNYMH), L-CDR2 (DTSKLAS), L-CDR3 (QQWSSNPLT) as shown in sequence nos. 89, 95, and 100;

[0084] (IV-2): H-CDR1 (GYTFTDSEMY), H-CDR2 (AIDPETGDTAFNQKFKG), H-CDR3 (AFDNDNDEGFAY) as shown in sequence nos. 59, 66, and 74; and L-CDR1 (SASSSVNYMH), L-CDR2 (DTSKLAS), L-CDR3 (QQWSSNPLT) as shown in sequence nos. 89, 95, and 100;

[0085] (IV-3): H-CDR1 (GYTFTDSEMY), H-CDR2 (AIDPETGDTAFNQKFKG), H-CDR3 (AFDNDNDDAFAY) as shown in sequence nos. 59, 66, and 75 in sequence; and L-CDR1 (SASSSVNYMH), L-CDR2 (DTSKLAS), L-CDR3 (QQWSSNPLT) as shown in sequence nos. 89, 95, and 100 in sequence;

[0086] (V-1): H-CDR1 (GYTFTDYELH), H-CDR2 (TIDPETGDTVYNQKFKA), H-CDR3 (AFYNDYDDGFAY) as shown in sequence nos. 60, 67, and 76 in sequence; and L-CDR1 (SVSSSVSYMH), L-CDR2 (DTSKLAS), L-CDR3 (QQWNSSPLT) as shown in sequence nos. 90, 95, and 101 in sequence;

[0087] (V-2): H-CDR1 (GYTFTDYELH), H-CDR2 (TIDPETGDTVYNQKFKA), H-CDR3 (AFYNDYDDGFAY) as shown in sequence nos. 60, 67, and 76 in sequence; and L-CDR1 (SVSSSVSYMH), L-CDR2 (DTSKLAS), L-CDR3 (QQWNTSPLT) as shown in sequence nos. 90, 95, and 102 in sequence;

[0088] (V-3): H-CDR1 (GYTFTDYELH), H-CDR2 (TIDPETGDTVYNQKFKA), H-CDR3 (AFYNDYDEGFAY) as shown in sequence nos. 60, 67, and 77 in sequence; and L-CDR1 (SVSSSVSYMH), L-CDR2 (DTSKLAS), L-CDR3 (QQWNSSPLT) as shown in sequence nos. 90, 95, and 101 in sequence;

[0089] (V-4): H-CDR1 (GYTFTDYELH), H-CDR2 (TIDPETGDTVYNQKFKA), H-CDR3 (AFYNDYDEGFAY) as shown in sequence nos. 60, 67, and 77 in sequence; and L-CDR1 (SVSSSVSYMH), L-CDR2 (DTSKLAS), L-CDR3 (QQWNTSPLT) as shown in sequence nos. 90, 95, and 102 in sequence;

[0090] (V-5): H-CDR1 (GYTFTDYELH), H-CDR2 (TIDPETGDTVYNQKFKA), H-CDR3 (AFYNDYDDAFAY) as shown in sequence nos. 60, 67, and 78 in sequence; and L-CDR1 (SVSSSVSYMH), L-CDR2 (DTSKLAS), L-CDR3 (QQWNSSPLT) as shown in sequence nos. 90, 95, and 101 in sequence;

[0091] (V-6): H-CDR1 (GYTFTDYELH), H-CDR2 (TIDPETGDTVYNQKFKA), H-CDR3 (AFYNDYDDAFAY) as shown in sequence nos. 60, 67, and 78 in sequence; and L-CDR1 (SVSSSVSYMH), L-CDR2 (DTSKLAS), L-CDR3 (QQWNTSPLT) as shown in sequence nos. 90, 95, and 102 in sequence;

[0092] (VI-1): H-CDR1 (GYRFTDSEMH), H-CDR2 (TIDPETGGTVYNQKFKG), H-CDR3 (AFYNDFDDGFAY) as shown in sequence nos. 61, 68, and 79; and L-CDR1 (SASTSVSYMH), L-CDR2 (DTSKLAS), L-CDR3 (QQWSSNPLT) as shown in sequence nos. 91, 95, and 100;

[0093] (VI-2): H-CDR1 (GYRFTDSEMH), H-CDR2 (TIDPETGGTVYNQKFKG), H-CDR3 (AFYNDFDEGFAY) as shown in sequence nos. 61, 68, and 80 in sequence; and L-CDR1 (SASTSVSYMH), L-CDR2 (DTSKLAS), L-CDR3 (QQWSSNPLT) as shown in sequence nos. 91, 95, and 100 in sequence;

[0094] (VI-3): H-CDR1 (GYRFTDSEMH), H-CDR2 (TIDPETGGTVYNQKFKG), H-CDR3 (AFYNDFDDAFAY) as shown in sequence nos. 61, 68, and 81 in sequence; and L-CDR1 (SASTSVSYMH), L-CDR2 (DTSKLAS), L-CDR3 (QQWSSNPLT) as shown in sequence nos. 91, 95, and 100 in sequence;

[0095] (VII-1): H-CDR1 (GYTFINYGMN), H-CDR2 (WINTYIGEPTYGDNFKG), H-CDR3 (EGDGFAY) as shown in sequence nos. 62, 69, and 82; and L-CDR1 (KSSQSLLYSGNQNNYLA), L-CDR2 (GASTRES), L-CDR3 (QNDHSYPYT) as shown in sequence nos. 92, 96, and 103;

[0096] (VII-2): H-CDR1 (GYTFINYGMN), H-CDR2 (WINTYIGEPTYGDNFKG), H-CDR3 (EGEGFAY) as shown in sequence nos. 62, 69, and 83 in sequence; and L-CDR1 (KSSQSLLYSGNQNNYLA), L-CDR2 (GASTRES), L-CDR3 (QNDHSYPYT) as shown in sequence nos. 92, 96, and 103 in sequence nos. 92, 96, and 103 in sequence; and

[0097] (VII-3): H-CDR1 (GYTFINYGMN), H-CDR2 (WINTYIGEPTYGDNFKG), H-CDR3 (EGDAFAY) as shown in sequence nos. 62, 69, and 84; and L-CDR1 (KSSQSLLYSGNQNNYLA), L-CDR2 (GASTRES), L-CDR3 (QNDHSYPYT) as shown in sequence nos. 92, 96, and 103;

[0098] In particular, the antibody or fragment thereof of the present invention comprises at least a heavy chain variable region and a light chain variable region, and both the heavy chain variable region and the light chain variable region comprise the CDR and framework regions (FRs) between them, and the domains of the heavy chain variable region and the light chain variable region are arranged as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Optionally, there may be up to 25% difference in amino acid sequences due to "at least 75% identity" within any framework region of the heavy chain variable region or the light chain variable region, or any domain or sequence within the antibody or fragment thereof of the present invention other than the heavy chain variable region and the light chain variable region. Such difference may be due to amino acid deletion, addition, or substitution at any position, and the substitution may be a conservative substitution or a non-conservative substitution.

[0099] Preferably, the heavy chain variable region contains an amino acid sequence as shown in any one of SEQ ID NOs 1 to 28 or an amino acid sequence having at least 75% identity with respect to the amino acid sequence as shown; and / or the light chain variable region contains an amino acid sequence as shown in any one of SEQ ID NOs 29 to 53 or an amino acid sequence having at least 75% identity with respect to the amino acid sequence as shown.

[0100] According to a specific embodiment of the present invention, the antibody or fragment thereof comprises a heavy chain variable region and a light chain variable region as indicated by any one selected from the following combinations:

[0101] (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;

[0102] (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;

[0103] (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;

[0104] (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;

[0105] (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;

[0106] (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;

[0107] (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;

[0108] (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;

[0109] (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;

[0110] (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;

[0111] (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;

[0112] (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;

[0113] (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;

[0114] (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;

[0115] (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;

[0116] (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;

[0117] (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;

[0118] (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;

[0119] (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;

[0120] (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;

[0121] (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;

[0122] (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;

[0123] (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;

[0124] (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;

[0125] (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;

[0126] (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;

[0127] (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;

[0128] (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;

[0129] (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;

[0130] (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;

[0131] (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;

[0132] (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;

[0133] (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;

[0134] (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;

[0135] (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;

[0136] (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;

[0137] (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;

[0138] (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;

[0139] (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;

[0140] (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;

[0141] (VI-2): Heavy chain variable region represented by SEQ ID NO. 21 and light chain variable region represented by SEQ ID NO. 47;

[0142] (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;

[0143] (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;

[0144] (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;

[0145] (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;

[0146] (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;

[0147] (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;

[0148] (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;

[0149] (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;

[0150] (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;

[0151] (VII-3): Heavy chain variable region represented by SEQ ID NO. 26 and light chain variable region represented by SEQ ID NO. 53;

[0152] (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;

[0153] (VII-5): Heavy chain variable region represented by SEQ ID NO. 26 and light chain variable region represented by SEQ ID NO. 51;

[0154] (VII-6): Heavy chain variable region represented by SEQ ID NO. 27 and light chain variable region represented by SEQ ID NO. 52;

[0155] (VII-7): Heavy chain variable region represented by SEQ ID NO. 27 and light chain variable region represented by SEQ ID NO. 53;

[0156] (VII-8): Heavy chain variable region represented by SEQ ID NO. 27 and light chain variable region represented by SEQ ID NO. 50;

[0157] (VII-9): Heavy chain variable region represented by SEQ ID NO. 27 and light chain variable region represented by SEQ ID NO. 51;

[0158] (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;

[0159] (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;

[0160] (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

[0161] (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;

[0162] The antibody or fragment thereof provided by the present invention binds to ST2, preferably mammalian ST2, more preferably primate ST2, even more preferably human or cynomolgus ST2, particularly human ST2. Experiments have demonstrated that the antibody provided by the present invention has the following activities: (1) specific binding to human ST2; (2) inhibition of IL-33 binding to human ST2; (3) blockade of the IL-33 / ST2 intracellular signaling pathway; (4) inhibition of the stimulating effect of various forms of IL-33 on the cellular production of IL5; (5) inhibition of the stimulating effect of IL-33 on the cellular production of IL5, IL6, and IL8; and (6) a long half-life in vivo.

[0163] Generally, the antibody or fragment thereof provided by the present invention is of any form, for example, a monoclonal antibody, a single chain antibody, a diabody, a single domain antibody, a nanobody, a wholly or partially humanized antibody, or a chimeric antibody and the like, or a fragment thereof. Preferably, the antibody is IgA, IgD, IgE, IgG, or IgM, and more preferably is an IgG1, IgG2, or IgG4 antibody.

[0164] Preferably, the fragment is a functionally active fragment of the antibody capable of specifically binding to ST2 or any part thereof. More preferably, the fragment is a short-chain variable fragment (scFv), a divalent short-chain variable fragment (BsFv), a disulfide-stabilized Fv fragment (dsFv), (disulfide-stabilized Fv fragment)2(dsFv)2, an antigen-binding fragment (Fab), a Fab' fragment, an F(ab')2 fragment, or a variable fragment (Fv) of the antibody.

[0165] More preferably, the antibody or fragment thereof further contains a human or mouse (murine) constant region, preferably a human or mouse heavy chain constant region (CH) and / or a light chain constant region (CL). Preferably, the antibody or fragment thereof contains a heavy chain and a light chain; more preferably, the antibody contains two heavy chains and two light chains.

[0166] Preferably, the antibody or a fragment thereof contains a heavy chain constant region selected from the group consisting of an constant region of IgG, IgA, IgM, IgD, and IgE and / or a light chain constant region of the kappa or lambda type. According to a specific embodiment of the present invention, the antibody contains a heavy chain constant region that is an IgG1, IgG2, or IgG4 subtype; or the antibody contains a light chain constant region that is a kappa subtype. More preferably, the heavy chain constant region contains an amino acid sequence as shown in SEQ ID NO. 54 or an amino acid sequence having at least 75% identity with the amino acid sequence as shown; and the light chain constant region contains an amino acid sequence as shown in SEQ ID NO. 55 or an amino acid sequence having at least 75% identity with the amino acid sequence as shown.

[0167] The at least 75% identity described in the context of the present invention may be any percentage identity such as 75% or more, for example, at least 75%, at least 80%, preferably at least 85%, more preferably at least 90%, and more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99% identity.

[0168] According to a specific embodiment of the present invention, the present invention particularly preferably provides an antibody as follows:

[0169] An antibody named "5888-116-H0L1" having a heavy chain constant region as shown in SEQ ID NO. 11 and a light chain constant region as shown in SEQ ID NO. 41;

[0170] An antibody named "5888-153-H0L1" having a heavy chain constant region as shown in SEQ ID NO. 16 and a light chain constant region as shown in SEQ ID NO. 44; and

[0171] An antibody named "5886-156-H1L0" having a heavy chain constant region as shown in SEQ ID NO. 3 and a light chain constant region as shown in SEQ ID NO. 30;

[0172] And each of the above antibodies has a heavy chain constant region as shown in SEQ ID NO. 54 and a light chain constant region as shown in SEQ ID NO. 55. The above antibodies are monoclonal antibodies containing two heavy chains and two light chains, respectively.

[0173] Based on the antibody or fragment thereof provided by the present invention, the present invention also provides a conjugate or fusion protein containing the antibody or fragment thereof of the present invention. The conjugate or fusion protein contains 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 antibody of the present invention, or even an active protein or polypeptide chemically or physically bound to the antibody or fragment thereof of the present invention.

[0174] In another aspect, the present invention provides a nucleic acid molecule comprising a heavy chain CDR, a light chain CDR, a heavy chain variable region, a light chain variable region, and a nucleotide sequence encoding a heavy chain or a light chain contained in any antibody or fragment thereof according to the present invention.

[0175] In another aspect, the present invention provides a vector containing a 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.

