Human anti-il-33 neutralizing monoclonal antibodies

Abstract

The present application relates to human anti-IL-33 neutralizing monoclonal antibodies. The present application aims to provide an antibody having an antagonistic effect on IL-33, particularly an isolated human anti-IL-33 neutralizing monoclonal antibody or a fragment of the antibody, in which the amino acid sequence of the framework region is the amino acid sequence of a germ line or a combination thereof. Epitopes of a plurality of anti-IL-33 monoclonal antibodies were identified, a human anti-IL-33 neutralizing monoclonal antibody was obtained, and a complementarity determining region having high binding to IL-33 was determined by introducing a mutation in the complementarity determining region of the antibody. By using the determined complementarity determining region, a human anti-IL-33 neutralizing monoclonal antibody having an amino acid sequence of the framework region being the amino acid sequence of a germ line or a combination thereof was obtained.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201480071117.7 (PCT Application No. PCT / JP2014 / 084695) filed on December 26, 2014, entitled "Human Anti-IL-33 Neutralizing Monoclonal Antibodies". TECHNICAL FIELD

[0002] The present application relates to human anti-IL-33 neutralizing monoclonal antibodies and antibodies that compete with the antibodies for binding, and to cytokine expression inhibitors containing these antibodies, and pharmaceutical compositions for the treatment, prevention or alleviation of IL-33 related diseases. BACKGROUND

[0003] Interleukin-33 (IL-33) is a cytokine belonging to the interleukin-1 family that is thought to play a role in inflammatory states. IL-33 is expressed constantly in the nucleus of epithelial cells, vascular endothelial cells, and is released as an alarmin accompanying cell destruction due to tissue damage caused by infection, physical and chemical stress. In addition, there can be a mechanism in which expression of IL-33 increases and is secreted due to stimulation by lipopolysaccharide or the like. IL-33 released to the extracellular space is able to activate intracellular signaling by binding to IL-33 receptors expressed on cells. IL-33 receptors are expressed in various immune system cells, epithelial cells, and the like, and in these cells, IL-33-inducible intracellular signal transduction is generated.

[0004] IL-33 is considered to induce allergic inflammation (asthma, atopic dermatitis, pollinosis, anaphylactic shock, etc.) by inducing production of Th2 cytokines (IL-4, IL-5, IL-6, IL-13, etc.) derived from Th2 cells, mast cells, eosinophils, basophils, NK (natural killer) T cells, type 2 innate lymphoid cells in immune system cells expressing IL-33 receptors (Non-Patent Literature 1: Tatsukuni Ohno et al., Allergy, 2012, Vol. 67, p1203). In addition, it has been reported that IL-33 stimulates induction of production of IL-1β, IL-6, TNF (tumor necrosis factor)-α in mast cells, macrophages among immune system cells expressing IL-33 receptors, and this phenomenon is associated with the onset of arthritis (a typical symptom of rheumatoid arthritis) induced by autoantibodies (Non-Patent Literature 2: Damo Xu et al., Journal of Immunology, 2010, Vol. 184, p2620). It has also been suggested that an antagonist of IL-33 is effective for acute kidney injury (Non-Patent Literature 3: Ali Akcay et al., Journal of American Society Nephrology, 2011, Vol. 22, p2057).In the case of humans, expression of IL-33 is confirmed to be increased in various inflammatory diseases (fibrosis of rheumatoid arthritis, asthma, systemic sclerosis, liver fibrosis, lung fibrosis, psoriasis, ulcerative colitis, Crohn's disease, multiple sclerosis, ankylosing spondylitis, etc.), and IL-33 is considered to be associated with the onset and maintenance of various diseases (Non-Patent Document 4: Yasushi Matsuyama et al., Journal of Rheumatology, 2010, Vol. 37, p 18; Non-Patent Document 5: David Pre'fontaine et al., Journal of Allergy and Clinical Immunology, 2010, Vol. 125, p 752; Non-Patent Document 6: Koichi Yanaba et al., Clinical Rheumatology, 2011, Vol. 30, p 825; Non-Patent Document 7: A. L. Rankin et al., Journal of Immunology, 2010, Vol. 184, p 1526; Non-Patent Document 8: Tamar Mchedlidze et al., Immunity, 2013, Vol. 39, p 357; Non-Patent Document 9: Liang-An Hu et al., Asian Pacific Journal of Cancer Prevention, 2013, Vol. 14, p 2563; Non-Patent Document 10: Luca Pastorelli et al., Proceedings of the National Academy of Sciences of the United States of America, 2010, vol. 107, p 8017).

