Anti-st2 antibodies and uses thereof

Abstract

Provided is an antibody or fragment thereof that binds ST2, which can specifically bind human ST2, inhibit the binding of IL-33 to human ST2, block the IL-33 / ST2 intracellular signaling pathway, and inhibit the promotion of cell IL5, IL6, IL8 production by different forms of IL-33, has higher biological activity than known anti-ST2 antibodies, and can be used for preventing, treating, or improving diseases associated with ST2 expression or IL-33 / ST2 pathway disorders.

Description

[0001] This patent application claims priority to Chinese invention patent application No. CN202010072085.X, filed on January 21, 2020, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of antibody drugs, and more specifically, to antibodies against human ST2 and their use in the preparation of drugs. Background Technology

[0003] Interleukin-33 (IL-33) is a cytokine associated with 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 ST2α) is a binding receptor for IL-33. It is a member of the Toll / IL-1 receptor family and is expressed on the cell surface of a variety of immune cells, including lymphocytes, especially helper T cells expressing IL-5 and IL-13, natural killer (NK) and natural killer-T (NKT) cells, as well as many so-called innate immune cells, such as mast cells, basophils, eosinophils, macrophages, and innate helper cells (also known as nuocytes) (Neill, Wong et al., 2010).

[0004] ST2 can downregulate the responsiveness of Toll-like receptors TLR2, TLR4, and TLR9, but it can also induce the release of type 2 cytokines through activation by its ligand IL-33 and association with the accessory protein IL-1RAcP. Models of the interactions between ST2, IL-33, and IL-1RAcP, as well as the interactions between IL-1R1 and IL-1RAcP, have been proposed in the literature (Lingel et al., Cell 17:1398-1410, 2009; Wang et al., Nat Immunol, 11:905-11, 2010).

[0005] IL-33 has been described as an "alarming factor" because it exists in its full-length form in the nuclei of epithelial and endothelial cells during homeostasis, but can be lysed and released during cell necrosis. Examples of IL-33-induced cellular responses include the production of inflammatory cytokines such as IL-5, IL-6, IL-13, TNF, IFN-γ, and GM-CSF, as well as the production of chemokines such as CXCL8, CCL17, and CCL24. IL-33 also demonstrates enhanced acute hypersensitivity responses by amplifying mast cell and basophil activation induced by IgE receptor signaling or other mast cell and basophil activators. IL-33 also enhances the recruitment, survival, and adhesion properties of ST2-expressing immune cells, thus playing a significant role in initiating and maintaining cellular inflammation in local tissues.

[0006] Dysregulation of the IL-33 / ST2 pathway has been shown to be associated with a variety of immune-mediated diseases, including asthma, rheumatoid arthritis, inflammatory bowel disease, atopic dermatitis, allergic rhinitis, nasal polyps, and systemic sclerosis. Therefore, therapeutic blockade of the IL-33 / ST2 pathway may help overcome hyperimmune responses. Inhibitors targeting this pathway mainly include IL-33 antibodies (such as MEDI3506, ANB020, REGN3500, MT-2990, LY-3375880, PF-06817024) and ST2 antibodies (such as CNTO7160, AMG-282). These investigational antibodies are all in Phase 1 and Phase 2 clinical trials, targeting indications including allergic rhinitis, atopic dermatitis, chronic obstructive pulmonary disease, and asthma.

[0007] The incidence of allergic inflammation and respiratory diseases is gradually increasing, while the drugs available on the market are still mainly glucocorticoids and β2 receptor agonists. Although the reported antibodies can all block the interaction between ST2 and its ligand, their biological activities vary. These differences in biological activity may lead to variations in clinical efficacy and dosage. Therefore, there is still a need in the field to provide ST2 antibodies with high affinity, high stability, and high biological activity. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to obtain a new high-affinity antibody that binds to ST by immunizing mice with human ST2 as an immunogen, obtaining mouse antibodies by B cell panning, and further obtaining new high-affinity antibodies that bind to ST through antibody engineering and humanization technology. The antibody is suitable for treating diseases or any indications related to the IL-33 / ST2 pathway.

[0009] To address the aforementioned technical problems, the present invention aims to provide an antibody or functional fragment thereof that specifically binds to ST2, and to provide its uses.

[0010] The technical solution of the present invention is as follows.

[0011] The “fragment” of the antibody described in this invention encompasses various functional or active fragments of the antibody, such as its antigen-binding portion, like Fab, F(ab')2, or scFv fragments.

[0012] On one hand, the present invention provides an antibody or a fragment thereof, the antibody or fragment thereof comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region and the light chain variable region comprise heavy chain CDR1 (H-CDR1), CDR2 (H-CDR2), CDR3 (H-CDR3) and light chain CDR1 (L-CDR1), CDR2 (L-CDR2), CDR3 (L-CDR3) selected from any of the following combinations:

[0013] (I-1): The heavy chain variable region shown in SEQ ID NO.1 and the light chain variable region shown in SEQ ID NO.29;

[0014] (I-2): The heavy chain variable region shown in SEQ ID NO.2 and the light chain variable region shown in SEQ ID NO.30;

[0015] (I-3): The heavy chain variable region shown in SEQ ID NO.2 and the light chain variable region shown in SEQ ID NO.31;

[0016] (I-4): The heavy chain variable region shown in SEQ ID NO.3 and the light chain variable region shown in SEQ ID NO.30;

[0017] (I-5): The heavy chain variable region shown in SEQ ID NO.3 and the light chain variable region shown in SEQ ID NO.31;

[0018] (II-1): The heavy chain variable region shown in SEQ ID NO.4 and the light chain variable region shown in SEQ ID NO.32;

[0019] (II-2): The heavy chain variable region shown in SEQ ID NO.5 and the light chain variable region shown in SEQ ID NO.33;

[0020] (II-3): The heavy chain variable region shown in SEQ ID NO.5 and the light chain variable region shown in SEQ ID NO.34;

[0021] (II-4): The heavy chain variable region shown in SEQ ID NO.5 and the light chain variable region shown in SEQ ID NO.35;

[0022] (II-5): The heavy chain variable region shown in SEQ ID NO. 6 and the light chain variable region shown in SEQ ID NO. 33;

[0023] (II-6): The heavy chain variable region shown in SEQ ID NO. 6 and the light chain variable region shown in SEQ ID NO. 34;

[0024] (II-7): The heavy chain variable region shown in SEQ ID NO.6 and the light chain variable region shown in SEQ ID NO.35;

[0025] (III-1): The heavy chain variable region shown in SEQ ID NO.7 and the light chain variable region shown in SEQ ID NO.36;

[0026] (III-2): The heavy chain variable region shown in SEQ ID NO.8 and the light chain variable region shown in SEQ ID NO.37;

[0027] (III-3): The heavy chain variable region shown in SEQ ID NO.8 and the light chain variable region shown in SEQ ID NO.38;

[0028] (III-4): The heavy chain variable region shown in SEQ ID NO.9 and the light chain variable region shown in SEQ ID NO.37;

[0029] (III-5): The heavy chain variable region shown in SEQ ID NO.9 and the light chain variable region shown in SEQ ID NO.38;

[0030] (IV-1): The heavy chain variable region shown in SEQ ID NO.10 and the light chain variable region shown in SEQ ID NO.39;

[0031] (IV-2): The heavy chain variable region shown in SEQ ID NO.11 and the light chain variable region shown in SEQ ID NO.40;

[0032] (IV-3): The heavy chain variable region shown in SEQ ID NO.11 and the light chain variable region shown in SEQ ID NO.41;

[0033] (IV-4): The heavy chain variable region shown in SEQ ID NO.12 and the light chain variable region shown in SEQ ID NO.40;

[0034] (IV-5): The heavy chain variable region shown in SEQ ID NO.12 and the light chain variable region shown in SEQ ID NO.41;

[0035] (IV-6): The heavy chain variable region shown in SEQ ID NO.13 and the light chain variable region shown in SEQ ID NO.40;

[0036] (IV-7): The heavy chain variable region shown in SEQ ID NO.13 and the light chain variable region shown in SEQ ID NO.41;

[0037] (IV-8): The heavy chain variable region shown in SEQ ID NO.14 and the light chain variable region shown in SEQ ID NO.40;

[0038] (IV-9): The heavy chain variable region shown in SEQ ID NO.14 and the light chain variable region shown in SEQ ID NO.41;

[0039] (V-1): The heavy chain variable region shown in SEQ ID NO.15 and the light chain variable region shown in SEQ ID NO.42;

[0040] (V-2): The heavy chain variable region shown in SEQ ID NO.16 and the light chain variable region shown in SEQ ID NO.43;

[0041] (V-3): The heavy chain variable region shown in SEQ ID NO.16 and the light chain variable region shown in SEQ ID NO.44;

[0042] (V-4): The heavy chain variable region shown in SEQ ID NO.16 and the light chain variable region shown in SEQ ID NO.45;

[0043] (V-5): The heavy chain variable region shown in SEQ ID NO.17 and the light chain variable region shown in SEQ ID NO.43;

[0044] (V-6): The heavy chain variable region shown in SEQ ID NO.17 and the light chain variable region shown in SEQ ID NO.44;

[0045] (V-7): The heavy chain variable region shown in SEQ ID NO.17 and the light chain variable region shown in SEQ ID NO.45;

[0046] (V-8): The heavy chain variable region shown in SEQ ID NO.18 and the light chain variable region shown in SEQ ID NO.43;

[0047] (V-9): The heavy chain variable region shown in SEQ ID NO.18 and the light chain variable region shown in SEQ ID NO.44;

[0048] (V-10): The heavy chain variable region shown in SEQ ID NO.18 and the light chain variable region shown in SEQ ID NO.45;

[0049] (V-11): The heavy chain variable region shown in SEQ ID NO.19 and the light chain variable region shown in SEQ ID NO.43;

[0050] (V-12): The heavy chain variable region shown in SEQ ID NO.19 and the light chain variable region shown in SEQ ID NO.44;

[0051] (V-13): The heavy chain variable region shown in SEQ ID NO.19 and the light chain variable region shown in SEQ ID NO.45;

[0052] (VI-1): The heavy chain variable region shown in SEQ ID NO.20 and the light chain variable region shown in SEQ ID NO.46;

[0053] (VI-2): The heavy chain variable region shown in SEQ ID NO.21 and the light chain variable region shown in SEQ ID NO.47;

[0054] (VI-3): The heavy chain variable region shown in SEQ ID NO.21 and the light chain variable region shown in SEQ ID NO.48;

[0055] (VI-4): The heavy chain variable region shown in SEQ ID NO.22 and the light chain variable region shown in SEQ ID NO.47;

[0056] (VI-5): The heavy chain variable region shown in SEQ ID NO.22 and the light chain variable region shown in SEQ ID NO.48;

[0057] (VI-6): The heavy chain variable region shown in SEQ ID NO.23 and the light chain variable region shown in SEQ ID NO.47;

[0058] (VI-7): The heavy chain variable region shown in SEQ ID NO.23 and the light chain variable region shown in SEQ ID NO.48;

[0059] (VI-8): The heavy chain variable region shown in SEQ ID NO.24 and the light chain variable region shown in SEQ ID NO.47;

[0060] (VI-9): The heavy chain variable region shown in SEQ ID NO.24 and the light chain variable region shown in SEQ ID NO.48;

[0061] (VII-1): The heavy chain variable region shown in SEQ ID NO.25 and the light chain variable region shown in SEQ ID NO.49;

[0062] (VII-2): The heavy chain variable region shown in SEQ ID NO.26 and the light chain variable region shown in SEQ ID NO.52;

[0063] (VII-3): The heavy chain variable region shown in SEQ ID NO.26 and the light chain variable region shown in SEQ ID NO.53;

[0064] (VII-4): The heavy chain variable region shown in SEQ ID NO.26 and the light chain variable region shown in SEQ ID NO.50;

[0065] (VII-5): The heavy chain variable region shown in SEQ ID NO.26 and the light chain variable region shown in SEQ ID NO.51;

[0066] (VII-6): The heavy chain variable region shown in SEQ ID NO.27 and the light chain variable region shown in SEQ ID NO.52;

[0067] (VII-7): The heavy chain variable region shown in SEQ ID NO.27 and the light chain variable region shown in SEQ ID NO.53;

[0068] (VII-8): The heavy chain variable region shown in SEQ ID NO.27 and the light chain variable region shown in SEQ ID NO.50;

[0069] (VII-9): The heavy chain variable region shown in SEQ ID NO.27 and the light chain variable region shown in SEQ ID NO.51;

[0070] (VII-10): The heavy chain variable region shown in SEQ ID NO.28 and the light chain variable region shown in SEQ ID NO.52;

[0071] (VII-11): The heavy chain variable region shown in SEQ ID NO.28 and the light chain variable region shown in SEQ ID NO.53;

[0072] (VII-12): The heavy chain variable region shown in SEQ ID NO.28 and the light chain variable region shown in SEQ ID NO.50;

[0073] (VII-13): The heavy chain variable region shown in SEQ ID NO.28 and the light chain variable region shown in SEQ ID NO.51.