[0176] The vector or nucleic acid molecule of the present invention may be used to transform or transfect a host cell for antibody preservation or expression, or to enter a host cell in any manner. Accordingly, in a further aspect, the present invention provides a host cell containing a nucleic acid molecule and / or vector according to the present invention, or transformed or transfected with a nucleic acid molecule and / or vector according to the present invention.

[0177] The host cell can be any prokaryotic or eukaryotic cell, such as bacteria, insects, fungi, plants, or animals.

[0178] According to the disclosure of the present invention, the antibody or fragment thereof, as well as the conjugate or fusion protein, nucleic acid molecule, vector, and / or host cell provided by the present invention accordingly, may be obtained using conventional techniques known in the art. The antibody or fragment thereof, the conjugate or fusion protein, nucleic acid molecule, vector, and / or host cell may be incorporated into a composition, particularly a pharmaceutical composition, e.g., a pharmaceutical formulation, and may be used for various purposes as practically required.

[0179] Accordingly, in another aspect, the present invention also provides a composition comprising an antibody or a fragment thereof, a conjugate or fusion protein, a nucleic acid molecule, a vector, and / or a host cell according to the present invention. Preferably, the composition is a pharmaceutical composition optionally containing a pharmaceutically acceptable excipient.

[0180] The present invention also provides the following related uses of the subject described above based on an antibody or a fragment thereof capable of specifically binding to ST2 or any part thereof.

[0181] In a further aspect, the present invention provides the use of an antibody or a fragment thereof, a conjugate or fusion protein, a nucleic acid molecule, a vector, a host cell and / or a composition in the manufacture of a medicine for the prevention, treatment or improvement of a disease; preferably, said disease is associated with the expression of ST2 or dysregulation of the IL-33 / ST2 pathway. Preferably, said 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.

[0182] Furthermore, the present invention provides a method for the prevention, treatment, or improvement of a disease comprising administering an antibody or a fragment thereof, a conjugate or fusion protein, a nucleic acid molecule, a vector, a host cell and / or a composition of the present invention, and optionally to a subject requiring additional drugs or means. Preferably, the disease is associated with the expression of ST2 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 above optional additional drug or means refers to any other hormonal or immunomodulatory drug or means that may be administered in combination with the antibody or its fragment, conjugate or fusion protein, nucleic acid molecule, vector, host cell and / or composition of the present invention, such as glucocorticoid, mepolizumab, dupilumab, tezepelumab, etc.The co-administration of the two may be carried out in any manner, including simultaneously, sequentially, or at intervals. The subject is a mammal, preferably a primate, more preferably a human or a cynomolgus monkey; preferably, the subject is a human.

[0183] Accordingly, the present invention also provides a pharmaceutical combination comprising the antibody or a fragment thereof, a conjugate or fusion protein, a nucleic acid molecule, a vector, a host cell and / or a composition of the present invention, and optionally an additional drug. The optional additional drug means any other hormonal or immunomodulatory drug or means that may be administered in combination with the antibody or a fragment thereof, a conjugate or fusion protein, a nucleic acid molecule, a vector, a host cell and / or a composition of the present invention, such as a glucocorticoid, mepolizumab, dupilumab, tezepelumab, etc.

[0184] The present invention also provides a method for detecting or diagnosing a disease comprising contacting a sample from a target with an antibody or a fragment thereof, a conjugate or fusion protein, a nucleic acid molecule, a vector, a host cell and / or composition. Preferably, the disease is associated with the expression of ST2 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; preferably, the subject is a human.

[0185] In another aspect, the present invention provides a kit containing the antibody or a fragment thereof, a conjugate or fusion protein, a nucleic acid molecule, a vector, a host cell and / or a composition of the present invention. The kit may be used for detection or diagnosis in a method for detecting or diagnosing a disease, for example, as described above.

[0186] In the present invention, mice were immunized with human ST2, culture supernatant was obtained through B cell panning, and positive clones were obtained through ELISA and additional functional assay screening; additionally, humanized antibodies were obtained by humanizing mouse antibodies (murine antibodies) through antibody engineering. Through activity screening experiments on in vitro ligand binding by antibodies, inhibition of in vitro ligand activation of effector cells by antibodies, inhibition of ligand promotion of effector cells to produce IL5, IL6, IL8, etc. by antibodies, determination of antibody affinity, and animal drug metabolism experiments, the antibodies of the present invention were proven to have higher biological activity compared to currently available anti-ST2 antibodies. Brief explanation of the drawing

[0187] Embodiments of the present invention are described in detail below with reference to the attached drawings, wherein: FIGS. 1 to 5 show inhibition rates in the analysis of the KU812-NF-κB reporter gene of B-cell clones from differently numbered mice, wherein FIG. 1: mouse number 5883; FIG. 2: mouse number 5884; FIG. 3: mouse number 5886; FIG. 4: mouse number 5887; and FIG. 5: mouse number 5888. Figure 6 shows the inhibitory activity of a mouse antibody against the promotion of KU812-IL5 production by IL-33. Figure 7 shows the binding of a mouse antibody to cyno ST2. Figure 8 shows the binding of a mouse antibody to mouse ST2. Figure 9 shows the blocking assay screening of humanized antibodies against the binding of IL33 to human ST2. Figure 10 shows the inhibitory activity of a humanized antibody against the promotion of KU812-IL5 production by IL-33. Figure 11 shows the inhibitory activity of a humanized antibody against the promotion of KU812-IL5 production by oxidized IL-33. Figure 12 shows the inhibitory activity of a humanized antibody against the promotion of KU812-IL5 production by reduced IL-33. Figure 13 shows the inhibitory activity of a humanized antibody against the promotion of HUVEC-IL6 production by IL33. Figure 14 shows the inhibitory activity of a humanized antibody against the promotion of HMC-1-IL8 production by IL33. Figure 15 shows the PK curves of humanized antibodies in mice, where panel 15-1: 5888-116-HOL1; panel 15-2: 5888-153-H0L1; panel 15-3: CNTO7160; panel 15-4: 5886-156-H1L0. Specific details for implementing the invention

[0188] The present invention will be described below with reference to specific embodiments. It will be understood by those skilled in the art that these embodiments are merely examples of the invention and do not limit the scope of the invention in any way.

[0189] The experimental procedures in the following examples are all standard procedures unless otherwise specified. The raw materials and reagents used in the following examples are all commercially available products unless otherwise specified.

[0190] - Human ST2: NP_003847.2 (Met1-Phe328)

[0191] - Human ST2-his: Human ST2 fused with a 6-histidine tag at the C-terminus

[0192] - Human ST2-fc: Human ST2 fused with a human IgG1 Fc tag at the C-terminus

[0193] - Human IL33: NP_254274.1(Ser112-Thr270)

[0194] - Human IL33-his: Human IL33 fused with a 6-histidine tag at the C-terminus

[0195] - Oxidized human IL-33-his: Human IL-33-his was diluted to 300 μg / ml with IMDM and incubated at 37°C for 18 hours, then purified using an S75 16:600 Superdex column (GE Healthcare).

[0196] - Reduced human IL33-his: Human IL33-his using Cys208Ser and Cys259Ser

[0197] - Control antibody CNTO7160: Has a heavy chain as shown in SEQ ID NO. 104 and a light chain as shown in SEQ ID NO. 105

[0198] - Cyno ST2: XP_005575214.1(Met1-Cys331)

[0199] - Cyno ST2-fc: Cyno ST2 fused with a human IgG1 Fc tag at the C-terminus

[0200] - Mouse ST2: NP_001020773.1(Met1-Arg332)

[0201] - Mouse ST2-fc: Mouse ST2 fused with a human IgG1 Fc tag at the C-terminus

[0202] Example 1 Mouse antibody screening

[0203] 1.1 Animal Immunization

[0204] After expressing human ST2-his using CHO-K1 cells, six BALB / c mice were immunized with Freund's adjuvant according to a standard immunization program. Immunization was performed in two mouse batches, each containing three mice, with a 2-week interval between batches. Each batch of mice was immunized four times and tested by ELISA using human ST2-his; final immunization was performed if any mouse had a serum titer of >1:100,000; spleens were collected 3-4 days later.

[0205] 1.2 B-cell panning and culture

[0206] Feeder cells were plated using culture medium on four 10 cm dishes (Corning, cat. 430167) two days prior to the inoculation experiment, treated with 25 μg / mL MMC for 6 hours one day prior to the inoculation experiment, and then plated on 96-well plates (Corning, cat. 3599) at 10,000 cells / well and 100 μL / well. Additionally, the human ST2-his antigen was coated on 6-well plates overnight at 4°C.

[0207] The spleen of the collected immune mouse was crushed, filtered, and centrifuged, and then erythrocyte lysate was added to remove erythrocytes. Treatment with said erythrocyte lysate was repeated several times until no apparent erythrocytes were present; then, DC cells were removed from the splenocytes. Splenocytes obtained from one spleen were uniformly plated onto a 6-well plate coated with the said antigen for panning, and after antigen panning, B cells were collected by trypsin, counted, and plated onto a 96-well plate coated with said feed cells. The cells were cultured at 37°C and 5% CO2 for 10–14 days, and the B cell culture supernatant was collected from wells where clear clones had formed for subsequent screening.

[0208] 1.3 Screening of supernatant containing mouse antibodies

[0209] (1) ELISA screening of mouse antibodies binding to human ST2-his:

[0210] The human ST2-his immunogen described above was diluted to 1 μg / mL in coating buffer, added to ELISA plates at a rate of 50 μL / well, and coated overnight at 4°C. The next day, the coated plates were taken, washed three times with PBST, and incubated at room temperature for 1 hour using blocking buffer. Subsequently, the plates were washed again three times with PBST; the B cell culture supernatant was added to the ELISA plates and incubated at room temperature for 1 hour. After washing the plates three times with PBST, goat anti-mouse secondary antibody (1:10000) was added to the plates at a rate of 50 μL / well and incubated at room temperature for 1 hour. The 96-well plates were washed three times with PBST, and TMB was added at a rate of 50 μL / well and color developed in the dark for 10 minutes. Afterward, 2M sulfuric acid was added to stop the reaction; and the absorbance value (OD value) at 450 nm was read using a microplate reader.

[0211] As a result of detection, B cell clones obtained from two mice, mouse numbers 5883 and 5884, showed a positive rate of over 97% in an ELISA for screening mouse antibodies that bind to human ST2-his, where an absorbance value (OD value) of more than 10 times that of the negative control (blank medium) was used as a positive criterion.

[0212] 1.4 KU812-NF-κB Reporter Gene Analysis Screening

[0213] 1 x 10⁻⁶ of the logarithmic growth phase 6KU812 cells were collected, washed once by centrifugation, and resuspended in 20 μL of Buffer R from the Neon Transfection System 10 μL Kit. 1 μg of the pGL4.32[luc2P / NF-κB-RE / Hygro] vector was added to the cells, followed by a single electric shock at 1000 V for 50 ms to induce transfection. After transfection, pressure screening was performed using Hygromycin B to finally obtain the KU812 / NF-κB-1# cell line.

[0214] Human IL33-his was diluted to 1 μg / mL in culture medium and mixed with B cell culture supernatant in a 1:1 ratio to obtain test samples. Additionally, negative control samples (diluted human IL33-his mixed with blank medium in a 1:1 ratio) and positive control samples (diluted human IL33-his mixed with 1 μg / mL CNTO7160 in a 1:1 ratio) were prepared. These samples were added to 384-well plates at a rate of 20 μL / well, respectively. KU812 / NF-κB-1# cells in the logarithmic growth phase were taken, added to 384-well plates at 20,000 / well and 20 μL / well, and cultured overnight (16-24 hours) at 37°C and 5% CO2. Next, the reagent Bright-glo was added to a 384-well plate at a rate of 40 μL / well, the plate was shaken for 3 minutes, detected with a microplate reader, and the RLU value was read.

[0215] The inhibition rate of each clone was calculated by referring to the values ​​of the negative and positive control groups above, and the results are shown in Figures 1 to 5.

[0216] Example 2 Antibody engineering

[0217] 2.1 Sequencing of antibodies in B cells

[0218] mRNA was extracted from B cell clones using the PureLink RNA Mini Kit according to the instructions, subpackaged, and stored at -80°C. The extracted mRNA was used as a template and reverse transcribed into cDNA using the PrimeScript II 1st Strand cDNA Synthesis Kit according to the instructions, subpackaged, and stored at -80°C.

[0219] VH and VL sequences were amplified using Ex Taq enzyme and the primers for heavy chain VH and light chain VL amplification shown in Table 1 (Tables 1-1 and 1-2) using the cDNA as a template; then linked to the pMD18T vector for sequencing.