[0005] Thus, since IL-33 is known to be associated with various diseases, especially inflammatory diseases, development of agonists, antagonists of IL-33 has continued to date (Patent Documents 1 to 4). Among them, antibodies against IL-33 have been attracting attention from the viewpoint of their specificity, strength of action. However, as for the antibodies developed so far, a mouse antibody which does not determine the epitope (Patent Document 1), or a commercially available goat polyclonal antibody which determines the cleavage site of IL-33 using caspase to determine the active form as non-cleaved IL-33, and takes the region containing the cleavage site (residues 155 to 198 of SEQ ID NO: 226 in the sequence listing) as the epitope (Patent Document 2) are known. On January 10, 2014, AnaptysBio, Inc. reported on the company's homepage that it had successfully produced a therapeutic development candidate antibody ANB020 against IL-33 using a unique somatic cell hypermutation technology (SHM-XEL) platform (Non-Patent Document 11: Hamza Suria, 'AnaptysBio announces development of novel anti-IL-33 therapeutic antibody', [on line], 2014, [retrieved on January 11, 2014], Retrieved from Internet <URL: http: / / www.anaptysbio.com / anti-il-33 / >). In addition, Murphy et al. obtained 20 human anti-IL-33 monoclonal antibodies using a mouse into which a variable region gene of a human antibody, i.e., a VelocImmune mouse (Patent Document 5). However, the epitope of this antibody has not been disclosed, and in addition, the amino acid sequence of the framework region of the 20 human anti-IL-33 monoclonal antibodies and the amino acid sequence of the human germ line differ by two or more amino acid residues. Therefore, when these antibodies are administered to humans, an immune response against the antibody is induced, and there are problems of reduction of the effect, induction of inflammation, and other side effects due to induction of human anti-human immunoglobulin antibody (HAHA).

[0006] Prior Art Documents

[0007] Patent Documents

[0008] Patent Document 1: International Publication No. 2005 / 079844

[0009] Patent Document 2: International Publication No. 2008 / 132709

[0010] Patent Document 3: International Publication No. 2011 / 031600

[0011] Patent Document 4: International Publication No. 2008 / 144610

[0012] Patent Literature 5: International Publication No. 2014 / 164959

[0013] Non-Patent Literature

[0014] Non-Patent Literature 1: Tatsukuni Ohno et al., Allergy, 2012, Vol. 67, p120

[0015] Non-Patent Literature 2: Damo Xu et al., Journal of Immunology, 2010, Vol. 184, p2620

[0016] Non-Patent Literature 3: Ali Akcay et al., Journal of American Society Nephrology, 2011, Vol. 22, p2057

[0017] Non-Patent Literature 4: Yasushi Matsuyama et al., Journal of Rheumatology, 2010, Vol. 37, p18

[0018] Non-Patent Literature 5: David Pre'fontaine et al., Journal of Allergy and Clinical Immunology, 2010, Vol. 125, p752

[0019] Non-Patent Literature 6: Koichi Yanaba et al., Clinical Rheumatology, 2011, Vol. 30, p825

[0020] Non-Patent Literature 7: A. L. Rankin et al., Journal of Immunology, 2010, Vol. 184, p1526

[0021] Non-Patent Literature 8: Tamar Mchedlidze et al., Immunity, 2013, Vol. 39, p357

[0022] Non-Patent Literature 9: Liang-An Hu et al., Asian Pacific Journal of Cancer Prevention, 2013, Vol. 14, p2563

[0023] Non-patent literature 10: Luca Pastorelli et al., Proceedings of the National Academy of Sciences of the United States of America, 2010, vol. 107, p8017

[0024] Non-patent literature 11: Hamza Suria, 'AnaptysBio announces development of novel anti-IL-33 therapeutic antibody', [on line], 2014, [retrieved on 11 January 2014], Retrieved from Internet: <URL: http: / / www.anaptysbio.com / anti-il-33 / > SUMMARY

[0025] In recent years, the relationship between IL-33 and diseases has been gradually elucidated, and there is a demand for anti-IL-33 neutralizing monoclonal antibodies having an antagonist action against IL-33. The action of an anti-IL-33 neutralizing monoclonal antibody is closely related to the region of the epitope to which the antibody binds. Since IL-33 is released to the extracellular space concomitantly with cell destruction, it has a high possibility of being cleaved by lysosome-derived proteolytic enzymes and the like, and a large number of fragments having the activity of IL-33 derived from the mature IL-33 can be produced. When a fragment contains an epitope consisting of a continuous amino acid sequence, a monoclonal antibody that binds to an epitope consisting of a continuous amino acid sequence of IL-33 binds firmly to one continuous amino acid sequence of the fragment, thereby hindering the binding of the fragment to an IL-33 receptor and the like, and is thus advantageous. However, it is still difficult to determine the epitope consisting of a continuous amino acid sequence described above, which is used to produce an IL-33 monoclonal antibody having a desired antagonist action.