[0074] Based on the given amino acid sequences of the light and heavy chain variable regions, those skilled in the art can conventionally determine the amino acid sequences of the heavy chain CDRs and light chain CDRs contained therein. For example, according to specific embodiments of the present invention, the Kabat, IMGT, ABM, and Chotia encoding methods are used to delineate the CDRs in the variable region amino acid sequences. Light and heavy chain CDRs and combinations thereof obtained by other methods known in the art are also covered within the scope of the present invention.

[0075] Preferably, the heavy chain variable region and the light chain variable region comprise a heavy chain CDR and a light chain CDR selected from any of the following combinations:

[0076] (I): H-CDR1 (GYSITSDYAWN), H-CDR2 (YIDYSGSTTYNPSLKS), and H-CDR3 (TVIDSMDY) are shown in sequence in SEQ ID NO.56, 63, and 70; and L-CDR1 (RASKSVSTSGHSYMH), L-CDR2 (LASNLES), and L-CDR3 (QHSREFPFT) are shown in sequence in SEQ ID NO.85, 93, and 97;

[0077] (II-1): H-CDR1 (GYSITSDYAWD), H-CDR2 (YIRYSGDTYYNPSLKS), and H-CDR3 (TMMDTMDY) are shown in sequence in SEQ ID NO.57, 64, and 71; and L-CDR1 (RASKSVSTSGNSYMH), L-CDR2 (LASNLES), and L-CDR3 (QHSREFPLT) are shown in sequence in SEQ ID NO.86, 93, and 98;

[0078] (II-2): H-CDR1 (GYSITSDYAWD), H-CDR2 (YIRYSGDTYYNPSLKS), and H-CDR3 (TMMDTMDY) are shown in sequence in SEQ ID NO.57, 64, and 71; and L-CDR1 (RASKSVSTSGNTYMH), L-CDR2 (LASNLES), and L-CDR3 (QHSREFPLT) are shown in sequence in SEQ ID NO.87, 93, and 98;

[0079] (III): H-CDR1 (GFSLSTSGMGVG), H-CDR2 (HIWWDDVKQYNPALKS), and H-CDR3 (IGGDYDYFDF) are shown in sequence in SEQ ID NO. 58, 65, and 72; and L-CDR1 (RASESVEYSGTSLMQ), L-CDR2 (VASNVES), and L-CDR3 (QQSRKVPWT) are shown in sequence in SEQ ID NO. 88, 94, and 99;

[0080] (IV-1): H-CDR1 (GYTFTDSEMY), H-CDR2 (AIDPETGDTAFNQKFKG), and H-CDR3 (AFDNDNDDGFAY) are shown in sequence in SEQ ID NO.59, 66, and 73; and L-CDR1 (SASSSVNYMH), L-CDR2 (DTSKLAS), and L-CDR3 (QQWSSNPLT) are shown in sequence in SEQ ID NO.89, 95, and 100;

[0081] (IV-2): H-CDR1 (GYTFTDSEMY), H-CDR2 (AIDPETGDTAFNQKFKG), and H-CDR3 (AFDNDNDEGFAY) are shown in sequence in SEQ ID NO.59, 66, and 74; and L-CDR1 (SASSSVNYMH), L-CDR2 (DTSKLAS), and L-CDR3 (QQWSSNPLT) are shown in sequence in SEQ ID NO.89, 95, and 100;

[0082] (IV-3): H-CDR1 (GYTFTDSEMY), H-CDR2 (AIDPETGDTAFNQKFKG), and H-CDR3 (AFDNDNDDAFAY) are shown in sequence in SEQ ID NO.59, 66, and 75; and L-CDR1 (SASSSVNYMH), L-CDR2 (DTSKLAS), and L-CDR3 (QQWSSNPLT) are shown in sequence in SEQ ID NO.89, 95, and 100;

[0083] (V-1): H-CDR1 (GYTFTDYELH), H-CDR2 (TIDPETGDTVYNQKFKA), and H-CDR3 (AFYNDYDDGFAY) are shown in sequence in SEQ ID NO. 60, 67, and 76; and L-CDR1 (SVSSSVSYMH), L-CDR2 (DTSKLAS), and L-CDR3 (QQWNSSPLT) are shown in sequence in SEQ ID NO. 90, 95, and 101;

[0084] (V-2): H-CDR1 (GYTFTDYELH), H-CDR2 (TIDPETGDTVYNQKFKA), and H-CDR3 (AFYNDYDDGFAY) are shown in sequence in SEQ ID NO. 60, 67, and 76; and L-CDR1 (SVSSSVSYMH), L-CDR2 (DTSKLAS), and L-CDR3 (QQWNTSPLT) are shown in sequence in SEQ ID NO. 90, 95, and 102;

[0085] (V-3): H-CDR1 (GYTFTDYELH), H-CDR2 (TIDPETGDTVYNQKFKA), and H-CDR3 (AFYNDYDEGFAY) are shown in sequence in SEQ ID NO. 60, 67, and 77; and L-CDR1 (SVSSSVSYMH), L-CDR2 (DTSKLAS), and L-CDR3 (QQWNSSPLT) are shown in sequence in SEQ ID NO. 90, 95, and 101;

[0086] (V-4): H-CDR1 (GYTFTDYELH), H-CDR2 (TIDPETGDTVYNQKFKA), and H-CDR3 (AFYNDYDEGFAY) are shown in sequence in SEQ ID NO. 60, 67, and 77; and L-CDR1 (SVSSSVSYMH), L-CDR2 (DTSKLAS), and L-CDR3 (QQWNTSPLT) are shown in sequence in SEQ ID NO. 90, 95, and 102;

[0087] (V-5): H-CDR1 (GYTFTDYELH), H-CDR2 (TIDPETGDTVYNQKFKA), and H-CDR3 (AFYNDYDDAFAY) are shown in sequence in SEQ ID NO. 60, 67, and 78; and L-CDR1 (SVSSSVSYMH), L-CDR2 (DTSKLAS), and L-CDR3 (QQWNSSPLT) are shown in sequence in SEQ ID NO. 90, 95, and 101;

[0088] (V-6): H-CDR1 (GYTFTDYELH), H-CDR2 (TIDPETGDTVYNQKFKA), and H-CDR3 (AFYNDYDDAFAY) are shown in sequence in SEQ ID NO. 60, 67, and 78; and L-CDR1 (SVSSSVSYMH), L-CDR2 (DTSKLAS), and L-CDR3 (QQWNTSPLT) are shown in sequence in SEQ ID NO. 90, 95, and 102;

[0089] (VI-1): H-CDR1 (GYRFTDSEMH), H-CDR2 (TIDPETGGTVYNQKFKG), and H-CDR3 (AFYNDFDDGFAY) are shown in sequence in SEQ ID NO. 61, 68, and 79; and L-CDR1 (SASTSVSYMH), L-CDR2 (DTSKLAS), and L-CDR3 (QQWSSNPLT) are shown in sequence in SEQ ID NO. 91, 95, and 100;

[0090] (VI-2): H-CDR1 (GYRFTDSEMH), H-CDR2 (TIDPETGGTVYNQKFKG), and H-CDR3 (AFYNDFDEGFAY) are shown in sequence in SEQ ID NO. 61, 68, and 80; and L-CDR1 (SASTSVSYMH), L-CDR2 (DTSKLAS), and L-CDR3 (QQWSSNPLT) are shown in sequence in SEQ ID NO. 91, 95, and 100;

[0091] (VI-3): H-CDR1 (GYRFTDSEMH), H-CDR2 (TIDPETGGTVYNQKFKG), and H-CDR3 (AFYNDFDDAFAY) are shown in sequence in SEQ ID NO. 61, 68, and 81; and L-CDR1 (SASTSVSYMH), L-CDR2 (DTSKLAS), and L-CDR3 (QQWSSNPLT) are shown in sequence in SEQ ID NO. 91, 95, and 100;

[0092] (VII-1): H-CDR1 (GYTFINYGMN), H-CDR2 (WINTYIGEPTYGDNFKG), and H-CDR3 (EGDGFAY) are shown in sequence in SEQ ID NO. 62, 69, and 82; and L-CDR1 (KSSQSLLYSGNQNNYLA), L-CDR2 (GASTRES), and L-CDR3 (QNDHSYPYT) are shown in sequence in SEQ ID NO. 92, 96, and 103;

[0093] (VII-2): H-CDR1 (GYTFINYGMN), H-CDR2 (WINTYIGEPTYGDNFKG), and H-CDR3 (EGEGFAY) are shown in sequence in SEQ ID NO. 62, 69, and 83; and L-CDR1 (KSSQSLLYSGNQNNYLA), L-CDR2 (GASTRES), and L-CDR3 (QNDHSYPYT) are shown in sequence in SEQ ID NO. 92, 96, and 103;

[0094] (VII-3): H-CDR1 (GYTFINYGMN), H-CDR2 (WINTYIGEPTYGDNFKG), and H-CDR3 (EGDAFAY) are shown in sequence in SEQ ID NO. 62, 69, and 84; and L-CDR1 (KSSQSLLYSGNQNNYLA), L-CDR2 (GASTRES), and L-CDR3 (QNDHSYPYT) are shown in sequence in SEQ ID NO. 92, 96, and 103.

[0095] Specifically, the antibody or fragment of the present invention comprises at least a heavy chain variable region and a light chain variable region, both of which include the aforementioned CDRs and spaced frame regions. The arrangement of the domains in the heavy chain variable region and the light chain variable region is as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Further optionally, the maximum 25% difference in amino acid sequence resulting from the "at least 75% identity" can exist in any frame region within the heavy chain variable region or the light chain variable region, or in any domain or sequence outside the heavy chain variable region and the light chain variable region in the antibody or fragment of the present invention. The difference can be caused by amino acid deletion, addition, or substitution at any position, wherein the substitution can be a conservative or non-conservative substitution.

[0096] Preferably, the heavy chain variable region comprises an amino acid sequence shown in any one of SEQ ID NO.1 to SEQ ID NO.28 or an amino acid sequence having at least 75% identity with said amino acid sequence; and / or,

[0097] The light chain variable region comprises an amino acid sequence shown in any one of SEQ ID NO.29 to SEQ ID NO.53 or an amino acid sequence having at least 75% identity with the amino acid sequence.