[0220] Primers for Heavy Chain VH Amplification VH-F-Mix1 OVH1 SAGGTCCAGCTGCAGCAGYYTGG OVH2 CAGGTRCAGCTGAAGSAGTCAGG OVH3 GAKGTGCAGCTTCAGCAGTCRGG OVH5 GAVGTGAWGCTGGTGGAGTCTGR OVH11 GAAGTGCAGCTGTTGGAGACTGG OVH14 GAGGTTCAGCTGCAGCAGTCTGK OVH15 CAGGTTCACCTACAACAGTCTGG VH-F-Mix2 OVH4 GAGGTGAAGCTTCTCGAGTCTGG OVH7 GAGGTGAAGCTGRTGGARTCTGR OVH8 CAGGTTACTCTGAAAGAGTCTGG OVH9 CAGATCCAGTTGGYGCAGTCTGG OVH10 GAGGTGCAGCTTGTTGAGWCTGG OVH12 CAGATGCAGCTTCAGGAGTCAGG OVH13 GAAGTGAAGCTTGAGGAGTCTGG VH-F-Mix3 OVH6a GAAGTGMAMTTKSWGCAGTCTGG OVH6b GAGGTGMAMTTKSWGCTGTCTGG OVH6c GATGTGMAMTTKSWGCAGTCTGG OVH6d GAAGTGMAMTTKSWGCTGTCTGG OVH6e GAGGTGMAMTTKSWGCAGTCTGG OVH6f GATGTGMAMTTKSWGCTGTCTGG OVH6g GAAGTGMAMTTKSWGGAGTCTGG OVH6h GAGGTGMAMTTKSWGGTGTCTGG OVH6i GATGTGMAMTTKSWGGAGTCTGG OVH6j GAAGTGMAMTTKSWGGTGTCTGG OVH6K GAGGTGMAMTTKSWGGAGTCTGG OVH6l GATGTGMAMTTKSWGGTGTCTGG VH-R-Mix HF-1 GAGGAAACGGTGACCGTGGT HF-2 GAGGAGACTGTGAGAGTGGT HF-3 GCAGAGACAGTGACCAGAGT HF-4 GAGGAGACGGTGACTGAGGT Primers for Light Chain VL Amplification VLk-F-Mix1 OVK1 GATGYTKTKVTGACCCAAACTCC OVK3 RACATTGTGCTGACMCAATCTCC OVK4a SAAAWTGTKCTCWCCCAGTCTCC OVK4b SAAAWTCTKCTCWCCCAGTCTCC OVK4c SAAAWTTTKCTCWCCCAGTCTCC OVK6a ARCATTGTGATGACCCAGWCTCA OVK6b ARCATTGTGATGACCCAGWCTCC OVK6c GRCATTGTGATGACCCAGWCTCA OVK6d GRCATTGTGATGACCCAGWCTCC OVK10 GATATCCAGATGACACAGACTAC OVK14 GAMATCMWGATGACCCARTCTCC VLk-F-Mix2 OVK2a GATATTGTGATRACBCAGGYTGA OVK2b GATATTGTGATRACBCAGGYTGC OVK5a GAYATYCTGATRACRCAGTCTCC OVK5b GAYATYGTGATRACRCAGTCTCC OVK5c GAYATYTTGATRACRCAGTCTCC OVK5d GAYATYCTGCTRACRCAGTCTCC OVK5e GAYATYGTGCTRACRCAGTCTCC OVK5f GAYATYTTGCTRACRCAGTCTCC OVK7 GACATTGTGATGACTCAGTCTCC OVK9 GACATCCAGATGAYYCAGTCTCC OVK11-16 GATGTCCAGATRAYYCAGTCTCC OVK12-13 GACATCCAGATGACWCARTCTYC OVK15-19 GACATCCAGATGAMMCAGTCTCC OVK17 GAAACAACTGTGACCCAGTCTCC OVK18 ACTGGAGAAACAACACAGGCTCC VLk-F-Mix3 OVK8a RAMATTATGWTGWCACAGTCTAC OVK8b RAMATTATGWTGWCACAGTCTAT OVK8c RAMATTGTGWTGWCACAGTCTAC OVK8d RAMATTGTGWTGWCACAGTCTAT OVK8e RAMATTTTGWTGWCACAGTCTAC OVK8f RAMATTTTGWTGWCACAGTCTAT OVK8g RAMATTATGWTGWCACAGTCTCC OVK8h RAMATTATGWTGWCACAGTCTCT OVK8i RAMATTGTGWTGWCACAGTCTCC OVK8j RAMATTGTGWTGWCACAGTCTCT OVK8k RAMATTTTGWTGWCACAGTCTCC OVK8l RAMATTTTGWTGWCACAGTCTCT VLk-R-Mix LF1 ACGTTTGATTTCCAGCTTGG LF2 ACGTTTTATTTCCAGCTTGG LF4 ACGTTTTATTTCCAACTTTG LF5 ACGTTTCAGCTCCAGCTTGG

[0221] 2.2 Recombinant Expression and Screening of Mouse Antibodies

[0222] (1) Recombinant expression of mouse antibodies

[0223] Light and heavy chains from the same clone (e.g., illustrated in FIGS. 1 to 5) were co-transfected into CHO-K1 cells in pairs, and 10 μg / mL MSX was added for pressure screening 24 hours after transfection. After cell density and viability were restored, the cells were inoculated for feed-batch expression, and the supernatant after expression was centrifuged to purify protein A. The obtained antibodies were used for quantitative screening after determining antibody concentrations using the BCA method.

[0224] Mouse antibodies are named after the cell clone number from which the light chain and heavy chain (H+L) originated. For example, the mouse antibody "5883-105H+L" represents a mouse antibody derived from a cell clone numbered 5883-105, in which the heavy chain is 5883-105H and the light chain is 5883-105L.

[0225] (2) Screening of binding activity of mouse antibodies against human ST2

[0226] Human ST2-his was diluted to 1 μg / mL in coating buffer, added to a plate at 50 μL / well, and coated overnight at 4°C. The next day, the coated plate was taken, washed three times with PBST, and incubated at room temperature for 1 hour using blocking buffer. Subsequently, the plate was washed again three times with PBST. Starting at 100 ng / mL, each mouse antibody was 3-fold diluted to obtain a total of 8 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 and washed three times with PBST; then, a goat anti-mouse secondary antibody (1:10000) was added to a 96-well plate at 50 μL / well and incubated at room temperature for 1 hour. A 96-well plate was washed three times with PBST, and TMB was added at a rate of 50 μL / well and color development was performed in the dark for 10 minutes. Afterward, 2M sulfuric acid was added to stop the reaction; and the absorbance value (OD value) at 450 nm was read using a microplate reader. The results are shown in Table 2.

[0227] Binding activity of mouse antibodies against human ST2 and relative activity of antibodies compared to CNTO7160 항체 EC 50 (ng / mL) 상대적 활성 (%) 5883-105H+L 13.52 84.99 5883-178H2+L1 9.64 119.19 5883-178H2+L2 4.93 233.02 5884-113H1+L2 9.61 149.71 5884-113H2+L1 12.56 114.57 5884-138H1+L1 16.33 88.12 5884-42H+L 9.71 136.87 5884-191H1+L1 9.43 140.95 5884-276H+L 7.49 177.51 5884-306H1+L3 6.05 225.66 5884-306H3+L1 8.99 151.82 5884-319H1+L1 4.80 284.20 5884-334H1+L1 11.89 134.57 5886-3H+L 9.79 163.40 5886-93H+L 10.94 146.25 5886-125H1+L1 7.46 171.94 5886-125H1+L2 68.70 18.66 5886-125H2+L2 6.86 186.88 5886-130H1+L1 25.52 66.26 5886-130H1+L2 22.89 73.88 5886-130H2+L2 11.08 152.62 5886-165H+L 21.10 100.14 5886-156H+L 16.12 131.08 5887-7H1+L2 17.16 151.57 5887-30H1+L1 140.30 18.54 5887-30H2+L2 1.73 1501.73 5887-41H+L 26.57 97.89 5887-83H2+L1 18.86 137.91 5887-91H+L 28.21 56.86 5887-98H1+L2 26.87 59.69 5887-267H3+L3 11.72 136.86 5887-280H1+L1 23.95 66.97 5887-98H3+L3 20.34 78.86 5887-98H5+L1 16.36 98.04 5887-172H3+L3 24.00 57.04 5887-221H2+L1 26.45 51.76 5887-240H+L 13.53 101.18 5887-257H2+L1 22.40 61.12 5887-267H1+L2 14.46 94.67 5887-318H2+L 5.11 211.59 5887-321H2+L1 5.52 195.80 5887-340H2+L1 8.54 126.57 5887-458H+L 11.45 94.41 5887-467H2+L1 12.25 88.24 5887-483H3+L2 15.32 58.02 5887-537H3+L1 12.28 72.38 5888-209H+L 4.45 128.75 5888-284H2+L 5.91 96.89 5888-344H3+L1 5.85 97.95 5888-378H+L 7.21 79.41 5887-127H3+L2 5.74 208.20 5887-167H+L 12.92 92.41 5887-220H+L 13.83 86.33 5887-357H1+L 10.74 111.17 5887-502H+L 43.91 35.00 5887-515H+L 42.68 36.01 5887-537H1+L2 46.62 32.97 5887-603H+L 35.35 32.73 5887-669H1+L2 24.16 47.89 5887-669H2+L1 17.89 64.67 5887-717H3+L1 25.27 45.79 5888-15H2+L2 15.72 116.35 5888-116H1+L1 28.84 63.42 5888-120H2+L1 27.04 67.64 5888-153H1+L2 20.93 87.39 5888-297H1+L1 19.22 95.16 5888-357H+L 11.30 123.54 5888-366H1+L2 17.23 65.82 5888-379H1+L2 29.49 33.90 5888-380H1+L2 23.29 42.93 5887-219H1+L1 21.26 39.78 5888-380H2+L1 58.62 14.43 5888-248H1+L1 25.54 33.11 5887-544H1+L1 17.65 47.92

[0228] (3) Blocking assay screening of mouse antibodies against IL33 binding to human ST2

[0229] Human ST2-fc was diluted to 10 μg / mL in coating buffer, added to a 96-well plate at a rate of 50 μL / well, and coated overnight in a refrigerator at 4°C. After washing the coated plates three times with PBST, blocking buffer was added to the plates at a rate of 100 μL / well and incubated at room temperature for 1 hour. Subsequently, the plates were washed again three times with PBST. Human IL33-his was diluted to 200 ng / mL in the dilution solution. Each mouse antibody was diluted to 200 μg / mL in the dilution solution, and then a total of eight serial dilutions were obtained by 3-fold dilution starting from 200 μg / mL. The diluted human IL33-his and each diluted mouse antibody were mixed in a 1:1 ratio, and the mixture was added to a 96-well plate at a rate of 50 μL / well and subsequently incubated at room temperature for 1 hour. After washing the above plate three times with PBST, a histidine-tagged secondary antibody (1:2500) was added at a rate of 50 μL / well, and the plate was incubated at room temperature for 1 hour. The above 96-well plate was washed again three times with PBST, and TMB was added at a rate of 50 μL / well, followed by color development in the dark for 10 minutes. Afterward, 2M sulfuric acid was added at a rate of 100 μL / well to stop the reaction; absorbance values ​​(OD values) at 450 nm and 650 nm were read using a microplate reader. The results are shown in Table 3.

[0230] Screening of blockade assays for mouse antibodies against IL33 binding to human ST2 and relative activity of antibodies compared to CNTO7160 항체 IC 50 (μg / mL) 상대적 활성 (%) 5883-105H+L 1.61 93.56 5883-178H2+L1 N / A N / A 5883-178H2+L2 N / A N / A 5884-113H1+L2 2.75 30.67 5884-113H2+L1 N / A N / A 5884-138H1+L1 1.18 71.23 5884-42H+L 1.48 56.99 5884-191H1+L1 1.97 42.73 5884-276H+L 1.15 66.66 5884-306H1+L3 0.69 110.69 5884-306H3+L1 N / A N / A 5884-319H1+L1 0.60 127.18 5884-334H1+L1 1.28 59.24 5886-3H+L 0.90 88.75 5886-93H+L 0.71 113.12 5886-125H1+L1 0.08 977.04 5886-125H1+L2 0.62 129.35 5886-125H2+L2 0.78 102.34 5886-130H1+L1 0.83 104.39 5886-130H1+L2 0.64 136.26 5886-130H2+L2 0.87 99.46 5886-165H+L 1.39 62.77 5886-156H+L 1.14 76.58 5887-7H1+L2 1.09 97.88 5887-30H1+L1 1.74 61.02 5887-30H2+L2 0.06 1792.88 5887-41H+L 1.29 82.72 5887-83H2+L1 1.22 86.99 5887-91H+L 1.92 58.53 5887-98H1+L2 N / A N / A 5887-267H3+L3 N / A N / A 5887-280H1+L1 1.41 79.80 5887-98H3+L3 0.39 290.15 5887-98H5+L1 1.00 112.54 5887-172H3+L3 0.95 117.88 5887-221H2+L1 1.27 88.49 5887-240H+L 0.87 128.60 5887-257H2+L1 1.75 64.19 5887-267H1+L2 N / A N / A 5887-318H2+L 0.88 104.58 5887-321H2+L1 0.93 99.16 5887-340H2+L1 N / A N / A 5887-458H+L 1.04 89.15 5887-467H2+L1 1.63 56.95 5887-483H3+L2 N / A N / A 5887-537H3+L1 0.99 93.54 5888-209H+L 0.95 97.63 5888-284H2+L N / A N / A 5888-344H3+L1 1.42 65.08 5888-378H+L 1.02 90.64 5887-127H3+L2 0.93 78.83 5887-167H+L 0.76 96.54 5887-220H+L 2.53 29.07 5887-357H1+L 1.13 64.78 5887-502H+L 0.91 80.81 5887-515H+L 0.97 75.35 5887-537H1+L2 0.49 149.34 5887-603H+L 0.80 92.19 5887-669H1+L2 N / A N / A 5887-669H2+L1 0.82 89.58 5887-717H3+L1 N / A N / A 5888-15H2+L2 0.87 67.02 5888-116H1+L1 0.90 65.22 5888-120H2+L1 1.03 56.63 5888-153H1+L2 0.75 78.36 5888-297H1+L1 0.85 68.97 5888-357H+L 0.62 94.69 5888-366H1+L2 N / A N / A 5888-379H1+L2 0.83 70.09 5888-380H1+L2 0.92 63.67 5887-219H1+L1 1.39 63.87 5888-380H2+L1 N / A N / A 5888-248H1+l1 1.43 62.13 5887-544H1+l1 0.96 92.26

[0231] (4) Screening of mouse antibodies against activation of the KU812-NF-κB reporter gene by IL33

[0232] Human IL33-his was diluted to 1 μg / mL in culture medium. Each mouse antibody and control antibody CNTO7160 was diluted to 50 μg / mL in culture medium and then 3-fold to obtain a total of 12 serial dilutions. Test samples were obtained by mixing the antibodies with the diluted human IL33-his in a 1:1 ratio. In addition, negative control samples (diluted human IL33-his mixed with blank medium in a 1:1 ratio) and positive control samples (blank medium) were prepared. The samples were added to 384-well plates at a rate of 20 μL / well.