[0026] As an anti-IL-33 neutralizing monoclonal antibody that binds to an epitope consisting of a continuous amino acid sequence of IL-33, it is desirable to have low antigenicity when administered to humans and the like. If it is a human antibody, it is desirable to have low antigenicity when administered to humans, and the amino acid sequence of the framework region is that of a human germline cell line or a combination thereof. However, when the SHM-XEL platform or the like is applied to a human antibody contained in a human antibody gene library, mutations in the amino acid sequence are introduced not only in the complementarity-determining region but also in the framework region. In addition, when a mouse into which a human antibody gene has been introduced is immunized with a human IL-33 protein and a human anti-IL-33 neutralizing monoclonal antibody is obtained, it is not possible to prevent mutations in the amino acid sequence in the framework region of the anti-IL-33 neutralizing monoclonal antibody. Therefore, it is still difficult to obtain an isolated human monoclonal antibody against IL-33, the amino acid sequence of the framework region of which is that of a human germline cell line or a combination thereof.

[0027] To solve the above problems, the present inventors conducted intensive studies, and as a result, found that an antibody that binds firmly to an epitope present at positions 155 to 198, which is considered to be a preferred epitope of the prior art, has little antagonist effect, on the other hand, an epitope consisting of a continuous amino acid sequence present at positions 101 to 154 or 199 to 270, particularly an epitope consisting of a continuous amino acid sequence present at positions 111 to 130, 131 to 150, 231 to 250, or 251 to 270, is important from the viewpoint of the antagonist effect of the antibody that binds to the epitope, thereby completing the present application.

[0028] Furthermore, the present inventors isolated a human anti-IL-33 neutralizing monoclonal antibody from a human antibody library, and further determined a complementarity-determining region having high binding and good physical properties by introducing mutations only in the complementarity-determining region thereof. As a result, a human antibody that binds to human IL-33 and neutralizes the function thereof was successfully obtained, the amino acid sequence of the framework region of the germline cell line of the human antibody not containing mutations. Therefore, the present application relates to the following invention:

[0029] [1] A monoclonal antibody that binds to an epitope consisting of a continuous amino acid sequence contained in positions 101 to 154 or 199 to 270 of SEQ ID NO: 226 of the sequence listing.

[0030] [2] The antibody according to item 1, wherein the epitope consisting of a continuous amino acid sequence contained in positions 101 to 154 or 199 to 270 of SEQ ID NO: 226 of the sequence listing is an epitope consisting of a continuous amino acid sequence contained in positions 111 to 130, 131 to 150, 231 to 250, or 251 to 270 of SEQ ID NO: 226 of the sequence listing.

[0031] [3] The antibody according to any one of items 1 or 2, wherein the epitope consisting of the consecutive amino acid sequence contained in positions 101 to 154 or 199 to 270 of SEQ ID NO: 226 in the sequence listing is an epitope consisting of an amino acid sequence comprising an amino acid selected from the group consisting of P118, 1119, T120, Y122, L123, R124, S125, L126, S127, Y129, N130, D131, Q132, S133, T135, A137, L138, E139, S142, Y143, E144, 1145, Y146, E148, D149, L150, D244, N245, H246, K266, L267, S268, and E269.

[0032] [4] The antibody according to any one of items 1 to 3, wherein the epitope consisting of the consecutive amino acid sequence contained in positions 101 to 154 or 199 to 270 of SEQ ID NO: 226 in the sequence listing is an epitope consisting of the consecutive amino acid sequence contained in positions 111 to 130, 131 to 150, 231 to 250, or 251 to 270 of SEQ ID NO: 226 in the sequence listing.

[0033] [5] The antibody according to any one of items 1 to 4, wherein the epitope consisting of the consecutive amino acid sequence contained in positions 101 to 154 or 199 to 270 of SEQ ID NO: 226 in the sequence listing is an epitope consisting of the consecutive amino acid sequence contained in positions 138 to 147 or 139 to 147 of SEQ ID NO: 226 in the sequence listing.

[0034] [6] The antibody according to any one of items 1 to 5, wherein the monoclonal antibody that binds to the epitope consisting of the consecutive amino acid sequence contained in positions 101 to 154 or 199 to 270 of SEQ ID NO: 226 in the sequence listing is an antagonist of IL-33.