[0098] 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 selected from any of the following combinations:

[0099] (I-1): The heavy chain variable region shown in SEQ ID NO.1 and the light chain variable region shown in SEQ ID NO.29;

[0100] (I-2): The heavy chain variable region shown in SEQ ID NO.2 and the light chain variable region shown in SEQ ID NO.30;

[0101] (I-3): The heavy chain variable region shown in SEQ ID NO.2 and the light chain variable region shown in SEQ ID NO.31;

[0102] (I-4): The heavy chain variable region shown in SEQ ID NO.3 and the light chain variable region shown in SEQ ID NO.30;

[0103] (I-5): The heavy chain variable region shown in SEQ ID NO.3 and the light chain variable region shown in SEQ ID NO.31;

[0104] (II-1): The heavy chain variable region shown in SEQ ID NO.4 and the light chain variable region shown in SEQ ID NO.32;

[0105] (II-2): The heavy chain variable region shown in SEQ ID NO.5 and the light chain variable region shown in SEQ ID NO.33;

[0106] (II-3): The heavy chain variable region shown in SEQ ID NO.5 and the light chain variable region shown in SEQ ID NO.34;

[0107] (II-4): The heavy chain variable region shown in SEQ ID NO.5 and the light chain variable region shown in SEQ ID NO.35;

[0108] (II-5): The heavy chain variable region shown in SEQ ID NO. 6 and the light chain variable region shown in SEQ ID NO. 33;

[0109] (II-6): The heavy chain variable region shown in SEQ ID NO. 6 and the light chain variable region shown in SEQ ID NO. 34;

[0110] (II-7): The heavy chain variable region shown in SEQ ID NO.6 and the light chain variable region shown in SEQ ID NO.35;

[0111] (III-1): The heavy chain variable region shown in SEQ ID NO.7 and the light chain variable region shown in SEQ ID NO.36;

[0112] (III-2): The heavy chain variable region shown in SEQ ID NO.8 and the light chain variable region shown in SEQ ID NO.37;

[0113] (III-3): The heavy chain variable region shown in SEQ ID NO.8 and the light chain variable region shown in SEQ ID NO.38;

[0114] (III-4): The heavy chain variable region shown in SEQ ID NO.9 and the light chain variable region shown in SEQ ID NO.37;

[0115] (III-5): The heavy chain variable region shown in SEQ ID NO.9 and the light chain variable region shown in SEQ ID NO.38;

[0116] (IV-1): The heavy chain variable region shown in SEQ ID NO.10 and the light chain variable region shown in SEQ ID NO.39;

[0117] (IV-2): The heavy chain variable region shown in SEQ ID NO.11 and the light chain variable region shown in SEQ ID NO.40;

[0118] (IV-3): The heavy chain variable region shown in SEQ ID NO.11 and the light chain variable region shown in SEQ ID NO.41;

[0119] (IV-4): The heavy chain variable region shown in SEQ ID NO.12 and the light chain variable region shown in SEQ ID NO.40;

[0120] (IV-5): The heavy chain variable region shown in SEQ ID NO.12 and the light chain variable region shown in SEQ ID NO.41;

[0121] (IV-6): The heavy chain variable region shown in SEQ ID NO.13 and the light chain variable region shown in SEQ ID NO.40;

[0122] (IV-7): The heavy chain variable region shown in SEQ ID NO.13 and the light chain variable region shown in SEQ ID NO.41;

[0123] (IV-8): The heavy chain variable region shown in SEQ ID NO.14 and the light chain variable region shown in SEQ ID NO.40;

[0124] (IV-9): The heavy chain variable region shown in SEQ ID NO.14 and the light chain variable region shown in SEQ ID NO.41;

[0125] (V-1): The heavy chain variable region shown in SEQ ID NO.15 and the light chain variable region shown in SEQ ID NO.42;

[0126] (V-2): The heavy chain variable region shown in SEQ ID NO.16 and the light chain variable region shown in SEQ ID NO.43;

[0127] (V-3): The heavy chain variable region shown in SEQ ID NO.16 and the light chain variable region shown in SEQ ID NO.44;

[0128] (V-4): The heavy chain variable region shown in SEQ ID NO.16 and the light chain variable region shown in SEQ ID NO.45;

[0129] (V-5): The heavy chain variable region shown in SEQ ID NO.17 and the light chain variable region shown in SEQ ID NO.43;

[0130] (V-6): The heavy chain variable region shown in SEQ ID NO.17 and the light chain variable region shown in SEQ ID NO.44;

[0131] (V-7): The heavy chain variable region shown in SEQ ID NO.17 and the light chain variable region shown in SEQ ID NO.45;

[0132] (V-8): The heavy chain variable region shown in SEQ ID NO.18 and the light chain variable region shown in SEQ ID NO.43;

[0133] (V-9): The heavy chain variable region shown in SEQ ID NO.18 and the light chain variable region shown in SEQ ID NO.44;

[0134] (V-10): The heavy chain variable region shown in SEQ ID NO.18 and the light chain variable region shown in SEQ ID NO.45;

[0135] (V-11): The heavy chain variable region shown in SEQ ID NO.19 and the light chain variable region shown in SEQ ID NO.43;

[0136] (V-12): The heavy chain variable region shown in SEQ ID NO.19 and the light chain variable region shown in SEQ ID NO.44;

[0137] (V-13): The heavy chain variable region shown in SEQ ID NO.19 and the light chain variable region shown in SEQ ID NO.45;

[0138] (VI-1): The heavy chain variable region shown in SEQ ID NO.20 and the light chain variable region shown in SEQ ID NO.46;

[0139] (VI-2): The heavy chain variable region shown in SEQ ID NO.21 and the light chain variable region shown in SEQ ID NO.47;

[0140] (VI-3): The heavy chain variable region shown in SEQ ID NO.21 and the light chain variable region shown in SEQ ID NO.48;

[0141] (VI-4): The heavy chain variable region shown in SEQ ID NO.22 and the light chain variable region shown in SEQ ID NO.47;

[0142] (VI-5): The heavy chain variable region shown in SEQ ID NO.22 and the light chain variable region shown in SEQ ID NO.48;

[0143] (VI-6): The heavy chain variable region shown in SEQ ID NO.23 and the light chain variable region shown in SEQ ID NO.47;

[0144] (VI-7): The heavy chain variable region shown in SEQ ID NO.23 and the light chain variable region shown in SEQ ID NO.48;

[0145] (VI-8): The heavy chain variable region shown in SEQ ID NO.24 and the light chain variable region shown in SEQ ID NO.47;

[0146] (VI-9): The heavy chain variable region shown in SEQ ID NO.24 and the light chain variable region shown in SEQ ID NO.48;

[0147] (VII-1): The heavy chain variable region shown in SEQ ID NO.25 and the light chain variable region shown in SEQ ID NO.49;

[0148] (VII-2): The heavy chain variable region shown in SEQ ID NO.26 and the light chain variable region shown in SEQ ID NO.52;

[0149] (VII-3): The heavy chain variable region shown in SEQ ID NO.26 and the light chain variable region shown in SEQ ID NO.53;

[0150] (VII-4): The heavy chain variable region shown in SEQ ID NO.26 and the light chain variable region shown in SEQ ID NO.50;

[0151] (VII-5): The heavy chain variable region shown in SEQ ID NO.26 and the light chain variable region shown in SEQ ID NO.51;

[0152] (VII-6): The heavy chain variable region shown in SEQ ID NO.27 and the light chain variable region shown in SEQ ID NO.52;

[0153] (VII-7): The heavy chain variable region shown in SEQ ID NO.27 and the light chain variable region shown in SEQ ID NO.53;

[0154] (VII-8): The heavy chain variable region shown in SEQ ID NO.27 and the light chain variable region shown in SEQ ID NO.50;

[0155] (VII-9): The heavy chain variable region shown in SEQ ID NO.27 and the light chain variable region shown in SEQ ID NO.51;

[0156] (VII-10): The heavy chain variable region shown in SEQ ID NO.28 and the light chain variable region shown in SEQ ID NO.52;

[0157] (VII-11): The heavy chain variable region shown in SEQ ID NO.28 and the light chain variable region shown in SEQ ID NO.53;

[0158] (VII-12): The heavy chain variable region shown in SEQ ID NO.28 and the light chain variable region shown in SEQ ID NO.50;

[0159] (VII-13): The heavy chain variable region shown in SEQ ID NO.28 and the light chain variable region shown in SEQ ID NO.51.

[0160] The antibody or fragment thereof provided by this invention binds to ST2, preferably mammalian ST2, more preferably primate ST2, and even more preferably human or cynomolgus monkey ST2, especially human ST2. Experiments have demonstrated that the antibody provided by this invention possesses the following activities:

[0161] (1) Specifically binds to human ST2;

[0162] (2) Inhibits the binding of IL-33 to human ST2;

[0163] (3) Blocking the intracellular signaling pathway of IL-33 / ST2;

[0164] (4) Inhibit the promoting effect of different forms of IL-33 on the production of IL5 in cells;

[0165] (5) Inhibits the promoting effect of IL-33 on the production of IL5, IL6 and IL8 in cells;

[0166] (6) It has a relatively long in vivo half-life.

[0167] Generally, the antibodies or fragments provided by the present invention are any form or fragments thereof, such as monoclonal antibodies, single-chain antibodies, bifunctional antibodies, single-domain antibodies, nanobodies, fully or partially humanized antibodies, or chimeric antibodies; preferably, the antibody is IgA, IgD, IgE, IgG, or IgM, more preferably IgG1, IgG2, or IgG4.

[0168] 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 scFv, BsFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2 or Fv fragment of the antibody.

[0169] More preferably, the antibody or fragment thereof further comprises a human or mouse 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 comprises a heavy chain and a light chain; more preferably, the antibody comprises two heavy chains and two light chains.

[0170] Preferably, the antibody or its fragment comprises a heavy chain constant region selected from IgG, IgA, IgM, IgD, or IgE and / or a κ or λ type light chain constant region. According to a specific embodiment of the present invention, the antibody further comprises a heavy chain constant region, which is an IgG1, IgG2, or IgG4 subtype; or, the antibody further comprises a light chain constant region, which is κ type; more preferably, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO. 54 or an amino acid sequence having at least 75% identity with the amino acid sequence; the light chain constant region comprises the amino acid sequence shown in SEQ ID NO. 55 or an amino acid sequence having at least 75% identity with the amino acid sequence.

[0171] In the context of this invention, "at least 75% identity" can refer to any percentage of identity, such as at least 75%, at least 80%, preferably at least 85%, more preferably at least 90%, further preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99%, which is ≥75%.

[0172] According to a specific embodiment of the present invention, and particularly preferably, the present invention provides the following antibody:

[0173] The antibody named "5888-116-H0L1" has its heavy chain variable region shown in SEQ ID NO.11 and its light chain variable region shown in SEQ ID NO.41.

[0174] The antibody named "5888-153-H0L1" has a heavy chain variable region shown in SEQ ID NO.16 and a light chain variable region shown in SEQ ID NO.44.

[0175] The antibody named "5886-156-H1L0" has a heavy chain variable region shown in SEQ ID NO.3 and a light chain variable region shown in SEQ ID NO.30.

[0176] The heavy chain constant regions of the above antibodies are shown in SEQ ID NO. 54, and the light chain constant regions are shown in SEQ ID NO. 55. The antibodies are monoclonal antibodies comprising two heavy chains and two light chains.

[0177] Based on the antibodies or fragments thereof of the present invention, the present invention also provides conjugates or fusion proteins comprising the antibodies or fragments thereof of the present invention. Such conjugates or fusion proteins may comprise other portions of the antibodies or fragments thereof of the present invention that are bound by chemical or physical methods, such as cell surface receptors, small molecule compounds such as amino acids and sugars, small molecule polymers, or any other portions that modify the antibodies of the present invention, or even active proteins or peptides.

[0178] On the other hand, the present invention also provides a nucleic acid molecule that encodes the heavy chain CDR, light chain CDR, heavy chain variable region, light chain variable region, heavy chain, or light chain in any antibody or fragment thereof of the present invention.

[0179] In another aspect, the present invention provides a vector comprising the nucleic acid molecule of the present invention. The vector may be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, or a bacteriophage vector, etc.

[0180] The vectors or nucleic acid molecules of the present invention can be used to transform or transfect host cells or enter host cells in any way for purposes such as preserving or expressing antibodies. Therefore, in another aspect, the present invention provides a host cell containing the nucleic acid molecules and / or vectors of the present invention, or the host cell being transformed or transfected by the nucleic acid molecules and / or vectors of the present invention. The host cell can be any prokaryotic or eukaryotic cell, such as bacterial or insect, fungal, plant, or animal cells.

[0181] Based on the disclosure of this invention, the antibodies or fragments thereof, as well as corresponding conjugates or fusion proteins, nucleic acid molecules, vectors, and / or host cells provided by this invention can be obtained using any conventional techniques known in the art. The antibodies or fragments thereof, conjugates or fusion proteins, nucleic acid molecules, vectors, and / or host cells can be included in compositions, more particularly in pharmaceutical compositions, such as pharmaceutical formulations, for use in various purposes as needed.

[0182] Therefore, in another aspect, the present invention also provides a composition comprising the antibody or fragment thereof, conjugate or fusion protein, nucleic acid molecule, vector and / or host cell described in this invention. Preferably, the composition is a pharmaceutical composition, which optionally further comprises pharmaceutically acceptable excipients.

[0183] The present invention also provides related applications of the above-mentioned subject matter, including: antibodies or fragments thereof that specifically bind to ST2 or any part thereof.

[0184] In another aspect, the present invention provides the use of the antibody or fragment thereof, conjugate or fusion protein, nucleic acid molecule, vector, host cell and / or composition in the preparation of a medicament for the prevention, treatment or improvement of a disease; preferably, the disease is associated with ST2 expression or dysregulation of the IL-33 / ST2 pathway; preferably, the disease is an inflammatory disease or an autoimmune disease; more preferably, the disease is heart failure, allergic rhinitis, nasal polyps, atopic dermatitis, chronic obstructive pulmonary disease, asthma, pulmonary fibrosis, sepsis, inflammatory bowel disease, systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, Wegener's granulomatosis or chemotherapy-related diarrhea.