[0233] KU812 / NF-κB-1# cells in the logarithmic growth phase were centrifuged, transferred to fresh medium, and added to the 384-well plate at 20,000 / well and 20 μL / well. The cells were cultured overnight (16-24 hours) at 37°C in 5% CO2. Subsequently, Bright-glo developer was added to the 384-well plate at 40 μL / well, and the plate was vibrated for 3 minutes, detected by a microplate reader, and the RLU value was read. The results are shown in Table 4.

[0234] Screening of mouse antibodies against IL33-induced activation of the KU812-NF-κB reporter gene and relative activity of antibodies compared to CNTO7160 항체 IC 50 (ng / mL) 상대적 활성 (%) 5883-105H+L 5.272 231.79 5883-178H2+L1 30.46 40.12 5883-178H2+L2 2.206 553.94 5884-113H1+L2 153.4 8.66 5884-113H2+L1 28111 0.05 5884-138H1+L1 171.1 7.76 5884-42H+L 219.8 6.04 5884-191H1+L1 1543 0.86 5884-276H+L 123.7 10.74 5884-306H1+L3 NA NA 5884-306H3+L1 NA NA 5884-319H1+L1 NA NA 5884-334H1+L1 459.5 2.89 5886-3H+L 83.54 15.90 5886-93H+L 82.94 16.01 5886-125H1+L1 30.22 43.94 5886-125H1+L2 NA NA 5886-125H2+L2 228.9 5.80 5886-130H1+L1 NA NA 5886-130H1+L2 NA NA 5886-130H2+L2 11.50 88.70 5886-165H+L 92.00 11.09 5886-156H+L 8.04 126.82 5887-7H1+L2 29.91 4.38 5887-30H1+L1 1.439 91.10 5887-30H2+L2 3.13 45.68 5887-41H+L 2.87 41.88 5887-83H2+L1 36.8 3.56 5887-91H+L N / A N / A 5887-280H1+L1 6603 0.62 5887-172H3+L3 N / A N / A 5887-221H2+L1 2797 1.46 5887-240H+L N / A N / A 5887-257H2+L1 123.2 33.07 5887-318H2+L N / A N / A 5887-321H2+L1 8894 0.46 5887-458H+L 1009 4.04 5887-467H2+L1 N / A N / A 5887-537H3+L1 101.3 40.22 5888-209H+L 119.9 33.98 5888-378H+L 103.3 39.44 5887-127H3+L2 154.70 162.12 5887-357H1+L 1296.00 19.35 5887-502H+L 3693.00 6.79 5887-515H+L 2584.00 9.71 5887-537H1+L2 2536.00 9.89 5887-603H+L 1131.00 22.18 5887-669H2+L1 332.40 75.45 5888-15H2+L2 173.20 144.80 5888-116H1+L1 206.40 121.51 5888-120H2+L1 161.90 154.91 5888-153H1+L2 49.52 506.46 5888-297H1+L1 56.56 443.42 5888-357H+L 55.20 454.35 5888-379H1+L2 390.80 64.18 5888-380H1+L2 124.60 201.28 5887-167H+L 147.90 133.54 5887-219H1+L1 N / A N / A 5887-544H1+l1 335.40 11.43

[0235] (5) Affinity screening of mouse antibodies

[0236] Based on the results of quantitative screening of the above mouse antibodies, 19 mouse antibodies were selected for affinity determination and in vitro pharmacological studies. Experiments on the interaction between the anti-human ST2 antibody and human ST2-his were performed using a Biacore X100 at 25°C in HBS-EP(1x) buffer (pH 7.4).

[0237] Each anti-human ST2 antibody was diluted to 10 nM and captured on the surface of a Protein A chip (for a capture time of 60 seconds). After antibody capture, human ST2-his solution (a two-fold gradient dilution from 11.8 nM to 0.7375 nM, with a total of 5 concentrations) was injected. Binding was monitored for 4 minutes and dissociation for 10 minutes, and the sensor surface was regenerated by injecting a glycine solution at pH 2.0. Data generated for kinetics and affinity analysis were analyzed using BIAevaluation software. Kinetic data were analyzed using a simple 1:1 binding model, and the results are shown in Table 5.

[0238] Affinity of mouse antibodies 항체 ka (1 / Ms) kd (1 / s) KD (M) 5883-105H+L 1.59E+07 3.79E-03 2.39E-10 5886-130H2+L2 2.62E+06 1.78E-03 6.79E-10 5886-156H+L 2.37E+06 1.09E-03 4.61E-10 5887-30H2+L2 6.95E+06 1.10E-02 1.58E-09 5887-41H+L 1.10E+06 6.13E-04 5.60E-10 5887-257H2+L1 1.09E+06 7.50E-04 6.89E-10 5887-537H3+L1 5.01E+06 8.40E-04 1.68E-10 5888-209H+L 9.85E+06 2.73E-03 2.77E-10 5888-378H+L 1.07E+07 1.42E-03 1.34E-10 5887-127H3+L2 4.23E+06 1.05E-03 2.47E-10 5887-167H+L 1.24E+06 6.99E-04 5.63E-10 5888-15H2+L2 5.16E+06 1.09E-03 2.10E-10 5888-116H1+L1 9.87E+06 1.11E-03 1.13E-10 5888-120H2+L1 1.29E+07 1.13E-03 8.75E-11 5888-153H1+L2 9.17E+06 8.64E-04 9.43E-11 5888-297H1+L1 1.02E+07 1.71E-03 1.68E-10 5888-357H+L 4.38E+06 7.00E-04 1.60E-10 5888-379H1+L2 9.57E+05 5.46E-04 5.71E-10 5888-380H1+L2 7.13E+06 1.03E-03 1.45E-10 CNTO7160 8.27E+05 5.39E-04 6.98E-10

[0239] (6) In vitro pharmacological study of mouse antibodies

[0240] Human IL33-his was diluted to 80 ng / mL in culture medium. Each mouse antibody was diluted to 40 µg / mL in culture medium and then diluted fourfold to obtain a total of eight serial dilutions. The diluted antibodies were mixed with the diluted human IL33-his in a 1:1 ratio, and the resulting mixture was added to a 96-well plate at a rate of 50 μL / well. KU812 cells in the logarithmic growth phase were centrifuged and added to a 96-well plate at a rate of 100,000 / well and 50 μL / well, and cultured for 48 hours.

[0241] The capture antibody included in the human IL-5 DuoSet ELISA kit was coated at a rate of 50 μL / well on ELISA plates at 4°C overnight, according to the instructions, using a 2 μg / mL working solution obtained by diluting the 240 μg / mL stock solution 120-fold with PBS one day prior. Then, the plates were blocked for 1 hour using blocking buffer and washed 3 times. A 120 ng / mL standard solution was diluted 400-fold to 300 pg / mL and then 2-fold to obtain a total of 7 serial dilutions of different concentrations. The cell culture supernatant and one of the diluted standard solutions (50 μL each) were pipetted into ELISA plates and incubated for 2 hours. The above plate was washed three times, and a 125 ng / mL working solution obtained by diluting the detection antibody (7.5 μg / mL stock solution) 60-fold was added to the plate at a rate of 50 μL / well and incubated for 2 hours. The above 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 a rate of 50 μL / well and incubated for 20-30 minutes. A substrate solution was added to the plate at a rate of 50 μL / well and color development was allowed in the dark for 5-10 minutes. Afterward, 2 M sulfuric acid was added at a rate of 100 μL / well to stop the reaction; absorbance values ​​(OD values) at 450 nm and 650 nm were read using a microplate reader. The results are shown in Table 6 and Figure 6.

[0242] Inhibitory activity of mouse antibody against IL-33-induced promotion of KU812-IL-5 production antibodies Relative activity (%) Maximum inhibition rate (%) 5883-105H+L 342.64 80.33 5886-130H2+L2 344.54 71.06 5886-156H+L 303.78 84.16 5887-30H2+L2 490.35 41.40 5887-41H+L 128.40 79.25 5887-257H2+L1 40.03 82.17 5887-537H3+L1 766.96 62.25 5888-209H+L 254.91 74.38 5888-378H+L 466.20 84.53 5887-127H3+L2 126.96 73.54 5887-167H+L 26.14 66.68 5888-15H2+L2 80.56 80.22 5888-116H1+L1 238.00 81.40 5888-120H2+L1 21.72 82.02 5888-153H1+L2 115.09 86.00 5888-297H1+L1 246.30 80.38 5888-357H+L 98.54 71.03 5888-379H1+L2 19.17 92.72 5888-380H1+L2 69.84 77.33 CNTO7160 89.00

[0243] (7) Cross-reaction experiment of mouse antibodies against mouse ST2 and cyno ST2

[0244] Cyno ST2-fc was diluted to 1 μg / mL in coating buffer, added to a 96-well plate at a rate of 50 μL / well, and coated overnight at 4°C. The next day, the coated plates were taken, washed three times with PBST, and incubated at room temperature for 1 hour using blocking buffer. Subsequently, the plates were washed again three times with PBST. Starting at 1000 ng / mL, each mouse antibody was 3-fold diluted to obtain a total of eight serial dilutions, which were then added to a 96-well plate at a rate of 50 μL / well. The plates were incubated at room temperature for 1 hour and washed three times with PBST; then, a goat anti-mouse secondary antibody (1:10000) was added to the 96-well plate at a rate of 50 μL / well and incubated at room temperature for 1 hour. The above 96-well plate was washed three times with PBST, and TMB was added at a rate of 50 μL / well and color development was performed in the dark for 10 minutes. Afterward, 2M sulfuric acid was added to stop the reaction; and the absorbance value (OD value) at 450 nm was read using a microplate reader. The results are shown in Table 7 and Figure 7. It can be seen that the mouse antibody can bind to Cyno ST2 with a binding tendency consistent with binding to human ST2.

[0245] Results of mouse antibody binding experiments against Cyno ST2 antibodies EC 50 (ng / mL) Relative activity (%) 5883-105H+L 29.45 45.67 5886-130H2+L2 19.30 69.69 5886-156H+L 20.26 66.39 5887-30H2+L2 2.31 582.76 5887-41H+L 61.51 21.87 5887-257H2+L1 17.33 77.61 5887-537H3+L1 17.14 78.47 5888-209H+L 15.23 88.31 5888-378H+L 11.91 112.93 5887-127H3+L2 15.78 85.23 5887-167H+L 54.87 44.51 5888-15H2+L2 20.95 116.56 5888-116H1+L1 38.36 63.66 5888-120H2+L1 47.71 51.18 5888-153H1+L2 30.26 80.70 5888-297H1+L1 28.17 86.69 5888-357H+L 19.25 126.86 5888-379H1+L2 60.18 40.58 5888-380H1+L2 30.59 79.83

[0246] Experiments on the interaction between anti-human ST2 antibodies and cyno ST2-fc were performed using a Biacore X100 and at 25 in HBS-EP(1x) buffer (pH 7.4). The procedure was performed in [location]. Each antibody was immobilized on the surface of a CM5 chip using an amino coupling kit. After antibody immobilization, Cyno ST2-fc solution (2-fold gradient dilution starting from a concentration of 8 nM, with a total of 6 concentrations obtained; the starting concentration may be increased if the signal of some samples is too low) was injected. Binding 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. Kinetic data were analyzed using a simple 1:1 binding model, and the results are shown in Table 8.

[0247] Affinity detection results of mouse antibodies against Cyno ST2 antibodies ka (1 / Ms) kd (1 / s) KD (M) CNTO7160 1.63E+06 2.21E-04 1.36E-10 5886-156H+L 3.62E+06 7.47E-04 2.06E-10 5887-41H+L 7.98E+06 7.21E-04 9.03E-11 5887-537H3+L1 6.33E+06 5.11E-04 8.07E-11 5888-116H1+L1 8.14E+06 3.82E-04 4.69E-11 5888-153H1+L2 9.68E+06 1.39E-03 1.43E-10 5888-357H+L 6.41E+06 8.26E-04 1.29E-10 5888-379H1+L2 4.78E+06 1.52E-04 3.17E-11

[0248] Mouse ST2-fc was diluted to 1 μg / mL in coating buffer, added to a 96-well plate at a rate of 50 μL / well, and coated overnight at 4°C. The next day, the coated plates were taken, washed three times with PBST, and incubated at room temperature for 1 hour using blocking buffer. Afterward, they were washed three times again with PBST. Starting at 1000 ng / mL, each mouse antibody was 3-fold diluted to obtain a total of 8 serial dilutions, which were then added to a 96-well plate at a rate of 50 μL / well. The plates were incubated at room temperature for 1 hour and washed three times with PBST; then, a goat anti-mouse secondary antibody (1:10000) was added to the 96-well plate at a rate of 50 μL / well and incubated at room temperature for 1 hour. The above 96-well plate was washed three times with PBST, and TMB was added at a rate of 50 μL / well and color development was allowed in the dark for 10 minutes. Afterwards, 2M sulfuric acid was added to stop the reaction; and the absorbance value (OD value) at 450 nm was read using a microplate reader. The results are shown in Figure 8. It can be seen that the mouse antibody can bind weakly to mouse ST2, similar to the control antibody CNTO7160.

[0249] 2.3 Humanization

[0250] Seven mouse antibodies 5886-156H+L, 5887-41H+L, 5887-537H3H+L1, 5888-116H1+L1, 5888-153H1+L2, 5888-357H+L, and 5888-379H1+L2 were selected for the humanization design.