[0185] Additionally, the present invention provides a method for preventing, treating, or improving a disease, the method comprising administering to a subject in need the antibody or fragment thereof, conjugate or fusion protein, nucleic acid molecule, vector, host cell and / or composition thereof, and optionally other drugs or means. Preferably, the disease is associated with ST2 expression or dysregulation of the IL-33 / ST2 pathway; preferably, the disease is an inflammatory disease or an autoimmune disease; more preferably, the disease is heart failure, allergic rhinitis, nasal polyps, atopic dermatitis, chronic obstructive pulmonary disease, asthma, pulmonary fibrosis, sepsis, inflammatory bowel disease, systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, Wegener's granulomatosis, or chemotherapy-related diarrhea. The optional other drugs or means refer to other hormonal drugs or immunomodulatory drugs or means that can be administered in combination with the antibody or fragment thereof, conjugate or fusion protein, nucleic acid molecule, vector, host cell and / or composition thereof, such as glucocorticoids, mepolilimab, dupilumab, tezepelumab, etc. The combined administration of the two can be carried out in any form, such as simultaneously, continuously, 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.

[0186] Accordingly, the present invention also provides a pharmaceutical combination comprising the antibody or fragment thereof described in this invention, conjugates or fusion proteins, nucleic acid molecules, vectors, host cells and / or compositions, and optional other pharmaceuticals. These optional other pharmaceuticals refer to other hormonal or immunomodulatory drugs that can be administered in combination with the antibody or fragment thereof, conjugates or fusion proteins, nucleic acid molecules, vectors, host cells and / or compositions of the present invention, such as glucocorticoids, mepolilimab, dupilumab, tezepelumab, etc.

[0187] The present invention also provides a method for detecting or diagnosing a disease, the method comprising contacting the antibody or a fragment thereof, conjugate or fusion protein, nucleic acid molecule, vector, host cell and / or composition with a sample from a subject. Preferably, the disease is associated with ST2 expression or dysregulation of the IL-33 / ST2 pathway; preferably, the disease is an inflammatory disease or an autoimmune disease; more preferably, the disease is heart failure, allergic rhinitis, nasal polyps, atopic dermatitis, chronic obstructive pulmonary disease, asthma, pulmonary fibrosis, sepsis, inflammatory bowel disease, systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, Wegener's granulomatosis, or chemotherapy-related diarrhea. The subject is a mammal, preferably a primate, more preferably a human or a cynomolgus monkey; preferably, the subject is a human.

[0188] In another aspect, the present invention provides a kit comprising the antibody or fragment thereof, conjugate or fusion protein, nucleic acid molecule, vector, host cell and / or composition described in the present invention. The kit can be used for detection or diagnostic purposes, such as in the methods described above for detecting or diagnosing diseases.

[0189] In this invention, mice were immunized with human ST2 antibodies, and the culture supernatant was obtained through B cell panning. Positive clones were then obtained through ELISA screening and further functional screening. Furthermore, humanized antibodies were obtained by humanizing the mouse antibodies through antibody engineering. Through in vitro ligand binding, in vitro inhibition of ligand-activated effector cells, in vitro inhibition of ligand-induced effector cell production of IL5, IL6, and IL8 activity screening experiments, antibody affinity assays, and in vivo drug metabolism experiments in animals, it was demonstrated that the antibody of this invention has higher biological activity compared to existing anti-ST2 antibodies. Attached Figure Description

[0190] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0191] Figures 1 to 5 The inhibition rates of KU812 NF-κB reporter gene activity are shown for B cell clones from different mouse numbers. Among them, Figure 1 : 5883 mice; Figure 2 : 5884 mice; Figure 3 : 5886 mice; Figure 4 : 5887 mice; Figure 5 :5888 mice.

[0192] Figure 6 The study demonstrated the activity of the murine antibody in inhibiting the IL33-induced KU812-IL5 production.

[0193] Figure 7 The binding of the murine antibody to cyno ST2 was demonstrated.

[0194] Figure 8 The binding of the mouse antibody to mouse ST2 was shown.

[0195] Figure 9 The study demonstrated that the humanized antibody inhibited the activity of IL33 binding to human ST2.

[0196] Figure 10 The study demonstrated that the humanized antibody inhibited the activity of IL33 in promoting KU812-IL5 production.

[0197] Figure 11 The study demonstrated the activity of the humanized antibody in inhibiting the KU812-IL5 production induced by oxidized IL33.

[0198] Figure 12 The humanized antibody was shown to inhibit the activity of reduced IL33 in promoting KU812-IL5 production.

[0199] Figure 13 The study demonstrated the activity of the humanized antibody in inhibiting IL33-induced HUVEC-IL6 production.

[0200] Figure 14 The study demonstrated the activity of the humanized antibody in inhibiting IL33-induced HMC-1IL8 production.

[0201] Figure 15 The mouse PK curves of the humanized antibody are shown, where 15-1: 5888-116-H0L1; 15-2: 5888-153-H0L1; 15-3: CNTO7160; 15-4: 5886-156-H1L0.

[0202] The best way to implement an invention

[0203] The present invention will be described below with reference to specific embodiments. Those skilled in the art will understand that these embodiments are for illustrative purposes only and do not limit the scope of the invention in any way.

[0204] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all medicinal materials and reagents used in the following examples are commercially available products.

[0205] Human ST2: NP_003847.2(Met1-Phe328)

[0206] Human ST2-his: Human ST2 with a 6-histidine tag fused to its C-terminus.

[0207] human ST2-fc: Human ST2 with a human IgG1 Fc tag fused to its C-terminus.

[0208] Human IL33: NP_254274.1(Ser112-Thr270)

[0209] Human IL33-his: Human IL33 with a 6-histidine tag fused to its C-terminus.

[0210] Oxidized human IL-33-his: Human IL-33-his was diluted to 300 μg / ml using IMDM, incubated at 37°C for 18 hours, and then purified using an S75 16:600 Superdex column (GE Healthcare).

[0211] Reduced human IL33-his: Human IL33-his, with Cys208Ser and Cys259Ser

[0212] Control antibody CNTO7160: Heavy chain shown in SEQ ID NO.104, light chain shown in SEQ ID NO.105.

[0213] Cyno ST2:XP_005575214.1(Met1-Cys331)

[0214] Cyno ST2-fc: Cyno ST2 with a human IgG1 Fc tag fused to its C-terminus.

[0215] Mouse ST2: NP_001020773.1(Met1-Arg332)

[0216] Mouse ST2-fc: Mouse ST2 with a human IgG1 Fc tag fused to its C-terminus.

[0217] Example 1: Mouse antibody screening

[0218] 1.1 Animal Immunization

[0219] Human ST2-his was expressed in CHO-K1 cells, and six BALB / c mice were immunized according to the standard Freund's adjuvant immunization protocol. Immunization was performed in two batches of three mice each, with a two-week interval between batches. Each batch of mice underwent four immunizations. ELISA was performed using the Human ST2-his expression. If the serum titer was >1:100,000, a final immunization was performed, and spleens were collected 3-4 days later.

[0220] 1.2 B cell selection and culture

[0221] Two days before the formal experiment, feeder cells were seeded into four 10cm culture dishes (Corning, cat. 430167) using culture medium. One day before the formal experiment, the feeder cells were treated with 25μg / mL MMC for 6h. Then, the cells were seeded into 96-well plates (Corning, cat. 3599) at a rate of 10,000 cells / well, 100μL / well. At the same time, 6-well plates were coated with the antigen Human ST2-his and incubated overnight at 4°C.

[0222] The collected spleens of immunized mice were ground, filtered, and centrifuged. Red blood cells were removed using erythrocyte lysis buffer, repeated multiple times until no obvious red blood cells were observed. Then, dendritic cells (DCs) were removed from the spleen cells. Spleen cells obtained from one spleen were evenly added to a 6-well plate pre-coated with the antigen for panning. B cells after antigen panning were collected and counted using trypsin and then transferred to a 96-well plate pre-coated with feeder cells. The plates were incubated at 37°C and 5% CO2 for approximately 10-14 days. The supernatant from the B cell culture that had formed distinct clones was then used for further screening.

[0223] 1.3 Screening with mouse anti-supernatant

[0224] (1) Mouse anti-binding Human ST2-his ELISA screening:

[0225] The above-mentioned immunogen Human ST2-his was diluted to 1 μg / mL with coating buffer, 50 μL / well, and coated overnight at 4°C. The next day, the coated plate was removed and washed three times with PBST, then incubated with blocking buffer at room temperature for 1 h, washed three more times with PBST, and the B cell supernatant was added to the ELISA plate. After incubation at room temperature for 1 h, the plate was washed three times with PBST, and 50 μL / well of goat anti-mouse secondary antibody (1:10000) was added. After incubation at room temperature for 1 h, the plate was washed three times with PBST, and 50 μL / well of TMB was added. The plate was incubated in the dark for 10 min, and the reaction was stopped with 100 μL / well of 2M sulfuric acid. The OD450 value was read on a microplate reader.

[0226] The test results showed that, with the OD value of the negative control (blank culture medium) being more than 10 times that of the negative control as the criterion for positive results, the positive rate of B cell clones from two mice numbered 5883 and 5884 in the screening results of mouse anti-human ST2-his ELISA reached more than 97%.

[0227] 1.4 Screening using the KU812 NF-κB reporter gene method

[0228] Take 1×10 during the logarithmic growth phase 6KU812 cells were centrifuged and washed once, resuspended in 20 μL of buffer R from the Neon Transfection System 10 μL Kit, and 1 μg of pGL4.32[luc2P / NF-κB-RE / Hygro] plasmid was added. Electroporation was performed at 1000V, 50ms, and 1 cycle. After transfection, pressure selection was performed using Hygromycin B to finally obtain the KU812 / NF-κB-1# cell line.

[0229] Human IL33-his was diluted to 1 μg / mL with culture medium and mixed 1:1 with B cell culture supernatant to prepare the test sample. A negative control sample (diluted human IL33-his mixed 1:1 with blank culture medium) and a positive control sample (diluted human IL33-his mixed 1 μg / mL CNTO7160 1:1) were also prepared, with 20 μL of each added to each well of a 384-well plate. KU812 / NF-κB-1# cells in logarithmic growth phase were added to each well at a rate of 20,000 cells / well, at a concentration of 20 μL / well, and incubated overnight (16-24 h) at 37°C with 5% CO2. Then, 40 μL of Bright-glo chromogenic reagent was added to each well, and the plate was shaken for 3 min. The RLU values ​​were then read using a microplate reader.

[0230] The inhibition rate of each clone was calculated based on the values ​​of the negative and positive controls. The results are shown in [the table below]. Figures 1 to 5 .

[0231] Example 2: Antibody engineering

[0232] 2.1 B-cell sequencing

[0233] According to PureLink TM The RNA Mini Kit instructions describe the extraction of B cell clone mRNA, which was then aliquoted and stored at -80°C. The extracted mRNA was used as a template in PrimeScript. TM II. The 1st Strand cDNA Synthesis Kit was used to reverse transcribe the DNA into cDNA, which was then aliquoted and stored at -80°C.

[0234] Using the heavy chain VH fishing primers and light chain VL fishing primers shown in Tables 1-1 and 1-2, VH and VL were amplified with the above cDNA as templates using the Ex Taq enzyme, and then ligated into the pMD18T vector for sequencing.

[0235] Table 1-1 Heavy Chain VH Fishing Primers

[0236]

[0237]

[0238] Table 1-2 Light Chain VL Fishing Primers

[0239]

[0240]

[0241] 2.2 Recombinant Expression and Screening of Mouse Antibodies

[0242] (1) Mouse anti-recombinant expression

[0243] Will come from the same clone (e.g.) Figures 1 to 5 The light and heavy chains (as shown in the figure) were transfected into CHO-K1 cells. After 24 hours of transfection, 10 μg / mL MSX was added for pressure selection. After the cell density and viability recovered, the cells were seeded for feed-batch expression. The supernatant after expression was centrifuged and purified by protein A. The antibody concentration was quantified using the BCA method and then used for quantitative selection.

[0244] Mouse antibodies are named according to the cell clone number from which the light and heavy chains (H+L) they contain originate. For example, mouse antibody "5883-105H+L" indicates that it comes from cell clone 5883-105, with the heavy chain being 5883-105H and the light chain being 5883-105L.