[0251] The heavy and light chain variable region sequences of seven mouse antibodies were compared with human germline sequences via a blast search in the IMGT database. Redundant genes and genes containing unpaired cysteine ​​were removed from the human germline genes. Among the remaining genes possessing the most matching framework and CDR regions, human germline genes were selected, and their framework regions were used as the human receptor framework. FR-4 was selected based on sequence similarity to IGHJ / IGJK germline genes. Tables 9 to 15 show the humanization sequences of seven mouse 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 the HZ0 version represents only the CDR-transplanted version, the HZ1 version has reverse mutation(s) introduced, and the HZ2 and additional versions have mutations attempted at PTM sites present in the sequence. Specific sequences after humanization are shown in Tables 9 to 15, with the corresponding CDR (defined by enhanced Chothia / AbM) underlined.

[0252] Humanized sequence of heavy and light chain variable regions number 5886-156 Heavy chain variable region (VH) order Light chain variable region (VL) order 5886-156H-VH(Sequence No. 1) DVQLQQSGPGLVKPSQSLSLTCTVT GYSITSDYAWN WIRQFPGNKLEWMG YIDYSGSTTYNPSLKS RFSITRDTSKNQFFLQLNSVTTEDTATYYCAS TVIDSMDY WGQGTSVTVSS 5886-156L-VL(Sequence No. 29) DIVLTQSPASLAVSLGQRATISC RASKSVSTSGHSYMH WYQQKPGQPPKLLIY LASNLES GVPARFSGSGSGTDFTLNINPMEEEDAATYYC QHSREFPFT FGSGTKLEIK IGHV4-30-4*01|IGHJ4*01 QVQLQESGPGLVKPSQTLSLTCTVS GGSISSGDYYWS WIRQPPGKGLEWIG YIYYSGSTYYNPSLKS RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR YFDY WGQGTLVTVSS IGKV7-3*01|IGKJ1*01 DIVLTQSPASLAVSPGQRATITC RASESVSFLGINLIH WYQQKPGQPPKLLIY QASNKDT GVPARFSGSGSGTDFTLTINPVEANDTANYYC LQSKNFPWT FGQGTKVEIK 5886-156H-VH-HZ0(Sequence No. 2) QVQLQESGPGLVKPSQTLSLTCTVS GYSITSDYAWN WIRQPPGKGLEWIG YIDYSGSTTYNPSLKS RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR TVIDSMDY WGQGTLVTVSS 5886-156L-VL-HZ0(Sequence No. 30) DIVLTQSPASLAVSPGQRATITC RASKSVSTSGHSYMH WYQQKPGQPPKLLIY LASNLES GVPARFSGSGSGTDFTLTINPVEANDTANYYC QHSREFPFT FGQGTKVEIK 5886-156H-VH-HZ1(Sequence No. 3) QVQLQESGPGLVKPSQTLSLTCTVS GYSITSDYAWN WIRQPPGKGLEWIG YIDYSGSTTYNPSLKS RVTISRDTSKNQFSLKLSSVTAADTAVYYCAS TVIDSMDY WGQGTLVTVSS 5886-156L-VL-HZ1(Sequence No. 31) DIVLTQSPASLAVSPGQRATITC RASKSVSTSGHSYMH WYQQKPGQPPKLLIY LASNLES GVPARFSGSGSGTDFTLTINPVEANDAANYYC QHSREFPFT FGQGTKVEIK

[0253] Humanized sequence of heavy and light chain variable regions number 5887-41 Heavy chain variable region (VH) order Light chain variable region (VL) order 5887-41H-VH(Sequence No. 4) DVQLQQSGPGLVKPSQSLSLTCTVT GYSITSDYAWD WIRQFPGNKLEWMG YIRYSGDTYYNPSLKS RISITRDTSKNQFFLQLNSVTTEDTATYYCAT TMMDTMDY WGQGTSVTVSS 5887-41L-VL(Sequence No. 32) DIVLTQSPASLTVSLGQRATISC RASKSVSTSGNSYMH WYQQKPGQPPKLLIY LASNLES GVPARFSGSGSGTDFTLNIHPVEEEDAATYYC QHSREFPLT FGAGTKLELK IGHV4-30-4*01|IGHJ4*01 QVQLQESGPGLVKPSQTLSLTCTVS GGSISSGDYYWS WIRQPPGKGLEWIG YIYYSGSTYYNPSLKS RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR YFDY WGQGTLVTVSS IGKV7-3*01|IGKJ2*01 DIVLTQSPASLAVSPGQRATITC RASESVSFLGINLIH WYQQKPGQPPKLLIY QASNKDT GVPARFSGSGSGTDFTLTINPVEANDTANYYC LQSKNFPYT FGQGTKLEIK 5887-41H-VH-HZ0(Sequence No. 5) QVQLQESGPGLVKPSQTLSLTCTVS GYSITSDYAWD WIRQPPGKGLEWIG YIRYSGDTYYNPSLKS RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR TMMDTMDY WGQGTLVTVSS 5887-41L-VL-HZ0(Sequence No. 33) DIVLTQSPASLAVSPGQRATITC RASKSVSTSGNSYMH WYQQKPGQPPKLLIY LASNLES GVPARFSGSGSGTDFTLTINPVEANDTANYYC QHSREFPLT FGQGTKLEIK 5887-41H-VH-HZ1(Sequence No. 6) QVQLQESGPGLVKPSQTLSLTCTVS GYSITSDYAWD WIRQPPGKGLEWIG YIRYSGDTYYNPSLKS RVTISRDTSKNQFSLKLSSVTAADTAVYYCAT TMMDTMDY WGQGTLVTVSS 5887-41L-VL-HZ1(Sequence No. 34) DIVLTQSPASLAVSPGQRATITC RASKSVSTSGNSYMH WYQQKPGQPPKLLIY LASNLES GVPARFSGSGSGTDFTLTINPVEANDAANYYC QHSREFPLT FGQGTKLEIK 5887-41L-VL-HZ2(Sequence No. 35) DIVLTQSPASLAVSPGQRATITC RASKSVSTSGNTYMH WYQQKPGQPPKLLIY LASNLES GVPARFSGSGSGTDFTLTINPVEANDAANYYC QHSREFPLT FGQGTKLEIK

[0254] Humanized sequence of heavy and light chain variable regions number 5887-537 Heavy chain variable region (VH) order Light chain variable region (VL) order 5887-537H3-VH(Sequence No. 7) QVTLKESGPGILQPSQTLSLTCSFS GFSLSTSGMGVG WIRQPSGKGLEWLA HIWWDDVKQYNPALKS RLTISKDTSSSQVFLKSASVDTADTATYYCAR IGGDYDYFDF WGQGTTLTVSS 5887-537L1-VL(Sequence No. 36) DIVLTQSPASLAVSLGQRATISC RASESVEYSGTSLMQ WYQQKPGQPPKLLIY VASNVES GVPARFSGSGSGTDFSLNIHPVEEDDIAMYFC QQSRKVPWT FGGGTKLEIK IGHV2-5*09| IGHD3-16*01 | IGHJ6*02 QVTLKESGPTLVKPTQTLTLTCTFS GFSLSTSGVGVG WIRQPPGKALEWLA LIYWDDDKRYGPSLKS RLTITKDTSKNQVVLTMTNMDPVDTATYYCAH RYYYYYGMDV WGQGTTVTVSS IGKV7-3*01|IGKJ2*01 DIVLTQSPASLAVSPGQRATITC RASESVSFLGINLIH WYQQKPGQPPKLLIY QASNKDT GVPARFSGSGSGTDFTLTINPVEANDTANYYC LQSKNFPYT FGQGTKLEIK 5887-537H3-VH-HZ0(Sequence No. 8) QVTLKESGPTLVKPTQTLTLTCTFS GFSLSTSGMGVG WIRQPPGKALEWLA HIWWDDVKQYNPALKS RLTITKDTSKNQVVLTMTNMDPVDTATYYCAH IGGDYDYFDF WGQGTTVTVSS 5887-537L1-VL-HZ0(Sequence No. 37) DIVLTQSPASLAVSPGQRATITC RASESVEYSGTSLMQ WYQQKPGQPPKLLIY VASNVES GVPARFSGSGSGTDFTLTINPVEANDTANYYC QQSRKVPWT FGQGTKLEIK 5887-537H3-VH-HZ1(Sequence No. 9) QVTLKESGPTLVKPTQTLTLTCTFS GFSLSTSGMGVG WIRQPPGKALEWLA HIWWDDVKQYNPALKS RLTITKDTSKSQVVLTMTNMDPVDTATYYCAR IGGDYDYFDF WGQGTTVTVSS 5887-537L1-VL-HZ1(Sequence No. 38) DIVLTQSPASLAVSPGQRATITC RASESVEYSGTSLMQ WYQQKPGQPPKLLIY VASNVES GVPARFSGSGSGTDFTLTINPVEANDIANYFC QQSRKVPWT FGQGTKLEIK

[0255] Humanized sequence of heavy and light chain variable regions number 5887-116 Heavy chain variable region (VH) order Light chain variable region (VL) order 5888-116H1-VH(Sequence No. 10) QVQLQQSGAELVRPGASVTLSCKAS GYTFTDSEMY WVRLTPVHGLEWIG AIDPETGDTAFNQKFKG KATLTADKSSSTAYMELRSLTSEDSVVYYCTR AFDNDNDDGFAY WGQGTLVTVSA 5888-116L1-VL(Sequence No. 39) QIVMTQSPAIMSASPGEKVTMTC SASSSVNYMH WYQQKSGTSPKRWIY DTSKLAS GVPARFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPLT FGAGTKLELK IGHV1-46*01|IGHJ3*01 QVQLVQSGAEVKKPGASVKVSCKAS GYTFTSYYMH WVRQAPGQGLEWMG IINPSGGSTSYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR DAFDV WGQGTMVTVSS IGKV1-33*01|IGKJ2*01 DIQMTQSPSSLSASVGDRVTITC QASQDISNYLN WYQQKPGKAPKLLIY DASNLET GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC QQYDNLPYT FGQGTKLEIK 5888-116H1-VH-HZ0(Sequence No. 11) QVQLVQSGAEVKKPGASVKVSCKAS GYTFTDSEMY WVRQAPGQGLEWMG AIDPETGDTAFNQKFKG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR AFDNDNDDGFAY WGQGTMVTVSS 5888-116L1-VL-HZ0(Sequence No. 40) DIQMTQSPSSLSASVGDRVTITC SASSSVNYMH WYQQKPGKAPKLLIY DTSKLAS GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC QQWSSNPLT FGQGTKLEIK 5888-116H1-VH-HZ1(Sequence No. 12) QVQLVQSGAEVKKPGASVKVSCKAS GYTFTDSEMY WVRQAPGQGLEWMG AIDPETGDTAFNQKFKG RVTMTADKSTSTAYMELSSLRSEDTAVYYCTR AFDNDNDDGFAY WGQGTMVTVSS 5888-116L1-VL-HZ1(Sequence No. 41) DIQMTQSPSSLSASVGDRVTITC SASSSVNYMH WYQQKPGKAPKRLIY DTSKLAS GVPSRFSGSGSGTDYTFTISSLQPEDAATYYC QQWSSNPLT FGQGTKLEIK 5888-116H1-VH-HZ2(Sequence No. 13) QVQLVQSGAEVKKPGASVKVSCKAS GYTFTDSEMY WVRQAPGQGLEWMG AIDPETGDTAFNQKFKG RVTMTADKSTSTAYMELSSLRSEDTAVYYCTR AFDNDNDEGFAY WGQGTMVTVSS 5888-116H1-VH-HZ3(Sequence No. 14) QVQLVQSGAEVKKPGASVKVSCKAS GYTFTDSEMY WVRQAPGQGLEWMG AIDPETGDTAFNQKFKG RVTMTADKSTSTAYMELSSLRSEDTAVYYCTR AFDNDNDDAFAY WGQGTMVTVSS

[0256] Humanized sequence of heavy and light chain variable regions number 5888-153 Heavy chain variable region (VH) order Light chain variable region (VL) order 5888-153H1-VH(Sequence No. 15) QVQLQQSGAELVRPGASVTLSCKAS GYTFTDYELH WVKQTPVHGLEWIG TIDPETGDTVYNQKFKA KAILTADKSSSTAYMEFRSLTSEDSAVCYCTR AFYNDYDDGFAY WGQGTLVTVSA 5888-153L2-VL(Sequence No. 42) QIVMTQTPAIMSASPGEKVTMTC SVSSSVSYMH WYQQKSGTSPKRWIY DTSKLAS GVPARFSGSGSGTSYSLTINNMEAEDAATYYC QQWNSSPLT FGAGTKLELK IGHV1-46*01|IGHJ1*01 QVQLVQSGAEVKKPGASVKVSCKAS GYTFTSYYMH WVRQAPGQGLEWMG IINPSGGSTSYAQKFQG RVTMTRDTSTSTTVYMELSSLRSEDATVYYCAR AEYFQH WGQGTLVTVSS IGKV3-11*01|IGKJ4*02 EIVLTQSPATLSLSPGERATLSC RASQSVSSYLA WYQQKPGQAPRLLIY DASNRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQRSNWPLT FGGGTKVEIK 5888-153H1-VH-HZ0(Sequence No. 16) QVQLVQSGAEVKKPGASVKVSCKAS GYTFTDYELH WVRQAPGQGLEWMG TIDPETGDTVYNQKFKA RVTMTRDTSTSTTVYMELSSLRSEDATVYYCAR AFYNDYDDGFAY WGQGTLVTVSS 5888-153L2-VL-HZ0(Sequence No. 43) EIVLTQSPATLSLSPGERATLSC SVSSSVSYMH WYQQKPGQAPRLLIY DTSKLAS GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQWNSSPLT FGGGTKVEIK 5888-153H1-VH-HZ1(Sequence No. 17) QVQLVQSGAEVKKPGASVKVSCKAS GYTFTDYELH WVRQAPGQGLEWMG TIDPETGDTVYNQKFKA RVTMTADKSTSTAYMELSSLRSEDTAVYYCTR AFYNDYDDGFAY WGQGTLVTVSS 5888-153L2-VL-HZ1(Sequence No. 44) EIVMTQSPATLSLSPGERATLSC SVSSSVSYMH WYQQKPGQAPRRLIY DTSKLAS GIPARFSGSGSGTDYTLTISSLEPEDAAVYC QQWNSSPLT FGGGTKVEIK 5888-153H1-VH-HZ2(Sequence No. 18) QVQLVQSGAEVKKPGASVKVSCKAS GYTFTDYELH WVRQAPGQGLEWMG TIDPETGDTVYNQKFKA RVTMTADKSTSTAYMELSSLRSEDTAVYYCTR AFYNDYDEGFAY WGQGTLVTVSS 5888-153L2-VL-HZ2(Sequence No. 45) EIVMTQSPATLSLSPGERATLSC SVSSSVSYMH WYQQKPGQAPRRLIY DTSKLAS GIPARFSGSGSGTDYTLTISSLEPEDAAVYC QQWNTSPLT FGGGTKVEIK 5888-153H1-VH-HZ3(Sequence No. 19) QVQLVQSGAEVKKPGASVKVSCKAS GYTFTDYELH WVRQAPGQGLEWMG TIDPETGDTVYNQKFKA RVTMTADKSTSTAYMELSSLRSEDTAVYYCTR AFYNDYDDAFAY WGQGTLVTVSS

[0257] Humanized sequence of heavy and light chain variable regions number 5888-357 Heavy chain variable region (VH) order Light chain variable region (VL) order 5888-357H-VH (Sequence No. 20) QVQLQQSVTELVRPGASVTLSCKAS GYRFTDSEMH WVKQTPVHGLEWIG TIDPETGGTVYNQKFKG KAKLTADRSSSTVSMELRSLTSEDSAVYYCTR AFYNDFDDGFAY WGQGTLVTVSA 5888-357L-VL(Sequence No. 46) QIVMTQSPAIMSASPGEKVTLTC SASTSVSYMH WYQQKSGTSPKRWIY DTSKLAS GVPARFSGSGSGTSYSLTISSMEAEDAATYYC QQWSSNPLT FGAGTKLELK IGHV1-46*01|IGHJ3*01 QVQLVQSGAEVKKPGASVKVSCKAS GYTFTSYYMH WVRQAPGQGLEWMG IINPSGGSTSYAQKFQG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR DAFDV WGQGTMVTVSS IGKV3-11*01|IGKJ4*02 EIVLTQSPATLSLSPGERATLSC RASQSVSSYLA WYQQKPGQAPRLLIY DASNRAT GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQRSNWPLT FGGGTKVEIK 5888-357H-VH-HZ0(Sequence No. 21) QVQLVQSGAEVKKPGASVKVSCKAS GYRFTDSEMH WVRQAPGQGLEWMG TIDPETGGTVYNQKFKG RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR AFYNDFDDGFAY WGQGTMVTVSS 5888-357L-VL-HZ0(Sequence No. 47) EIVLTQSPATLSLSPGERATLSC SASTSVSYMH WYQQKPGQAPRLLIY DTSKLAS GIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQWSSNPLT FGGGTKVEIK 5888-357H-VH-HZ1(Sequence No. 22) QVQLVQSGAEVKKPGASVKVSCKAS GYRFTDSEMH WVRQAPGQGLEWMG TIDPETGGTVYNQKFKG RVTMTADRSTSTVYMELSSLRSEDTAVYYCTR AFYNDFDDGFAY WGQGTMVTVSS 5888-357L-VL-HZ1(Sequence No. 48) EIVMTQSPATLSLSPGERATLSC SASTSVSYMH WYQQKPGQAPRRLIY DTSKLAS GIPARFSGSGSGTDYTLTISSLEPEDAAVYC QQWSSNPLT FGGGTKVEIK 5888-357H-VH-HZ2(Sequence No. 23) QVQLVQSGAEVKKPGASVKVSCKAS GYRFTDSEMH WVRQAPGQGLEWMG TIDPETGGTVYNQKFKG RVTMTADRSTSTVYMELSSLRSEDTAVYYCTR AFYNDFDEGFAY WGQGTMVTVSS 5888-357H-VH-HZ3(Sequence No. 24) QVQLVQSGAEVKKPGASVKVSCKAS GYRFTDSEMH WVRQAPGQGLEWMG TIDPETGGTVYNQKFKG RVTMTADRSTSTVYMELSSLRSEDTAVYYCTR AFYNDFDDAFAY WGQGTMVTVSS

[0258] Humanized sequence of heavy and light chain variable regions number 5888-379 Heavy chain variable region (VH) order Light chain variable region (VL) order 5888-379H1-VH(Sequence No. 25) QIQLAQSGPELKKPGETVKISCKAS GYTFINYGMN WVKQAPGKDLKWMG WINTYIGEPTYGDNFKG RFAFSLETSASTVYLQINNLKNEDTATYFCAR EGDGFAY WGQGTLVTVSA 5888-379L2-VL(Sequence No. 49) DIVMTQSPPSLSVSVGEKVTMSC KSSQSLLYSGNQNNYLA WYQQKPGQPPKLLIY GASTRES GVPDRFTGSGSGTDFTLTISSVQAEDLAVYYC QNDHSYPYT FGGGTKLEIK IGHV1-3*01 | IGHJ4*01 QVQLVQSGAEVKKPGASVKVSCKAS GYTFTSYAMH WVRQAPGQRLEWMG WINAGNGNTKYSQKFQG RVTITRDTSASTAYMELSSLRSEDTAVYYCAR YFDY WGQGTLVTVSS IGKV4-1*01 | IGKJ2*01 DIVMTQSPDSLAVSLGERATINC KSSQSVLYSSNNKNYLA WYQQKPGQPPKLLIY WASTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC QQYYSTPYT FGQGTKLEIK 5888-379H1-HZ0(Sequence No. 26) QVQLVQSGAEVKKPGASVKVSCKAS GYTFINYGMN WVRQAPGQRLEWMG WINTYIGEPTYGDNFKG RVTITRDTSASTAYMELSSLRSEDTAVYYCAR EGDGFAY WGQGTLVTVSS 5888-379L2-VK4-HZ0(Sequence No. 50) DIVMTQSPDSLAVSLGERATINC KSSQSLLYSGNQNNYLA WYQQKPGQPPKLLIY GASTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC QNDHSYPYT FGGGTKVEIK 5888-379H1-VH-HZ1(Sequence No. 27) QVQLVQSGAEVKKPGASVKVSCKAS GYTFINYGMN WVRQAPGQRLEWMG WINTYIGEPTYGDNFKG RVTITRDTSASTAYMELSSLRSEDTAVYYCAR EGEGFAY WGQGTLVTVSS 5888-379L2-VK4-HZ1(Sequence No. 51) DIVMTQSPDSLAVSLGERATINC KSSQSLLYSGNQNNYLA WYQQKPGQPPKLLIY GASTRES GVPDRFSGSGSGTDFTLTISSLQAEDLAVYYC QNDHSYPYT FGGGTKVEIK 5888-379H1-VH-HZ2(Sequence No. 28) QVQLVQSGAEVKKPGASVKVSCKAS GYTFINYGMN WVRQAPGQRLEWMG WINTYIGEPTYGDNFKG RVTITRDTSASTAYMELSSLRSEDTAVYYCAR EGDAFAY WGQGTLVTVSS IGKV1-27*01 | IGKJ2*01 DIQMTQSPSSLSASVGDRVTITC RASQGISNYLA WYQQKPGKVPKLLIY AASTLQS GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC QKYNSAPYT FGQGTKLEIK 5888-379L2-VK1-HZ0(Sequence No. 52) DIQMTQSPSSLSASVGDRVTITC KSSQSLLYSGNQNNYLA WYQQKPGKVPKLLIY GASTRES GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC QNDHSYPYT FGGGTKVEIK 5888-379L2-VK1-HZ1(Sequence No. 53) DIQMTQSPSSLSASVGDRVTITC KSSQSLLYSGNQNNYLA WYQQKPGKVPKLLIY GASTRES GVPSRFSGSGSGTDFTLTISSLQPEDLATYYC QNDHSYPYT FGGGTKVEIK

[0259] 2.4 Recombinant Expression and Screening of Humanized Antibodies

[0260] (1) Recombinant expression of humanized antibodies

[0261] Humanized light and heavy chains derived from the same mouse 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 in pairs into CHO-K1 cells. 24 hours after transfection, 10 µg / mL MSX was added for pressure screening. After cell density and viability were restored, cells were inoculated for feed-batch expression, and the supernatant after expression was centrifuged to purify it to Protein A. The obtained antibodies were used for quantitative screening after determining antibody concentrations using the BCA method.

[0262] Humanized antibodies and the paired light and heavy chain variable regions contained in the antibodies are shown in Tables 16 to 22. Antibodies having names with the suffix "ix" are accordingly chimeric antibodies.

[0263] Light and heavy chain variable regions of the humanized antibody pair No. 5886-156 5886-156L-VL(Sequence No. 29) 5886-156L-VL-HZ0(Sequence No. 30) 5886-156L-VL-HZ1(Sequence No. 31) 5886-156H-VH(Sequence No. 1) 5886-156-ix 5886-156H-VH-HZ0(Sequence No. 2) 5886-156-H0L0 5886-156-H0L1 5886-156H-VH-HZ1(Sequence No. 3) 5886-156-H1L0 5886-156-H1L1

[0264] Light and heavy chain variable regions of the humanized antibody pair No. 5887-41 5887-41L-VL(Sequence No. 32) 5887-41L-VL-HZ0(Sequence No. 33) 5887-41L-VL-HZ1(Sequence No. 34) 5887-41L-VL-HZ2(Sequence No. 35) 5887-41H-VH(Sequence No. 4) 5887-41-ix 5887-41H-VH-HZ0(Sequence No. 5) 5887-41-H0L0 5887-41-H0L1 5887-41-H0L2 5887-41H-VH-HZ1(Sequence No. 6) 5887-41-H1L0 5887-41-H1L1 5887-41-H1L2

[0265] Light and heavy chain variable regions of the humanized antibody pair numbered 5887-537 5887-537L1-VL(Sequence No. 36) 5887-537L1-VL-HZ0(Sequence No. 37) 5887-537L1-VL-HZ1(Sequence No. 38) 5887-537H3-VH(Sequence No. 7) 5887-537-ix 5887-537H3-VH-HZ0(Sequence No. 8) 5887-537-H0L0 5887-537-H0L1 5887-537H3-VH-HZ1(Sequence No. 9) 5887-537-H1L0 5887-537-H1L1

[0266] Light and heavy chain variable regions of the humanized antibody pair No. 5888-116 5888-116L1-VL(Sequence No. 39) 5888-116L1-VL-HZ0(Sequence No. 40) 5888-116L1-VL-HZ1(Sequence No. 41) 5888-116H1-VH(Sequence No. 10) 5888-116-ix 5888-116H1-VH-HZ0(Sequence No. 11) 5888-116-H0L0 5888-116-H0L1 5888-116H1-VH-HZ1(Sequence No. 12) 5888-116-H1L0 5888-116-H1L1 5888-116H1-VH-HZ2(Sequence No. 13) 5888-116-H2L0 5888-116-H2L1 5888-116H1-VH-HZ3(Sequence No. 14) 5888-116-H3L0 5888-116-H3L1

[0267] Light and heavy chain variable regions of the humanized antibody pair number 5888-153 5888-153L2-VL(Sequence No. 42) 5888-153L2-VL-HZ0(Sequence No. 43) 5888-153L2-VL-HZ1(Sequence No. 44) 5888-153L2-VL-HZ2(Sequence No. 45) 5888-153H1-VH(Sequence No. 15) 5888-153-ix 5888-153H1-VH-HZ0(Sequence No. 16) 5888-153-H0L0 5888-153-H0L1 5888-153-H0L2 5888-153H1-VH-HZ1(Sequence No. 17) 5888-153-H1L0 5888-153-H1L1 5888-153-H1L2 5888-153H1-VH-HZ2(Sequence No. 18) 5888-153-H2L0 5888-153-H2L1 5888-153-H2L2 5888-153H1-VH-HZ3(Sequence No. 19) 5888-153-H3L0 5888-153-H3L1 5888-153-H3L2

[0268] Light and heavy chain variable regions of the humanized antibody pair number 5888-357 5888-357L-VL(Sequence No. 46) 5888-357L-VL-HZ0(Sequence No. 47) 5888-357L-VL-HZ1(Sequence No. 48) 5888-357H-VH (Sequence No. 20) 5888-357-ix 5888-357H-VH-HZ0(Sequence No. 21) 5888-357-H0L0 5888-357-H0L1 5888-357H-VH-HZ1(Sequence No. 22) 5888-357-H1L0 5888-357-H1L1 5888-357H-VH-HZ2(Sequence No. 23) 5888-357-H2L0 5888-357-H2L1 5888-357H-VH-HZ3(Sequence No. 24) 5888-357-H3L0 5888-357-H3L1

[0269] Light and heavy chain variable regions of the humanized antibody pair number 5888-379 5888-379L2-VL(Sequence No. 49) 5888-379L2-VK1-HZ0(Sequence No. 52) 5888-379L2-VK1-HZ1(Sequence No. 53) 5888-379L2-VK4-HZ0(Sequence No. 50) 5888-379L2-VK4-HZ1(Sequence No. 51) 5888-379H1-VH(Sequence No. 25) 5888-379-ix 5888-379H1-VH-HZ0(Sequence No. 26) 5888-379-H0L0 5888-379-H0L1 5888-379-H0L2 5888-379-H0L3 5888-379H1-VH-HZ1(Sequence No. 27) 5888-379-H1L0 5888-379-H1L1 5888-379-H1L2 5888-379-H1L3 5888-379H1-VH-HZ2(Sequence No. 28) 5888-379-H2L0 5888-379-H2L1 5888-379-H2L2 5888-379-H2L3

[0270] (2) Blocking assay screening of humanized antibodies against the binding of IL33 to human ST2

[0271] Detection was performed according to the experimental procedure described in Section 2.2, "(3) Screening of blocking assays of mouse antibodies against the binding of IL33 to human ST2." The results are shown in Figure 9 and Table 23. In Figure 9, the negative control is a mixture of blank medium and diluted human IL33-his in a 1:1 ratio, and the positive control is blank medium.