[0245] (2) Screening for mouse anti-binding human ST2 activity

[0246] Human ST2-his was diluted to 1 μg / mL with coating buffer and added to 96-well plates at 50 μL / well. The plates were incubated overnight at 4°C. The next day, the coated plates were removed and washed three times with PBST. They were then incubated with blocking buffer at room temperature for 1 h, followed by three more washes with PBST. Mouse antibody was initially diluted 3-fold from 100 ng / mL, resulting in eight concentrations. Each concentration was added to a 96-well plate at 50 μL / well. The plates were incubated at room temperature for 1 h, washed three times with PBST, and then 50 μL / well of goat anti-mouse secondary antibody (1:10000) was added. The plates were incubated at room temperature for 1 h, washed three times with PBST, and then 50 μL / well of TMB was added. The plates were incubated in the dark for 10 min, and the incubation was terminated with 100 μL / well of 2M sulfuric acid. OD450 values ​​were read using a microplate reader. The results are shown in Table 2.

[0247] Table 2. Murine antibody binding activity against human ST2 and its relative activity compared to CNTO7160.

[0248]

[0249]

[0250]

[0251] (3) Screening of mouse anti-inhibition of IL33 binding to human ST2 activity

[0252] The human ST2-fc protein was diluted to 10 μg / mL with coating buffer and coated into 96-well plates at a rate of 50 μL / well. The plates were incubated overnight at 4°C. The plates were washed three times with PBST, and blocking buffer was added at a rate of 100 μL / well. The plates were incubated at room temperature for 1 h. The plates were then washed three times with PBST. Human IL33-his was diluted to 200 ng / mL using dilution buffer. Mouse antibody was diluted to an initial concentration of 200 μg / mL using the same dilution buffer, followed by 3-fold dilutions to obtain a total of 8 concentrations. The diluted human IL33-his and diluted mouse antibody were mixed 1:1 and added to 50 μL / well of a 96-well plate. The plate was incubated at room temperature for 1 h. After washing three times with PBST, histidine-tagged secondary antibody (1:2500) was added at 50 μL / well, and the plate was incubated at room temperature for 1 h. After washing three times with PBST, TMB was added at 50 μL / well of the 96-well plate. The plate was incubated in the dark for 10 min, and the incubation was terminated with 100 μL / well of 2M sulfuric acid. OD450 and OD650 values ​​were read using a microplate reader. The results are shown in Table 3.

[0253] Table 3. Inhibition of IL33-to-human ST2 activity by murine antibodies and their relative activity compared to CNTO7160.

[0254]

[0255]

[0256]

[0257]

[0258] (4) Screening of mouse anti-IL33-activated KU812 NF-κB reporter gene activity

[0259] Human IL33-his was diluted to 1 μg / mL with culture medium, and mouse antibody and control antibody CNTO7160 were diluted to 50 μg / mL with culture medium. Then, they were diluted 3-fold to obtain a total of 12 concentrations. The antibody was mixed with diluted human IL33-his at a 1:1 ratio to form the test sample. In addition, negative control (blank culture medium mixed with diluted human IL33-his at a 1:1 ratio) and positive control (blank culture medium) were prepared, and 20 μL of each was added to a 384-well plate.

[0260] Take KU812 / NF-κB-1# cells in the logarithmic growth phase, centrifuge and change the medium, add 20,000 cells / well and 20 μL / well to the above 384-well plate, incubate at 37℃ and 5% CO2 overnight (16-24h), then add 40 μL of chromogenic reagent Bright-glo to the well, shake for 3 min, and detect on a microplate reader to read the RLU value. The results are shown in Table 4.

[0261] Table 4. Inhibition of IL33-activated KU812 NF-κB reporter gene activity by murine antibodies and their relative activity compared to CNTO7160.

[0262]

[0263]

[0264]

[0265] (5) Screening for mouse anti-affinity

[0266] Based on the quantitative screening results of the above murine antibodies, 19 murine antibodies were selected for affinity assays and in vitro pharmacological studies. The interaction experiment between anti-human ST2 antibody and human ST2-his was performed using a Biacore X100. All experiments were conducted at 25°C in HBS-EP(1×) buffer (pH 7.4).

[0267] Anti-human ST2 antibody was diluted to 10 nM and captured on the surface of a protein A chip (capture time 60 s). After antibody capture, human ST2-his solution (diluted 2-fold from 11.8 nM to 0.7375 nM, a total of 5 concentrations) was injected. Association was monitored for 4 minutes, dissociation for 10 minutes, and regeneration of the sensor surface was achieved by injecting a glycine solution at pH 2.0. Data generated from kinetic and affinity assays were analyzed using BIAevaluation software. Kinetic data were analyzed using a simple 1:1 binding model; the results are shown in Table 5.

[0268] Table 5 Results of mouse antibody affinity assay

[0269] 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

[0270] (6) In vitro pharmacological studies of mouse antibodies

[0271] Human IL33-his was diluted to 80 ng / mL with culture medium, and the antibody was diluted to 40 μg / mL with culture medium. These were diluted four times to obtain eight concentrations. The two were mixed 1:1, and 50 μL / well was added to each well of a 96-well plate. KU812 cells in logarithmic growth phase were centrifuged, and 100,000 cells / well were added at 50 μL / well to the same 96-well plate. The plates were incubated for 48 h.

[0272] Following the Human IL-5 DuoSet ELISA kit instructions, coat the capture antibody one day in advance (by diluting 240 μg / mL stock solution 120-fold (PBS) to 2 μg / mL working solution), coat overnight at 4°C, 50 μL / well. Block with blocking buffer for 1 h, wash three times. Dilute 120 ng / mL standard solution 400-fold to 300 pg / mL, then 2-fold, for a total of 7 concentrations. Add 50 μL of cell culture supernatant and the diluted standard solution to each well of the ELISA plate and incubate for 2 h. Wash three times, add detection antibody (7.5 μg / mL stock solution) diluted 60-fold to 125 ng / mL working solution, 50 μL / well, and incubate for 2 h. Wash three times, add SA-HRP diluted 40-fold to 125 ng / mL working solution, 50 μL / well, and incubate for 20-30 min. Add 50 μL / well of chromogenic buffer, incubate in the dark for 5-10 min, then add 100 μL / well of 2M sulfuric acid to stop the reaction. Read the OD450 and OD650 values ​​using a microplate reader. Results are shown in Table 6. Figure 6 .

[0273] Table 6 Results of the activity of murine antibody in inhibiting IL33-promoting KU812-IL5 production

[0274] 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

[0275] (7) Crossover experiment of mouse and monkey ST2 antibodies

[0276] Cyno ST2-fc was diluted to 1 μg / mL with coating buffer and added to 96-well plates at 50 μL / well. The plates were incubated overnight at 4°C. The next day, the coated plates were removed and washed three times with PBST. They were then incubated with blocking buffer at room temperature for 1 h, followed by three more washes with PBST. Mouse antibody was initially diluted 3-fold from 1000 ng / mL, resulting in eight concentrations. Each concentration was added to the 96-well plates at 50 μL / well. The plates were incubated at room temperature for 1 h, washed three times with PBST, and then 50 μL / well of goat anti-mouse secondary antibody (1:10000) was added. The plates were incubated at room temperature for 1 h, washed three times with PBST, and then 50 μL / well of TMB was added. The plates were incubated in the dark for 10 min, and the incubation was terminated with 100 μL / well of 2M sulfuric acid. OD450 values ​​were read using a microplate reader. Results are shown in Table 7. Figure 7It can be seen that mouse antibodies can bind to cyno ST2, and the binding trend is consistent with that of human ST2.

[0277] Table 7. Results of the binding experiment between murine antibody and cyno ST2.

[0278] 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

[0279] Interaction experiments between anti-human ST2 antibody and human ST2-his were performed using a Biacore X100. All experiments were conducted at 25°C in HBS-EP (1.1×) buffer (pH 7.4). The antibody was immobilized on the surface of a CM5 chip using an amino-coupling kit. After immobilization, cyno ST2-fc solution (initial concentration 8 nM, 2-fold dilution for 6 concentrations; for individual samples with low signal values, the initial concentration could be increased) was injected. Association was monitored for 2 minutes, dissociation for 10 minutes, and regeneration of the sensor surface was achieved by injecting a glycine solution at pH 1.5. Kinetic data were analyzed using a simple 1:1 binding model; the results are shown in Table 8.

[0280] Table 8. Results of Affinity Measurement of Mouse Antibody with CynoST2

[0281] 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

[0282] Mouse ST2-fc was diluted to 1 μg / mL with coating buffer and added to 96-well plates at 50 μL / well. The plates were incubated overnight at 4°C. The next day, the coated plates were removed and washed three times with PBST. They were then incubated with blocking buffer at room temperature for 1 hour, followed by three more washes with PBST. Mouse antibody was initially diluted 3-fold from 1000 ng / mL to eight different concentrations, and added to 96-well plates at 50 μL / well. The plates were incubated at room temperature for 1 hour, washed three times with PBST, and then 50 μL / well of goat anti-mouse secondary antibody (1:10000) was added. The plates were incubated at room temperature for 1 hour, washed three times with PBST, and then 50 μL / well of TMB was added. The plates were incubated in the dark for 10 minutes, and the incubation was terminated with 100 μL / well of 2M sulfuric acid. The OD450 values ​​were read using a microplate reader. Results are shown in the attached table. Figure 8 It can be seen that the mouse antibody binds weakly to Mouse ST2, consistent with the control antibody CNTO7160.

[0283] 2.3 Humanization

[0284] Seven mouse antibodies, namely 5886-156H+L, 5887-41H+L, 5887-537H3+L1, 5888-116H1+L1, 5888-153H1+L2, 5888-357H+L, and 5888-379H1+L2, were selected for humanization design.

[0285] The heavy and light chain variable region sequences of seven murine antibodies were compared with human germline sequences using a BLAST search of the IMGT database. Redundant genes and those with unpaired cysteine ​​residues were removed from this group of human germline genes. The remaining closest matching human germline genes in both the frame and CDR regions were selected as the recipient human frame. FR-4 was selected based on sequence similarity of the IGHJ / IGJK germline genes. Tables 9 to 15 show the humanized sequence forms of seven murine antibodies: 5886-156H+L, 5887-41H+L, 5887-537H3+L1, 5888-116H1+L1, 5888-153H1+L2, 5888-357H+L, and 5888-379H1+L2. Version HZ0 represents CDR transplantation only, version HZ1 introduces a reversion mutation, and versions HZ2 and above attempt to mutate PTM sites appearing in the sequence. The specific humanized sequences are shown in Tables 9 to 15, where the corresponding CDRs are indicated by underlined lines (according to the definition of enhanced Chothia / AbM CDRs).

[0286] Table 9. Humanized sequence forms of the variable region of the 5886-156 heavy-light chain.

[0287]

[0288] Table 10. Humanized sequence forms of the variable region of the heavy-light chain in 5887-41

[0289]

[0290]

[0291] Table 11. Humanized sequence forms of the variable regions of heavy and light chains in 5887-537

[0292]

[0293]

[0294] Table 12 Humanized sequence forms of the variable region of the heavy-light chain in 5888-116

[0295]

[0296]

[0297] Table 13 Humanized sequence forms of the variable region of the heavy-light chain in 5888-153

[0298]

[0299]

[0300] Table 14. Humanized sequence forms of the variable region of the heavy-light chain in 5888-357

[0301]

[0302]

[0303] Table 15 Humanized sequence forms of the variable region of the heavy-light chain in 5888-379

[0304]

[0305]

[0306] 2.4 Recombinant Expression and Screening of Humanized Antibodies

[0307] (1) Recombinant expression of humanized antibodies

[0308] Humanized light and heavy chains derived from the same mouse antibody were constructed using the heavy chain constant region shown in SEQ ID NO.54 and the light chain constant region shown in SEQ ID NO.55. These were then combined and transfected into CHO-K1 cells. After 24 hours of transfection, 10 μg / mL MSX was added for pressure selection. Once cell density and viability recovered, the cells were seeded for feed-batch expression. The supernatant from the centrifuged cells after expression was purified by protein A, and the antibody concentration was quantified using the BCA method for quantitative selection.

[0309] The pairing of humanized antibodies and their heavy and light chain variable regions is shown in Tables 16 to 22. The suffix "ix" indicates that the antibody is a corresponding chimeric antibody.

[0310] Table 16. Light and heavy chain variable region sequence pairings of humanized antibody 5886-156

[0311]

[0312] Table 17. Light and heavy chain variable region sequence matching of humanized antibodies 5887-41

[0313]

[0314] Table 18. Light and heavy chain variable region sequence matching of humanized antibodies 5887-537

[0315]

[0316]

[0317] Table 19. Light and heavy chain variable region sequence pairings of the humanized antibody 5888-116

[0318]

[0319] Table 20. Light and heavy chain variable region sequence matching of humanized antibody 5888-153

[0320]

[0321] Table 21. Light and heavy chain variable region sequence pairings of the humanized antibody 5888-357

[0322]

[0323]

[0324] Table 22. Light and heavy chain variable region sequence matching of humanized antibody 5888-379

[0325]

[0326] (2) Screening for humanized antibody inhibition of IL33 binding to human ST2 activity

[0327] The test was conducted according to the experimental procedure described in Section 2.2 above, "(3) Screening for mouse anti-inhibition of IL33 binding to human ST2 activity". Results are shown below. Figure 9 And Table 23, Figure 9 The negative control was a mixture of blank culture medium and diluted human IL33-his at a 1:1 ratio, while the positive control was a blank culture medium.

[0328] Table 23. Activity of humanized antibodies in inhibiting IL33 binding to human ST2 and their relative activity compared to CNTO7160.

[0329]

[0330]

[0331] (3) Screening for the activity of humanized antibodies in inhibiting IL33 and promoting KU812-IL5 production

[0332] Human IL33-his was diluted to 80 ng / mL with culture medium, and the humanized antibody was diluted to 640 μg / mL with culture medium. These were diluted four times to obtain 11 concentrations. The two were mixed 1:1 and 50 μL / well was added to a 96-well plate. The subsequent experimental procedure was the same as in Section 2.2, “(6) In vitro pharmacological study of mouse antibody”. The results are shown in Table 24 and… Figure 10 , Figure 10 The negative control was a mixture of blank culture medium and diluted human IL33-his at a 1:1 ratio, while the positive control was a blank culture medium.

[0333] Table 24. Results of the humanized antibody's activity in inhibiting IL33-induced KU812-IL5 production and its relative activity compared to CNTO7160.

[0334]

[0335]

[0336] (4) Study on the activity of humanized antibody in inhibiting IL33 activation of KU812 NF-κB reporter gene

[0337] The experimental procedure described in Section 2.2 above, "(4) Screening for mouse anti-IL33-activated KU812 NF-κB reporter gene activity", was followed. The results are shown in Table 25.

[0338] Table 25. Inhibition of IL33-activated KU812 NF-κB reporter gene activity by humanized antibodies and their relative activity compared to CNTO7160.

[0339] 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％

[0340] (5) Study on the inhibitory activity of humanized antibody against oxidized IL33 promoting KU812-IL5 production

[0341] Oxidized human IL33-his was diluted to 200 ng / mL with culture medium, and the humanized antibody was diluted to 640 μg / mL with culture medium. These were diluted four times to obtain 11 concentrations. The two were mixed 1:1 and 50 μL / well was added to a 96-well plate. The subsequent experimental procedure was the same as in Section 2.2, “(6) In vitro pharmacological study of mouse antibody”. The results are shown below. Figure 11 And Table 26, Figure 11 The negative control was a mixture of blank culture medium and diluted human IL33-his at a 1:1 ratio, while the positive control was a blank culture medium.

[0342] Table 26 Results of the activity of humanized antibodies in inhibiting the production of oxidized IL33 and promoting KU812-IL5.

[0343] 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％

[0344] (6) Study on the inhibitory activity of humanized antibody against reduced IL33 promoting KU812-IL5 production

[0345] Reduced human IL33-his was diluted to 6 ng / mL with culture medium, and the humanized antibody was diluted to 640 μg / mL with culture medium. Eleven spots were prepared using a 4-fold dilution. The two were then mixed 1:1, and 50 μL / well was added to each well of a 96-well plate. The subsequent experimental procedure was the same as in Section 2.2, “(6) In vitro pharmacological study of mouse antibody”. Results are shown below. Figure 12 And Table 27, Figure 12 The negative control was a mixture of blank culture medium and diluted human IL33-his at a 1:1 ratio, while the positive control was a blank culture medium.

[0346] Table 27 Results of the activity of humanized antibodies in inhibiting the reduction of IL33 and promoting KU812-IL5 production.

[0347] 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％

[0348] (7) Study on the inhibitory activity of humanized antibody against IL33-promoted HUVEC-IL6 production

[0349] HUVEC cells were incubated at 10,000 cells / well, 100 μL, at 37°C and 5% CO2 for 18-24 h. Human IL33-his was diluted to 10 ng / mL with culture medium, and the humanized antibody was diluted to 400 μg / mL with culture medium. These were diluted 4-fold to obtain 11 concentrations. The two were mixed 1:1, and 100 μL / well was added to each well of the 96-well plate. The plates were then incubated at 37°C and 5% CO2 for 18-24 h.

[0350] Following the Human IL-6 DuoSet ELISA kit instructions, coat the capture antibody one day in advance (by diluting 240 μg / mL stock solution 120-fold (PBS) to 2 μg / mL working solution), coat overnight at 4°C, 50 μL / well. Block with blocking buffer for 1 h, wash three times. Dilute 180 ng / mL standard solution 300-fold to 600 pg / mL, then 2-fold, for a total of 7 concentrations. Add 50 μL of cell culture supernatant and the diluted standard solution to each well of the ELISA plate and incubate for 2 h. Wash three times, add detection antibody (3 μg / mL stock solution) diluted 60-fold to 50 ng / mL working solution, 50 μL / well, and incubate for 2 h. Wash three times, add SA-HRP diluted 40-fold to 125 ng / mL working solution, 50 μL / well, and incubate for 20-30 min. Add 50 μL of chromogenic buffer per well, incubate in the dark for 5-10 min, then add 100 μL of 2M sulfuric acid per well to stop the reaction. Read the OD450 and OD650 values ​​using a microplate reader. Results are shown below. Figure 13 And Table 28, Figure 13 The negative control was a mixture of blank culture medium and diluted human IL33-his at a 1:1 ratio, while the positive control was a blank culture medium.

[0351] Table 28 Results of the activity of humanized antibodies in inhibiting IL33-promoted HUVEC-IL6 production.

[0352] 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％

[0353] (8) Study on the inhibitory activity of humanized antibody against IL33-induced HMC-1 IL8 production

[0354] The final concentration of human IL33-his was 1000 ng / mL. Humanized antibody was initially 640 μg / mL, diluted 4-fold (totaling 11 concentrations), and the mixture was added 50 μL / well to each well of a 96-well plate. Logarithmic growth phase HMC-1 cells were added at a rate of 50,000 cells / well, at 50 μL / well, and incubated at 37°C and 5% CO2 for 18-24 h.

[0355] According to the Human IL-8 DuoSet ELISA kit instructions, one day in advance, coat the capture antibody (using a 120-fold dilution (PBS) working solution) and incubate overnight at 4°C, 50 μL / well. Block with blocking buffer for 1 h, then wash three times. Dilute the standard solution 40-fold to 2000 pg / mL, then 2-fold, for a total of 7 concentrations. Add 50 μL of cell culture supernatant and the diluted standard solution to each well in an ELISA plate and incubate for 2 h. Wash three times, add 50 μL / well of the 60-fold dilution of the detection antibody working solution, and incubate for 2 h. Wash three times, add 50 μL / well of SA-HRP working solution (40-fold dilution to 125 ng / mL), and incubate for 20-30 min. Add 50 μL / well of chromogenic buffer, incubate in darkness for 5-10 min, and stop with 100 μL / well of 2M sulfuric acid. Read the OD450 and OD650 values ​​using a microplate reader. Results are shown in the figure. Figure 14 And Table 29, Figure 4 The negative control was a 1:1 mixture of blank culture medium and human IL33-his, and the positive control was a blank culture medium.

[0356] Table 29 Results of the activity of humanized antibodies in inhibiting IL33-promoting HMC-1 IL8 production.

[0357] 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％

[0358] (9) Humanized antibody affinity assay

[0359] The interaction between the anti-human ST2 antibody and human ST2-his was investigated using a Biacore X100.

[0360] (9-1) Dissociation kinetics and affinity experiment of human ST2 and humanized antibody at pH 7.4: All experiments were performed at 25°C in HBS-EP(1×) buffer (pH 7.4).

[0361] Anti-human ST2 antibody was diluted to 2 μg / mL and captured on the surface of a Protein A chip for 60 s. After antibody capture, human ST2-his solution (starting concentration 20 nM, serially diluted 2-fold to 6 concentrations) was injected. Association was monitored for 180 s, dissociation for 700 s, and regeneration of the sensor surface was achieved by injecting a glycine solution at pH 2.0. Kinetic data were analyzed using a 1:1 binding model.

[0362] (9-2) Dissociation kinetics and affinity experiment of human ST2 and humanized antibody at pH 5.5:

[0363] The experimental procedure described in (9-1) was followed, except that dissociation was carried out in HBS-EP(1×) buffer at pH 5.5 and the dissociation was monitored for 600 s.

[0364] The results are shown in Table 30.

[0365] Table 30 Affinity of humanized antibodies at pH 7.4 and pH 5.5

[0366]

[0367] Antibody Ab2 in CN104334582B

[0368] Example 3 Antibody mouse PK

[0369] After the adaptation period, 20 mice were randomly divided into 4 groups of 5 mice each. The drug administration information is shown in Table 31.

[0370] Table 31 Drug administration information for each group of mice

[0371] A 5888-116-H0L1 5mg / kg ip 0.1mL / 10g B 5888-153-H0L1 5mg / kg ip 0.1mL / 10g C CNTO7160 5mg / kg ip 0.1mL / 10g D 5886-156-H1L0 5mg / kg ip 0.1mL / 10g

[0372] Mice were administered drugs according to their respective groups, and the administration time was recorded. Blood samples were collected from each group of mice before administration (0hr) and at 4hr, 8hr, 24hr (1d), 72hr (3d), 120hr (5d), 168hr (7d), 240hr (10d), 288hr (12d), and 336hr (14d) after administration. Serum was collected and stored at -60 to -80℃.

[0373] The method for detecting drug concentration in mouse pharmacokinetic (PK) blood is as follows:

[0374] (1) Coating: Dilute human ST2-his to 1 μg / mL with PBS (7.2-7.4) and add 50 μL / well to a 96-well ELISA plate. Seal the plate. Incubate at 2-8℃ for 15-20 hours. Wash three times with PBST (pH 7.2-7.4, Tween-20 0.05% v / v).

[0375] (2) Sealing and drying: Use N502 for sealing, 200 μL / well, at room temperature for 1-2 hours. Aspirate the sealing solution. Dry in a 25°C incubator for >1 hour. Use immediately or seal and store at 2-8°C.

[0376] (3) Preparation of standard curves and quality control samples: The antibody samples were diluted with mouse plasma (EDTA-K) to 1000 ng / mL, and then serially diluted 2-fold to 15 ng / mL (7 points including 1000 ng / mL). In addition, the antibody samples of the standard curves were quantitatively diluted to the quantitative range as quality control samples (QC) at concentrations of 1000 ng / mL, 100 ng / mL, and 20 ng / mL.

[0377] (4) Sample processing: Obtain samples from each blood collection point and dilute them to the standard curve range using pure mouse plasma.

[0378] (5) Standard curve, QC, and sample dilution: Dilute 0.1% casein (pH 6.2, casein stock solution diluted with PBS 6.2) 10-fold (e.g., 10 μL + 90 μL) into the dried plate, 50 μL / well, with two replicates per sample. Seal the plate and incubate at room temperature for 2 hours. Wash three times with PBST (pH 7.2-7.4, Tween-20 0.05% v / v).

[0379] (6) Secondary antibody incubation: Dilute Goat anti-Human IgG Fc-HRP 50,000 times with 10% goat serum, add 50 μL / well to the plate, seal the plate, and incubate at room temperature for 1 hour. Wash 3 times with PBST (pH 7.2-7.4, Tween-20 0.05% v / v).

[0380] (7) Color development: Add 50 μL of TMB that has been incubated to room temperature into the plate and react in the dark for 20 min.

[0381] (8) Termination and reading: 100 μL of 2M sulfuric acid per well, tap the plate gently to mix. Take a reading at 450 nm (reference 650 nm) and calculate the concentration of the sample.

[0382] The experimental results showed that the half-lives of all three antibodies exceeded 100 hours, specifically in the order of 5886-156-H1L0 > 5888-153-H0L1 > 5888-116-H0L1. The half-life of 5886-156H1L0 was comparable to that of CNTO7160, reaching 10 days. (See results below.) Figure 15 And Table 32.