[0272] Screening of blockade assays for humanized antibodies against IL33 binding to human ST2 and relative activity of antibodies compared to CNTO7160 antibodies IC 50 (μg / mL) Relative activity Maximum inhibition rate 5886-156-H1L0 1.217 103.12% 89.24% 5886-156-H1L1 1.004 125.00% 88.05% 5887-41-H1L0 0.5802 216.30% 92.76% 5887-41-H1L1 2.231 56.25% 92.41% 5887-537-H1L0 0.08819 1205.35% 88.29% 5888-116-H0L1 0.02045 5198.04% 94.09% 5888-116-H1L1 0.4863 218.59% 94.89% 5888-116-H3L1 0.6112 186.52% 90.65% 5888-153-H0L0 2.571 44.34% 92.89% 5888-153-H0L1 0.3003 379.62% 94.33% 5888-153-H0L2 0.4742 240.40% 93.53% 5888-153-H1L0 1.322 86.23% 92.52% 5888-153-H1L1 0.6241 182.66% 93.75% 5888-153-H1L2 0.852 133.80% 93.95% 5888-153-H3L1 0.5531 227.81% 93.67% 5888-153-H3L2 0.8284 152.10% 93.67% 5888-357-H1L1 0.4896 257.35% 93.24% 5888-379-H0L0 0.4878 179.56% 90.55% 5888-379-H1L0 0.4653 188.24% 89.14% 5888-379-H1L1 0.3173 276.05% 89.48% 5888-379-H2L1 0.4273 206.06% 84.83% 5888-379-H2L2 0.4022 218.92% 86.05% CNTO7160 92.55%

[0273] (3) Inhibitory activity of humanized antibodies against the promotion of KU812-IL5 production by IL-33

[0274] 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 fourfold to obtain a total of 11 serial dilutions of different concentrations. The diluted antibodies were mixed with the diluted human IL33-his in a 1:1 ratio, and the resulting mixture was added to a 96-well plate at a rate of 50 μL / well. The subsequent experimental procedure was identical to that described in Section 2.2, "(6) In vitro pharmacological study of mouse antibodies." The results are shown in Fig. 10 and Table 24. In Fig. 10, the negative control is a mixture of blank medium and diluted human IL33-his in a 1:1 ratio, and the positive control is blank medium.

[0275] Inhibitory activity of humanized antibodies against IL-33-induced promotion of KU812-IL5 production and relative activity of antibodies compared to CNTO7160 antibodies IC 50 (μg / mL) Relative activity Maximum inhibition rate 5886-156-H1L0 0.03539 181.12% 98.07% 5886-156-H1L1 0.02893 221.57% 94.95% 5887-41-H1L0 0.0699 91.70% 97.27% 5888-116-H0L1 0.009289 656.37% 98.31% 5888-153-H0L1 0.03033 201.02% 99.11% 5888-153-H3L1 0.008149 741.93% 97.02% 5888-153-H3L2 0.07554 142.04% 94.24% 5888-379-H1L0 0.0874 131.01% 99.40% 5888-379-H2L1 0.08393 121.17% 97.95% 5888-379-H2L2 0.09478 107.30% 97.88% CNTO7160 97.55%

[0276] (4) Screening of humanized antibodies against activation of the KU812-NF-κB reporter gene by IL33

[0277] Detection was performed according to the experimental procedure described in Section 2.2, "(4) Screening of mouse antibodies for activation of the KU812-NF-κB reporter gene by IL33". The results are shown in Table 25.

[0278] Screening of humanized antibodies against IL33-induced activation of the KU812-NF-κB reporter gene and relative activity of the antibody compared to CNTO7160 antibodies IC 50 (ng / mL) Relative activity Maximum inhibition rate 5886-156-H1L0 477.9 41.91% 92.49% 5887-41-H1L0 55.35 361.88% 89.24% 5888-116-H0L1 13.03 1537.22% 95.74% 5888-153-H0L1 43.78 457.51% 88.24% 5888-153-H1L1 12.36 1620.55% 91.36% 5888-153-H1L2 44.07 454.50% 84.98% 5888-153-H3L1 39.42 508.12% 86.11% 5888-153-H3L2 55.28 362.34% 88.11% CNTO7160 200.3 88.24%

[0279] (5) Inhibitory activity of humanized antibody against promotion of KU812-IL5 production by oxidized IL-33

[0280] 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 fourfold to obtain a total of 11 serial dilutions. The diluted antibodies were mixed with diluted human IL33-his in a 1:1 ratio, and the resulting mixture was added to a 96-well plate at a rate of 50 μL / well. The subsequent experimental procedure was identical to that described in Section 2.2, "(6) In vitro pharmacological study of mouse antibodies" above. The results are shown in Figure 11 and Table 26. In Figure 11, the negative control is a mixture of blank medium and diluted human IL33-his in a 1:1 ratio, and the positive control is blank medium.

[0281] Inhibitory activity of humanized antibodies against the promotion of KU812-IL5 production by oxidized IL33 antibodies IC 50 (μg / mL) Relative activity Maximum inhibition rate 5886-156-H1L0 0.0285 77.49% 101.11% 5887-41-H1L0 0.0337 65.40% 101.12% 5888-116-H0L1 0.0036 614.14% 101.56% 5888-153-H0L1 0.0147 150.07% 101.47% 5888-153-H3L1 0.0136 161.97% 101.38% 5888-153-H3L2 0.0173 127.44% 101.37% 5888-379-H1L0 0.0161 137.27% 101.34% 5888-379-H2L1 0.0228 96.84% 101.14% CNTO7160 0.0221 100.00% 98.90%

[0282] (6) Inhibitory activity of humanized antibody against promotion of KU812-IL5 production by reduced IL-33

[0283] 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 fourfold to obtain a total of 11 serial dilutions. The diluted antibodies were mixed with diluted human IL33-his in a 1:1 ratio, and the resulting mixture was added to a 96-well plate at a rate of 50 μL / well. The subsequent experimental procedure was the same as described in Section 2.2, "(6) In vitro pharmacological study of mouse antibodies." The results are shown in Fig. 12 and Table 27. In Fig. 12, the negative control is a mixture of blank medium and diluted human IL33-his in a 1:1 ratio, and the positive control is blank medium.

[0284] Inhibitory activity of humanized antibodies against the promotion of KU812-IL5 production by reduced IL33 antibodies IC 50 (μg / mL) Relative activity Maximum inhibition rate 5886-156-H1L0 0.7524 194.71% 93.38% 5887-41-H1L0 0.7056 207.62% 78.62% 5888-116-H0L1 0.1245 1176.71% 75.06% 5888-153-H0L1 0.3891 376.51% 83.56% 5888-153-H3L1 0.2646 553.67% 86.93% 5888-153-H3L2 0.5218 280.76% 87.24% 5888-379-H1L0 2.8650 51.13% 96.68% 5888-379-H2L1 2.8650 51.13% 96.68% CNTO7160 1.4650 100.00% 97.30%

[0285] (7) Inhibitory activity of humanized antibodies against the promotion of HUVEC-IL6 production by IL33

[0286] HUVEC cells were cultured in 96-well plates at 37°C and 5% CO2 for 18–24 hours at 10,000 cells / well and 100 μL / well. 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 4-fold to obtain a total of 11 serial dilutions. The diluted antibodies were mixed with the diluted human IL33-his in a 1:1 ratio, and the resulting mixture was added to the 96-well plates at 50 μL / well and cultured at 37°C and 5% CO2 for 18–24 hours.

[0287] The capture antibody included in the Human IL-6 DuoSet ELISA Kit was coated at a rate of 50 μL / well on ELISA plates at 4°C overnight, according to the instructions, using a 2 μg / mL working solution obtained by diluting the 240 μg / mL stock solution 120-fold with PBS one day prior. Then, the plates were blocked for 1 hour using blocking buffer and washed 3 times. A 180 ng / mL standard solution was diluted 300-fold to 600 pg / mL and then 2-fold to obtain a total of 7 serial dilutions of different concentrations. The cell culture supernatant and one of the diluted standard solutions (50 μL each) were pipetted into ELISA plates and incubated for 2 hours. The plate was washed three times, and a 50 ng / mL working solution of the detection antibody (3 μg / mL stock solution) diluted 60-fold was added to the plate at a rate of 50 μL / well and incubated 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 a rate of 50 μL / well and incubated for 20-30 minutes. A substrate solution was added to the plate at a rate of 50 μL / well and color development was allowed in the dark for 5-10 minutes. Subsequently, 2 M sulfuric acid was added at a rate of 100 μL / well to stop the reaction; absorbance values ​​(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 is a mixture of blank medium and diluted human IL33-his in a 1:1 ratio, and the positive control is blank medium.

[0288] Inhibitory activity of humanized antibodies against the promotion of HUVEC-IL6 production by IL33 antibodies IC50 (μg / mL) Relative activity Maximum inhibition rate 5886-156-H1L0 4.051 67.29% 87.89% 5887-41-H1L0 1.308 208.41% 93.20% 5888-116-H0L1 0.8321 327.60% 92.61% 5888-153-H0L1 0.8435 323.18% 92.99% 5888-153-H3L1 1.636 166.63% 93.05% 5888-153-H3L2 3.688 73.92% 91.85% 5888-379-H1L0 16.1 16.93% 83.05% 5888-379-H2L1 26.14 10.43% 71.60% CNTO7160 2.726 100.00% 66.59%

[0289] (8) Inhibitory activity of humanized antibodies against the promotion of HMC-1 IL8 production by IL33

[0290] Human IL33-his was diluted to a final concentration of 1000 ng / mL. Starting at 640 µg / mL, each humanized antibody was diluted fourfold to obtain a total of 11 serial dilutions. The diluted antibodies were mixed with the diluted human IL33-his in a 1:1 ratio, and the resulting mixture was added to 96-well plates at a rate of 50 μL / well. HMC-1 cells in the logarithmic growth phase were taken, added to 96-well plates at 50,000 / well and 50 μL / well, and cultured for 18–24 hours at 37°C and 5% CO2.

[0291] The capture antibody included in the Human IL-8 DuoSet ELISA Kit was coated onto ELISA plates at a rate of 50 μL / well using a working solution obtained by diluting 120-fold with PBS, overnight at 4°C according to the instructions one day prior. Then, the plates were blocked for 1 hour using a blocking buffer and washed 3 times. A standard solution was diluted 40-fold to 2000 pg / mL and then diluted 2-fold to obtain a total of 7 serial dilutions. The cell culture supernatant and one of the diluted standard solutions (50 μL each) were pipetted onto ELISA plates and incubated for 2 hours. The plates were washed 3 times, and a working solution containing the detection antibody diluted 60-fold was added to the plates at a rate of 50 μL / well and incubated for 2 hours. The above plates were washed three more times, and a 125 ng / mL working solution obtained by diluting SA-HRP 40-fold was added to the plates at a rate of 50 μL / well and incubated for 20-30 minutes. Substrate solution was added to the plates at a rate of 50 μL / well and color development was allowed in the dark for 5-10 minutes. Subsequently, 2 M sulfuric acid was added at a rate of 100 μL / well to stop the reaction; absorbance values ​​(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 is a mixture of blank medium and diluted human IL33-his in a 1:1 ratio, and the positive control is blank medium.

[0292] Inhibitory activity of humanized antibodies against the promotion of HMC-1 IL8 production by IL33 antibodies IC50 (μg / mL) Relative activity Maximum inhibition rate 5886-156-H1L0 0.1771 56.86% 81.01% 5887-41-H1L0 0.1234 81.60% 77.81% 5888-116-H0L1 0.005512 1826.92% 90.57% 5888-153-H0L1 0.03592 280.35% 87.17% 5888-153-H3L1 0.04719 213.39% 86.75% 5888-153-H3L2 0.09443 106.64% 83.30% 5888-379-H1L0 0.334 30.15% 85.60% 5888-379-H2L1 0.2298 43.82% 90.15% CNTO7160 0.1007 82.34%

[0293] (9) Measurement of affinity of humanized antibodies

[0294] Experiments on the interaction between anti-human ST2 antibodies and human ST2-his were performed using Biacore X100.

[0295] (9-1) Affinity test of dissociation kinetics of human ST2 from humanized antibody at pH 7.4: The above test was performed at 25°C in HBS-EP(1x) buffer (pH 7.4).

[0296] Each anti-human ST2 antibody was diluted to 2 µg / mL and captured on the surface of a Protein A chip for a capture time of 60 seconds. After antibody capture, a human ST2-his solution (gradual dilution from a starting concentration of 20 nM, yielding a total of 6 concentrations) was injected. Binding was monitored for 180 seconds and dissociation 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.

[0297] (9-2) Affinity experiment of dissociation kinetics of human ST2 from humanized antibody at pH 5.5:

[0298] The above experiment was performed according to the experimental procedure described in (9-1), with the difference that dissociation was performed in HBS-EP(1x) buffer, pH 5.5 and dissociation was monitored for 600 seconds. The results are shown in Table 30.