[0383] Table 32 Mouse PK parameters of the antibody

[0384] <![CDATA[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

[0385] The above description of specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention, and as long as they do not depart from the spirit of the present invention, they should all fall within the scope of the appended claims. sequence list <110> Maiwei (Shanghai) Biotechnology Co., Ltd. <120> Anti-ST2 antibodies and their applications <130> LC21210001P-CN <140> CN2021800107562 <141> 2021-01-21 <150> CN202010072085.X <151> 2020-01-21 <160> 105 <170> PatentIn version 3.3 <210> 1 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> 5886-156H-VH <400> 1 Asp Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45 Met Gly Tyr Ile Asp Tyr Ser Gly Ser Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Phe Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Ser Thr Val Ile Asp Ser Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser 115 <210> 2 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> 5886-156H-VH-HZ0 <400> 2 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Asp Tyr Ser Gly Ser Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Val Ile Asp Ser Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 3 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> 5886-156H-VH-HZ1 <400> 3 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Asp Tyr Ser Gly Ser Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Thr Val Ile Asp Ser Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 4 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> 5887-41H-VH <400> 4 Asp Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp 20 25 30 Tyr Ala Trp Asp Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45 Met Gly Tyr Ile Arg Tyr Ser Gly Asp Thr Tyr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Thr Thr Met Met Asp Thr Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser 115 <210> 5 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> 5887-41H-VH-HZ0 <400> 5 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 Tyr Ala Trp Asp Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Arg Tyr Ser Gly Asp Thr Tyr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Met Met Asp Thr Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 6 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> 5887-41H-VH-HZ1 <400> 6 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 Tyr Ala Trp Asp Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Arg Tyr Ser Gly Asp Thr Tyr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Thr Met Met Asp Thr Met Asp Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 <210> 7 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> 5887-537H3-VH <400> 7 Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Gln Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala His Ile Trp Trp Asp Asp Val Lys Gln Tyr Asn Pro Ala 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Ser Ser Gln Val 65 70 75 80 Phe Leu Lys Ser Ala Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Ile Gly Gly Asp Tyr Asp Tyr Phe Asp Phe Trp Gly Gln 100 105 110 Gly Thr Thr Leu Thr Val Ser Ser 115 120 <210> 8 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> 5887-537H3-VH-HZ0 <400> 8 Gln Val Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Met Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45 Trp Leu Ala His Ile Trp Trp Asp Asp Val Lys Gln Tyr Asn Pro Ala 50 55 60 Leu Lys Ser Arg Leu Thr Ile Thr Lys Asp Thr Ser Lys Asn Gln Val 65 70 75 80 Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala His Ile Gly Gly Asp Tyr Asp Tyr Phe Asp Phe Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 9 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> 5887-537H3-VH-HZ1 <400> 9 Gln Val Thr Leu Lys Glu Ser Gly Pro Thr Leu Val Lys Pro Thr Gln 1 5 10 15 Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Met Gly Val Gly Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu 35 40 45 Trp Leu Ala His Ile Trp Trp Asp Asp Val Lys Gln Tyr Asn Pro Ala 50 55 60 Leu Lys Ser Arg Leu Thr Ile Thr Lys Asp Thr Ser Lys Ser Gln Val 65 70 75 80 Val Leu Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Ile Gly Gly Asp Tyr Asp Tyr Phe Asp Phe Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 10 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> 5888-116H1-VH <400> 10 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Thr Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30 Glu Met Tyr Trp Val Arg Leu Thr Pro Val His Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Asp Pro Glu Thr Gly Asp Thr Ala Phe Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Val Val Tyr Tyr Cys 85 90 95 Thr Arg Ala Phe Asp Asn Asp Asn Asp Asp Gly Phe Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ala 115 120 <210> 11 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> 5888-116H1-VH-HZ0 <400> 11 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30 Glu Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ala Ile Asp Pro Glu Thr Gly Asp Thr Ala Phe Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Phe Asp Asn Asp Asn Asp Asp Gly Phe Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 12 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> 5888-116H1-VH-HZ1 <400> 12 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30 Glu Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ala Ile Asp Pro Glu Thr Gly Asp Thr Ala Phe Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Ala Phe Asp Asn Asp Asn Asp Asp Gly Phe Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 13 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> 5888-116H1-VH-HZ2 <400> 13 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30 Glu Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ala Ile Asp Pro Glu Thr Gly Asp Thr Ala Phe Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Ala Phe Asp Asn Asp Asn Asp Glu Gly Phe Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 14 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> 5888-116H1-VH-HZ3 <400> 14 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30 Glu Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ala Ile Asp Pro Glu Thr Gly Asp Thr Ala Phe Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Ala Phe Asp Asn Asp Asn Asp Asp Ala Phe Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 15 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> 5888-153H1-VH <400> 15 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Thr Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Leu His Trp Val Lys Gln Thr Pro Val His Gly Leu Glu Trp Ile 35 40 45 Gly Thr Ile Asp Pro Glu Thr Gly Asp Thr Val Tyr Asn Gln Lys Phe 50 55 60 Lys Ala Lys Ala Ile Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Glu Phe Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Cys Tyr Cys 85 90 95 Thr Arg Ala Phe Tyr Asn Asp Tyr Asp Asp Gly Phe Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ala 115 120 <210> 16 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> 5888-153H1-VH-HZ0 <400> 16 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Leu His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Thr Ile Asp Pro Glu Thr Gly Asp Thr Val Tyr Asn Gln Lys Phe 50 55 60 Lys Ala Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Phe Tyr Asn Asp Tyr Asp Asp Gly Phe Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 17 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> 5888-153H1-VH-HZ1 <400> 17 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Leu His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Thr Ile Asp Pro Glu Thr Gly Asp Thr Val Tyr Asn Gln Lys Phe 50 55 60 Lys Ala Arg Val Thr Met Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Ala Phe Tyr Asn Asp Tyr Asp Asp Gly Phe Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 18 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> 5888-153H1-VH-HZ2 <400> 18 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Leu His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Thr Ile Asp Pro Glu Thr Gly Asp Thr Val Tyr Asn Gln Lys Phe 50 55 60 Lys Ala Arg Val Thr Met Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Ala Phe Tyr Asn Asp Tyr Asp Glu Gly Phe Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 19 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> 5888-153H1-VH-HZ3 <400> 19 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Glu Leu His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Thr Ile Asp Pro Glu Thr Gly Asp Thr Val Tyr Asn Gln Lys Phe 50 55 60 Lys Ala Arg Val Thr Met Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Ala Phe Tyr Asn Asp Tyr Asp Asp Ala Phe Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 20 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> 5888-357H-VH <400> 20 Gln Val Gln Leu Gln Gln Ser Val Thr Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Thr Leu Ser Cys Lys Ala Ser Gly Tyr Arg Phe Thr Asp Ser 20 25 30 Gly Met His Trp Val Lys Gln Thr Pro Val His Gly Leu Glu Trp Ile 35 40 45 Gly Thr Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Lys Leu Thr Ala Asp Arg Ser Ser Ser Thr Val Ser 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Ala Phe Tyr Asn Asp Phe Asp Asp Gly Phe Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ala 115 120 <210> 21 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> 5888-357H-VH-HZ0 <400> 21 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Arg Phe Thr Asp Ser 20 25 30 Gly Met Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe 35 40 45 Gly Thr Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Phe Tyr Asn Asp Phe Asp Asp Gly Phe Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 22 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> 5888-357H-VH-HZ1 <400> 22 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Arg Phe Thr Asp Ser 20 25 30 Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Thr Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Ala Asp Arg Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Ala Phe Tyr Asn Asp Phe Asp Asp Gly Phe Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 23 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> 5888-357H-VH-HZ2 <400> 23 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Arg Phe Thr Asp Ser 20 25 30 Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Thr Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Ala Asp Arg Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Ala Phe Tyr Asn Asp Phe Asp Glu Gly Phe Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 24 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> 5888-357H-VH-HZ3 <400> 24 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Arg Phe Thr Asp Ser 20 25 30 Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Thr Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Ala Asp Arg Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Ala Phe Tyr Asn Asp Phe Asp Asp Ala Phe Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 25 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> 5888-379H1-VH <400> 25 Gln Ile Gln Leu Ala Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Asn Tyr 20 25 30 Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Asp Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Ile Gly Glu Pro Thr Tyr Gly Asp Asn Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Val Tyr 65 70 75 80 Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Glu Gly Asp Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ala 115 <210> 26 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> 5888-379H1-HZ0 <400> 26 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Ile Gly Glu Pro Thr Tyr Gly Asp Asn Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Asp Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 27 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> 5888-379H1-VH-HZ1 <400> 27 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Ile Gly Glu Pro Thr Tyr Gly Asp Asn Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Glu Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 28 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> 5888-379H1-VH-HZ2 <400> 28 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ile Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Ile Gly Glu Pro Thr Tyr Gly Asp Asn Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Asp Ala Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 29 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> 5886-156L-VL <400> 29 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly His Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile Asn 65 70 75 80 Pro Met Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg 85 90 95 Glu Phe Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 30 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> 5886-156L-VL-HZ0 <400> 30 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly 1 5 10 15 Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly His Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn 65 70 75 80 Pro Val Glu Ala Asn Asp Thr Ala Asn Tyr Tyr Cys Gln His Ser Arg 85 90 95 Glu Phe Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 31 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> 5886-156L-VL-HZ1 <400> 31 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly 1 5 10 15 Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly His Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn 65 70 75 80 Pro Val Glu Ala Asn Asp Ala Ala Asn Tyr Tyr Cys Gln His Ser Arg 85 90 95 Glu Phe Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 32 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> 5887-41L-VL <400> 32 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Thr Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly Asn Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ser Arg 85 90 95 Glu Phe Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 110 <210> 33 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> 5887-41L-VL-HZ0 <400> 33 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly 1 5 10 15 Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly Asn Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn 65 70 75 80 Pro Val Glu Ala Asn Asp Thr Ala Asn Tyr Tyr Cys Gln His Ser Arg 85 90 95 Glu Phe Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 34 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> 5887-41L-VL-HZ1 <400> 34 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly 1 5 10 15 Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly Asn Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn 65 70 75 80 Pro Val Glu Ala Asn Asp Ala Ala Asn Tyr Tyr Cys Gln His Ser Arg 85 90 95 Glu Phe Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 35 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> 5887-41L-VL-HZ2 <400> 35 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly 1 5 10 15 Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly Asn Thr Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn 65 70 75 80 Pro Val Glu Ala Asn Asp Ala Ala Asn Tyr Tyr Cys Gln His Ser Arg 85 90 95 Glu Phe Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 36 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> 5887-537L1-VL <400> 36 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Glu Tyr Ser 20 25 30 Gly Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Val Ala Ser Asn Val Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Ile His 65 70 75 80 Pro Val Glu Glu Asp Asp Ile Ala Met Tyr Phe Cys Gln Gln Ser Arg 85 90 95 Lys Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 37 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> 5887-537L1-VL-HZ0 <400> 37 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly 1 5 10 15 Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Glu Tyr Ser 20 25 30 Gly Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Val Ala Ser Asn Val Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn 65 70 75 80 Pro Val Glu Ala Asn Asp Thr Ala Asn Tyr Tyr Cys Gln Gln Ser Arg 85 90 95 Lys Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 38 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> 5887-537L1-VL-HZ1 <400> 38 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly 1 5 10 15 Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Glu Tyr Ser 20 25 30 Gly Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Val Ala Ser Asn Val Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn 65 70 75 80 Pro Val Glu Ala Asn Asp Ile Ala Asn Tyr Phe Cys Gln Gln Ser Arg 85 90 95 Lys Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 39 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> 5888-116L1-VL <400> 39 Gln Ile Val Met Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Asn Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 40 <211> 106 <212> PRT <213> Artificial Sequence(Artificial Sequence) <220> <223> 5888-116L1-VL-HZ0 <400> 40 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Asn Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 41 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> 5888-116L1-VL-HZ1 <400> 41 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Asn Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 42 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> 5888-153L2-VL <400> 42 Gln Ile Val Met Thr Gln Thr Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Val Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Asn Asn Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Asn Ser Ser Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 43 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> 5888-153L2-VL-HZ0 <400> 43 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Val Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Asn Ser Ser Pro Leu Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 44 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> 5888-153L2-VL-HZ1 <400> 44 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Val Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Arg Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Ala Ala Val Tyr Tyr Cys Gln Gln Trp Asn Ser Ser Pro Leu Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 45 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> 5888-153L2-VL-HZ2 <400> 45 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Val Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Arg Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Ala Ala Val Tyr Tyr Cys Gln Gln Trp Asn Thr Ser Pro Leu Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 46 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> 5888-357L-VL <400> 46 Gln Ile Val Met Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Leu Thr Cys Ser Ala Ser Thr Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu 65 70 75 80 Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 47 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> 5888-357L-VL-HZ0 <400> 47 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Thr Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Phe Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 48 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> 5888-357L-VL-HZ1 <400> 48 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Thr Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Arg Leu Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu 65 70 75 80 Asp Ala Ala Val Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 49 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> 5888-379L2-VL <400> 49 Asp Ile Val Met Thr Gln Ser Pro Pro Ser Leu Ser Val Ser Val Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Gly Asn Gln Asn Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp His Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 50 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> 5888-379L2-VK4-HZ0 <400> 50 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Gly Asn Gln Asn Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp His Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 100 105 110 Lys <210> 51 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> 5888-379L2-VK4-HZ1 <400> 51 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Gly Asn Gln Asn Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95 Asp His Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 100 105 110 Lys <210> 52 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> 5888-379L2-VK1-HZ0 <400> 52 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Gly Asn Gln Asn Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Val Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys Gln Asn 85 90 95 Asp His Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 100 105 110 Lys <210> 53 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> 5888-379L2-VK1-HZ1 <400> 53 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Gly Asn Gln Asn Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Val Pro Lys Leu Leu Ile Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Leu Ala Thr Tyr Tyr Cys Gln Asn 85 90 95 Asp His Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 100 105 110 Lys <210> 54 <211> 327 <212> PRT <213> Artificial Sequence <220> <223> Human IgG4 <400> 54 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> 55 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Human kappa <400> 55 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 56 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> H-CDR1 <400> 56 Gly Tyr Ser Ile Thr Ser Asp Tyr Ala Trp Asn 1 5 10 <210> 57 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> H-CDR1 <400> 57 Gly Tyr Ser Ile Thr Ser Asp Tyr Ala Trp Asp 1 5 10 <210> 58 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR1 <400> 58 Gly Phe Ser Leu Ser Thr Ser Gly Met Gly Val Gly 1 5 10 <210> 59 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> H-CDR1 <400> 59 Gly Tyr Thr Phe Thr Asp Ser Glu Met Tyr 1 5 10 <210> 60 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> H-CDR1 <400> 60 Gly Tyr Thr Phe Thr Asp Tyr Glu Leu His 1 5 10 <210> 61 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> H-CDR1 <400> 61 Gly Tyr Arg Phe Thr Asp Ser Glu Met His 1 5 10 <210> 62 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> H-CDR1 <400> 62 Gly Tyr Thr Phe Ile Asn Tyr Gly Met Asn 1 5 10 <210> 63 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> H-CDR2 <400> 63 Tyr Ile Asp Tyr Ser Gly Ser Thr Thr Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 64 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> H-CDR2 <400> 64 Tyr Ile Arg Tyr Ser Gly Asp Thr Tyr Tyr Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 <210> 65 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> H-CDR2 <400> 65 His Ile Trp Trp Asp Asp Val Lys Gln Tyr Asn Pro Ala Leu Lys Ser 1 5 10 15 <210> 66 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> H-CDR2 <400> 66 Ala Ile Asp Pro Glu Thr Gly Asp Thr Ala Phe Asn Gln Lys Phe Lys 1 5 10 15 Gly <210> 67 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> H-CDR2 <400> 67 Thr Ile Asp Pro Glu Thr Gly Asp Thr Val Tyr Asn Gln Lys Phe Lys 1 5 10 15 Ala <210> 68 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> H-CDR2 <400> 68 Thr Ile Asp Pro Glu Thr Gly Gly Thr Val Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly <210> 69 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> H-CDR2 <400> 69 Trp Ile Asn Thr Tyr Ile Gly Glu Pro Thr Tyr Gly Asp Asn Phe Lys 1 5 10 15 Gly <210> 70 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 70 Thr Val Ile Asp Ser Met Asp Tyr 1 5 <210> 71 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 71 Thr Met Met Asp Thr Met Asp Tyr 1 5 <210> 72 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 72 Ile Gly Gly Asp Tyr Asp Tyr Phe Asp Phe 1 5 10 <210> 73 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 73 Ala Phe Asp Asn Asp Asn Asp Asp Gly Phe Ala Tyr 1 5 10 <210> 74 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 74 Ala Phe Asp Asn Asp Asn Asp Glu Gly Phe Ala Tyr 1 5 10 <210> 75 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 75 Ala Phe Asp Asn Asp Asn Asp Asp Ala Phe Ala Tyr 1 5 10 <210> 76 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 76 Ala Phe Tyr Asn Asp Tyr Asp Asp Gly Phe Ala Tyr 1 5 10 <210> 77 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 77 Ala Phe Tyr Asn Asp Tyr Asp Glu Gly Phe Ala Tyr 1 5 10 <210> 78 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 78 Ala Phe Tyr Asn Asp Tyr Asp Asp Ala Phe Ala Tyr 1 5 10 <210> 79 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 79 Ala Phe Tyr Asn Asp Phe Asp Asp Gly Phe Ala Tyr 1 5 10 <210> 80 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 80 Ala Phe Tyr Asn Asp Phe Asp Glu Gly Phe Ala Tyr 1 5 10 <210> 81 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 81 Ala Phe Tyr Asn Asp Phe Asp Asp Ala Phe Ala Tyr 1 5 10 <210> 82 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 82 Glu Gly Asp Gly Phe Ala Tyr 1 5 <210> 83 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 83 Glu Gly Glu Gly Phe Ala Tyr 1 5 <210> 84 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> H-CDR3 <400> 84 Glu Gly Asp Ala Phe Ala Tyr 1 5 <210> 85 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> L-CDR1 <400> 85 Arg Ala Ser Lys Ser Val Ser Thr Ser Gly His Ser Tyr Met His 1 5 10 15 <210> 86 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> L-CDR1 <400> 86 Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Asn Ser Tyr Met His 1 5 10 15 <210> 87 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> L-CDR1 <400> 87 Arg Ala Ser Lys Ser Val Ser Thr Ser Gly Asn Thr Tyr Met His 1 5 10 15 <210> 88 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> L-CDR1 <400> 88 Arg Ala Ser Glu Ser Val Glu Tyr Ser Gly Thr Ser Leu Met Gln 1 5 10 15 <210> 89 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> L-CDR1 <400> 89 Ser Ala Ser Ser Ser Val Asn Tyr Met His 1 5 10 <210> 90 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> L-CDR1 <400> 90 Ser Val Ser Ser Ser Val Ser Tyr Met His 1 5 10 <210> 91 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> L-CDR1 <400> 91 Ser Ala Ser Thr Ser Val Ser Tyr Met His 1 5 10 <210> 92 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> L-CDR1 <400> 92 Lys Ser Ser Gln Ser Leu Leu Tyr Ser Gly Asn Gln Asn Asn Tyr Leu 1 5 10 15 Ala <210> 93 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> L-CDR2 <400> 93 Leu Ala Ser Asn Leu Glu Ser 1 5 <210> 94 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> L-CDR2 <400> 94 Val Ala Ser Asn Val Glu Ser 1 5 <210> 95 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> L-CDR2 <400> 95 Asp Thr Ser Lys Leu Ala Ser 1 5 <210> 96 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> L-CDR2 <400> 96 Gly Ala Ser Thr Arg Glu Ser 1 5 <210> 97 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> L-CDR3 <400> 97 Gln His Ser Arg Glu Phe Pro Phe Thr 1 5 <210> 98 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> L-CDR3 <400> 98 Gln His Ser Arg Glu Phe Pro Leu Thr 1 5 <210> 99 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> L-CDR3 <400> 99 Gln Gln Ser Arg Lys Val Pro Trp Thr 1 5 <210> 100 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> L-CDR3 <400> 100 Gln Gln Trp Ser Ser Asn Pro Leu Thr 1 5 <210> 101 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> L-CDR3 <400> 101 Gln Gln Trp Asn Ser Ser Pro Leu Thr 1 5 <210> 102 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> L-CDR3 <400> 102 Gln Gln Trp Asn Thr Ser Pro Leu Thr 1 5 <210> 103 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> L-CDR3 <400> 103 Gln Asn Asp His Ser Tyr Pro Tyr Thr 1 5 <210> 104 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> CNTO7160, H <400> 104 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Asp Met Ile Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Arg Gly Glu Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Pro Trp Ser Thr Glu Gly Ser Phe Phe Val Leu Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val 195 200 205 Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys 210 215 220 Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu 260 265 270 Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 435 440 445 Gly Lys 450 <210> 105 <211> 214 <212> PRT <213> Artificial Sequence <220> CNTO7160, L <400> 105 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Asp Asp Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ile Thr Ala Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210

Claims

1. An antibody or antigen-binding fragment thereof that binds to ST2, said antibody or antigen-binding fragment comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein said heavy chain variable region and light chain variable region comprise heavy chain CDR and light chain CDR as shown below: H-CDR1, H-CDR2, and H-CDR3 are shown in SEQ ID NO. 56, 63, and 70, respectively; and L-CDR1, L-CDR2, and L-CDR3 are shown in SEQ ID NO. 85, 93, and 97, respectively.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The heavy chain variable region comprises an amino acid sequence shown in any one of SEQ ID NO. 1 to SEQ ID NO. 3 or an amino acid sequence having at least 75% identity with said amino acid sequence; and / or, The light chain variable region comprises an amino acid sequence shown in any one of SEQ ID NO. 29 to SEQ ID NO. 31 or an amino acid sequence having at least 75% identity with the amino acid sequence.

3. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region selected from any of the following combinations: (1): The heavy chain variable region shown in SEQ ID NO. 1 and the light chain variable region shown in SEQ ID NO. 29; (2): The heavy chain variable region shown in SEQ ID NO. 2 and the light chain variable region shown in SEQ ID NO. 30; (3): The heavy chain variable region shown in SEQ ID NO. 2 and the light chain variable region shown in SEQ ID NO. 31; (4): The heavy chain variable region shown in SEQ ID NO. 3 and the light chain variable region shown in SEQ ID NO. 30; (5): The heavy chain variable region shown in SEQ ID NO. 3 and the light chain variable region shown in SEQ ID NO.

31.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that, The ST2 mentioned is the mammalian ST2.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that, The ST2 mentioned is a primate ST2.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that, The ST2 is either human ST2 or cynomolgus monkey ST2.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that, The antibody is a monoclonal antibody, a single-chain antibody, a fully or partially humanized antibody, or a chimeric antibody.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that, The antibody is IgA, IgD, IgE, IgG, or IgM.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that, The antibody is IgG1, IgG2, or IgG4.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that, The antigen-binding fragment is an scFv, BsFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2, or Fv fragment of the antibody.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that, The antibody also contains a human or mouse-derived heavy chain constant region and / or light chain constant region.

12. The antibody or its antigen-binding fragment according to claim 11, characterized in that, The antibody comprises a heavy chain and a light chain.

13. The antibody or its antigen-binding fragment according to claim 11, characterized in that, The antibody comprises two heavy chains and two light chains.

14. The antibody or its antigen-binding fragment according to claim 11, characterized in that, The antibody comprises a heavy chain constant region selected from IgG, IgA, IgM, IgD or IgE and / or a κ or λ type light chain constant region.

15. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, characterized in that, The antibody further comprises a heavy chain constant region, which is an IgG1, IgG2, or IgG4 subtype; or, the antibody further comprises a light chain constant region, which is a κ type.

16. The antibody or its antigen-binding fragment according to claim 15, characterized in that, The heavy chain constant region contains the amino acid sequence shown in SEQ ID NO. 54 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO. 55 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO.

55.

17. A nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 16.

18. A carrier comprising the nucleic acid molecule of claim 17.

19. A host cell comprising the nucleic acid molecule of claim 17 and / or the vector of claim 18, or the host cell being transformed or transfected by the nucleic acid molecule of claim 17 and / or the vector of claim 18.

20. A composition comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 16, a nucleic acid molecule as described in claim 17, a vector as described in claim 18, and / or a host cell as described in claim 19.

21. The composition according to claim 20, characterized in that, The composition is a pharmaceutical composition, which optionally further comprises pharmaceutically acceptable excipients.

22. A pharmaceutical combination comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 16, a nucleic acid molecule as described in claim 17, a vector as described in claim 18, a host cell as described in claim 19, and / or a composition as described in claim 20 or 21, and optionally other pharmaceuticals.

23. A kit comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 16, a nucleic acid molecule as described in claim 17, a vector as described in claim 18, a host cell as described in claim 19, and / or a composition as described in claim 20 or 21.

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