[0299] Affinity of humanized antibodies at pH 7.4 and pH 5.5 antibodies Affinity (pH 7.4) Affinity (pH 5.5) kd ratio of pH 5.5 / pH 7.4 ka (1 / Ms) kd (1 / s) KD (M) ka (1 / Ms) kd (1 / s) KD (M) 5886-156-H1L0 9.90E+05 2.72E-04 2.75E-10 1.01E+06 2.79E-04 2.76E-10 1.03 5887-41-H1L0 4.67E+05 9.98E-05 2.14E-10 6.22E+05 1.00E-04 1.61E-10 1.00 5888-116-H0L1 1.79E+06 4.64E-04 2.59E-10 2.95E+06 1.40E-03 4.74E-10 3.02 5888-153-H0L1 1.76E+06 1.76E-04 9.97E-11 2.15E+06 1.52E-04 7.06E-11 0.86 5888-153-H3L1 1.53E+06 1.48E-04 9.70E-11 1.90E+06 2.32E-04 1.22E-10 1.57 5888-153-H3L2 1.65E+06 1.71E-04 1.04E-10 1.87E+06 2.45E-04 1.31E-10 1.43 5888-379-H1L0 7.77E+05 3.51E-04 4.52E-10 1.79E+06 2.55E-03 1.43E-09 7.26 5888-379-H2L1 9.79E+05 4.85E-04 4.95E-10 3.46E+06 5.75E-03 1.67E-09 11.86 CNTO7160 7.63E+05 1.75E-04 2.29E-10 6.76E+05 2.67E-04 3.95E-10 1.53 Ab2* 7.58E+05 8.64E-05 1.14E-10 7.45E+05 3.93E-04 5.28E-10 4.55 5888-379-H2L2 1.36E+06 7.02E-04 5.15E-10 1.01E+07 2.69E-02 2.67E-09 38.38 5886-156-H1L1 1.45E+06 3.77E-04 2.61E-10

[0300] * Antibody Ab2 of CN104334582B

[0301] Example 3 : Pharmacokinetics of antibodies in mice

[0302] After the acclimatization period, 20 mice were randomly divided into four groups of five mice each. Administration information is shown in Table 31.

[0303] Administration information for each mouse group antibodies Method of administration Dosage A 5888-116-H0L1 5 mg / kg, intraperitoneal injection 0.1 mL / 10g B 5888-153-H0L1 5 mg / kg, intraperitoneal injection 0.1 mL / 10g C CNTO7160 5 mg / kg, intraperitoneal injection 0.1 mL / 10g D 5886-156-H1L0 5 mg / kg, intraperitoneal injection 0.1 mL / 10g

[0304] Mice were administered according to the group, and the time of administration was recorded. Blood from mice in each group was collected before administration (0 hours), and at 4, 8, 24 hours (1 day), 72 hours (3 days), 120 hours (5 days), 168 hours (7 days), 240 hours (10 days), 288 hours (12 days), and 336 hours (14 days) after administration, and serum was collected and stored at -60 to -80°C.

[0305] Blood drug concentrations for PK studies in mice were detected as follows:

[0306] (1) Coating: Human ST2-his was diluted to 1 μg / mL in PBS (pH 7.2-7.4), added to 96-well ELSIA plates at 50 μL / well, and then sealed with film. The plates were stewed 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).

[0307] (2) Blocking and drying: The plates were blocked with 200 μL / well of N502 at room temperature for 1-2 hours. After aspirating the blocking solution, the plates were dried in an incubator at 25°C for >1 hour, then used immediately or sealed with film and stored at 2-8°C.

[0308] (3) Standard preparations used for plotting standard curves and quality control: Antibody samples were diluted to 1000 ng / mL using mouse plasma (EDTA-K) and then gradient-diluted to 15 ng / mL (7 concentrations including 1000 ng / mL). Additionally, the same antibody samples used to plot standard curves were quantitatively diluted to concentrations within the concentration range for quantitative analysis and used for quality control (QC). Specifically, antibody samples were diluted to 1000 ng / mL, 100 ng / mL, and 20 ng / mL, respectively.

[0309] (4) Preparation of sample to be detected: Obtain a sample from a different blood sampling point and dilute it to a concentration within the concentration range used to plot the standard curve.

[0310] (5) Dilution standards, quality control, and samples: Standards, quality control, and samples were diluted 10-fold (e.g., 10 μL diluted to 90 μL) with 0.1% casein (pH 6.2, obtained by diluting stock casein with PBS, pH 6.2) and added to dried plates at 50 μL / well, and this was repeated twice per sample. The plates were sealed with film and incubated at room temperature for 2 hours, after which PBST (0.05% (v / v) Tween-2

[0311] (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 a rate of 50 μL / well, then sealed and incubated at room temperature for 1 hour. Afterward, the plate was washed 3 times with PBST (containing 0.05% (v / v) Tween-20, pH 7.2-7.4).

[0312] (7) Color development: TMB heated to room temperature was added to the plate at a rate of 50 μL / well and color developed in a dark room for 20 minutes.

[0313] (8) Reaction cessation and OD value reading: 2M sulfuric acid was added to the plate at a rate of 100 μL / well, and then the sulfuric acid was tapped to ensure uniform mixing. The concentration of each sample tested was calculated using the OD reading at 450 nm relative to 650 nm.

[0314] The experimental results showed that three antibodies had half-lives longer than 100 hours, specifically in the order of 5886-156-H1L0 > 5888-153-H0L1 > 5888-116-H0L1. Among the three antibodies, antibody 5886-156H1L0 had a half-life of 10 days, which is the same as CNTO7160. The results are shown in 15 and Table 32.

[0315] PK parameters of antibodies in mice parameters 5888-116-H0L1 5888-153-H0L1 CNTO7160 5886-156-H1L0 t 1 / 2 (h) 174.78 191.46 241.97 241.18 Cmax (kg*ng / ml / mg) 10972.58 9380.59 15733.80 11319.84 AUC (h*ng / ml) 11509209.16 11404571.90 17097590.16 12860587.84 CL (ml / h / kg) 0.31 0.30 0.18 0.26 MRT (h) 139.03 146.65 146.40 147.33

[0316] The above description of the embodiments of the present invention is not intended to limit the invention, and those skilled in the art may make various changes and modifications to the invention without departing from the spirit of the invention, and such changes and modifications must be within the scope of the appended claims of the present invention.

Claims

Claim 1 An antibody or an antigen-binding fragment thereof that binds to ST2, wherein the antibody or the antigen-binding fragment thereof contains a heavy chain variable region (VH) and a light chain variable region (VL), and the heavy chain variable region and the light chain variable region contain a heavy chain CDR (H-CDR) and a light chain CDR (L-CDR) as shown in any one of the combinations selected from the following: (I): H-CDR1, H-CDR2, H-CDR3 as shown sequentially in SEQ ID NOs 56, 63, and 70; and L-CDR1, L-CDR2, L-CDR3 as shown sequentially in SEQ ID NOs 85, 93, and 97; (II): H-CDR1, H-CDR2, H-CDR3 as shown sequentially in SEQ ID NOs 57, 64, and 71; and sequentially L-CDR1, L-CDR2, L-CDR3 as shown in SEQ ID NOs 86, 93, 98; (III): sequentially H-CDR1, H-CDR2, H-CDR3 as shown in SEQ ID NOs 58, 65, 72; and sequentially L-CDR1, L-CDR2, L-CDR3 as shown in SEQ ID NOs 88, 94, 99; (IV-1): sequentially H-CDR1, H-CDR2, H-CDR3 as shown in SEQ ID NOs 59, 66, 73; and sequentially L-CDR1, L-CDR2, L-CDR3 as shown in SEQ ID NOs 89, 95, 100; (IV-2): sequentially H-CDR1, H-CDR2, H-CDR3 as shown in SEQ ID NOs 59, 66, 75; and sequentially L-CDR1, L-CDR2, L-CDR3 as shown in SEQ ID NOs 89, 95, 100; (V-1): sequentially H-CDR1, H-CDR2, H-CDR3 as shown in SEQ ID NOs 60, 67, 76; and sequentially L-CDR1, L-CDR2, L-CDR3 as shown in SEQ ID NOs 90, 95, 101; (V-2): sequentially H-CDR1, H-CDR2, H-CDR3 as shown in SEQ ID NOs 60, 67, 76; and sequentially L-CDR1, L-CDR2, L-CDR3 as shown in SEQ ID NOs 90, 95, 102;(V-3): H-CDR1, H-CDR2, H-CDR3 as shown in sequence nos. 60, 67, and 78 in sequence; and L-CDR1, L-CDR2, L-CDR3 as shown in sequence nos. 90, 95, and 101 in sequence; (V-4): H-CDR1, H-CDR2, H-CDR3 as shown in sequence nos. 60, 67, and 78 in sequence; and L-CDR1, L-CDR2, L-CDR3 as shown in sequence nos. 90, 95, and 102 in sequence; (VI-1): H-CDR1, H-CDR2, H-CDR3 as shown in sequence nos. 61, 68, and 79 in sequence; and sequentially L-CDR1, L-CDR2, L-CDR3 as shown in SEQ ID NOs 91, 95, and 100; (VII-1): H-CDR1, H-CDR2, H-CDR3 as shown in SEQ ID NOs 62, 69, and 82; and sequentially L-CDR1, L-CDR2, L-CDR3 as shown in SEQ ID NOs 92, 96, and 103; (VII-2): H-CDR1, H-CDR2, H-CDR3 as shown in SEQ ID NOs 62, 69, and 83; and sequentially L-CDR1, L-CDR2, L-CDR3 as shown in SEQ ID NOs 92, 96, and 103; and (VII-3): H-CDR1, H-CDR2, H-CDR3 as shown in sequence nos. 62, 69, and 84 in sequence; and L-CDR1, L-CDR2, L-CDR3 as shown in sequence nos. 92, 96, and 103 in sequence.; Claim 2 delete Claim 3 In claim 1, the antibody or its antigen-binding fragment is characterized by containing a heavy chain variable region and a light chain variable region as shown in any one of 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. 3 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. 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. 6 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. 6 and a light chain variable region as shown in SEQ ID NO. 34; (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. 9 and Light chain variable region shown in SEQ ID NO. 37; (IV-1-a): Heavy chain variable region shown in SEQ ID NO. 10 and light chain variable region shown in SEQ ID NO. 39; (IV-1-b): Heavy chain variable region shown in SEQ ID NO. 11 and light chain variable region shown in SEQ ID NO. 41; (IV-1-c): Heavy chain variable region shown in SEQ ID NO. 12 and light chain variable region shown in SEQ ID NO. 41; (IV-2-a): Heavy chain variable region shown in SEQ ID NO. 14 and light chain variable region shown in SEQ ID NO. 41; (V-1-a): Heavy chain variable region shown in SEQ ID NO. 15 and light chain variable region shown in SEQ ID NO. 42; (V-1-b): Heavy chain variable region shown in SEQ ID NO. 16 and light chain variable region shown in SEQ ID NO. 43; (V-1-c): Heavy chain variable region shown in SEQ ID NO. 16 and light chain variable region shown in SEQ ID NO. 44; (V-2-a): Heavy chain variable region shown in SEQ ID NO. 16 and light chain variable region shown in SEQ ID NO. 45 Region; (V-1-d): heavy chain variable region shown in SEQ ID NO. 17 and light chain variable region shown in SEQ ID NO. 44;(V-2-b): The heavy chain variable region shown in SEQ ID NO. 17 and the light chain variable region shown in SEQ ID NO. 45; (V-3-a): The heavy chain variable region shown in SEQ ID NO. 19 and the light chain variable region shown in SEQ ID NO. 44; (V-4-a): The heavy chain variable region shown in SEQ ID NO. 19 and the light chain variable region shown in SEQ ID NO. 45; (VI-1-a): The heavy chain variable region shown in SEQ ID NO. 20 and the light chain variable region shown in SEQ ID NO. 46; (VI-1-b): The heavy chain variable region shown in SEQ ID NO. 22 and the light chain variable region shown in SEQ ID NO. 48; (VII-1-a): The heavy chain variable region shown in SEQ ID NO. 25 and the light chain variable region shown in SEQ ID NO. 49; (VII-1-b): The heavy chain variable region shown in SEQ ID NO. 26 and the light chain variable region shown in SEQ ID NO. 52; (VII-2-a): The heavy chain variable region shown in SEQ ID NO. 27 and the light chain variable region shown in SEQ ID NO. 52; (VII-2-b): A heavy chain variable region represented by SEQ ID NO. 27 and a light chain variable region represented by SEQ ID NO. 53; (VII-3-a): a heavy chain variable region represented by SEQ ID NO. 28 and a light chain variable region represented by SEQ ID NO. 53; and (VII-3-b): a heavy chain variable region represented by SEQ ID NO. 28 and a light chain variable region represented by SEQ ID NO. 50.; Claim 4 The antibody or antigen-binding fragment thereof according to claim 1, characterized in that the antibody or its antigen-binding fragment binds to ST2. Claim 5 An antibody or an antigen-binding fragment thereof, characterized in that, in claim 1, the antibody is a monoclonal antibody, a single-chain antibody, a diabody, a completely or partially humanized antibody, or a chimeric antibody. Claim 6 delete Claim 7 A nucleic acid molecule characterized by containing a nucleotide sequence encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1, 3 to 5. Claim 8 A vector containing a nucleic acid molecule according to paragraph 7. Claim 9 A non-human host cell containing a nucleic acid molecule according to paragraph 7, or transformed or transfected with a nucleic acid molecule according to paragraph 7. Claim 10 delete Claim 11 A pharmaceutical composition for the prevention, treatment, or improvement of a disease comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1, 3 to 5; 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-associated diarrhea. Claim 12 delete Claim 13 delete Claim 14 A method for detecting a disease, characterized by including the step of contacting an antibody or an antigen-binding fragment thereof according to any one of claims 1, 3 to 5 with a sample from a target. in vitro A method for detection or diagnosis in which 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. Claim 15 A kit for detecting a disease, characterized by comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1, 3 to 5, 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-associated diarrhea. Claim 16 delete

Images