[0035] [7] The antibody according to any one of items 1 to 6, wherein the monoclonal antibody that binds to the epitope consisting of the consecutive amino acid sequence contained in positions 101 to 154 or 199 to 270 of SEQ ID NO: 226 in the sequence listing hinders the binding of an IL-33 receptor to IL-33.

[0036] [8] A pharmaceutical composition for treating, preventing, or alleviating an IL-33- related disease, the composition containing the antibody according to any one of items 1 to 7.

[0037] [9] A cytokine expression inhibitor containing the antibody according to any one of items 1 to 7.

[0038]

[10] The inhibitor according to item 9, which inhibits the expression of TNF-α, IFN-γ, IL-1β, IL-4, IL-5, IL-6 or IL-13.

[0039]

[11] The inhibitor according to item 9 or 10, which inhibits the expression of IFN-γ, IL-5, IL-6 or IL-13.

[0040]

[12] An epitope selected from the group consisting of:

[0041] 1) the epitope described in any one of items 1 to 5;

[0042] 2) an epitope consisting of an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to the sequence consisting of consecutive amino acids of the epitope described in 1);

[0043] 3) an epitope consisting of an amino acid sequence having at least 90% sequence identity to the sequence consisting of consecutive amino acids of the epitope described in 1).

[0044]

[13] An antibody produced or screened using the epitope described in item 12.

[0045]

[14] The antibody according to any one of items 1 to 7, wherein the monoclonal antibody that binds to the epitope consisting of the sequence of consecutive amino acids contained in positions 101 to 154 or positions 199 to 270 of SEQ ID NO: 226 in the sequence listing is a chimeric antibody, a humanized antibody, or a human antibody.

[0046]

[15] The antibody according to item 14, wherein the amino acid sequence of the framework region is the amino acid sequence of the framework region of a human germline or a combination thereof.

[0047]

[16] The antibody according to item 15, wherein the amino acid sequence of the light chain framework region 1 is residues 1 to 22 of SEQ ID NO: 317 in the sequence listing, the amino acid sequence of the light chain framework region 2 is residues 36 to 50 of SEQ ID NO: 317 in the sequence listing, the amino acid sequence of the light chain framework region 3 is residues 58 to 89 of SEQ ID NO: 317 in the sequence listing, and,

[0048] The amino acid sequence of the heavy chain framework region 1 is residues 1 to 30 of SEQ ID NO: 367 or residues 1 to 30 of SEQ ID NO: 368, the amino acid sequence of the heavy chain framework region 2 is residues 36 to 49 of SEQ ID NO: 367 or residues 36 to 49 of SEQ ID NO: 368, the amino acid sequence of the heavy chain framework region 3 is residues 67 to 98 of SEQ ID NO: 367 or residues 67 to 98 of SEQ ID NO: 368, and the amino acid sequence of the heavy chain framework region 4 is residues 5 to 15 of SEQ ID NO: 407.

[0049]

[17] The antibody of item 15 or 16, wherein the amino acid sequence of the light chain framework region 1 is residues 1 to 22 of SEQ ID NO: 317, the amino acid sequence of the light chain framework region 2 is residues 36 to 50 of SEQ ID NO: 317, the amino acid sequence of the light chain framework region 3 is residues 58 to 89 of SEQ ID NO: 317, and the amino acid sequence of the light chain framework region 4 is residues 3 to 12 of SEQ ID NO: 401, and the amino acid sequence of the heavy chain framework region 1 is residues 1 to 30 of SEQ ID NO: 367, the amino acid sequence of the heavy chain framework region 2 is residues 36 to 49 of SEQ ID NO: 367, the amino acid sequence of the heavy chain framework region 3 is residues 67 to 98 of SEQ ID NO: 368, and the amino acid sequence of the heavy chain framework region 4 is residues 5 to 15 of SEQ ID NO: 407.

[0050]

[18] An isolated human anti-IL-33 neutralizing monoclonal antibody, wherein the combination of the amino acid sequences of each of the light chain complementarity determining region 1 (LCDR1), the light chain complementarity determining region 2 (LCDR2), the light chain complementarity determining region 3 (LCDR3), the heavy chain complementarity determining region 1 (HCDR1), the heavy chain complementarity determining region 2 (HCDR2), and the heavy chain complementarity determining region 3 (HCDR3) is selected from the combinations shown in Table 1 as C1 to C30.

[0051] [Table 1]

[0052] The following sequence numbers in Table 1 refer to the sequence numbers in the sequence listing

[0053] LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3 C1 SEQ ID NO: 1 SEQ ID NO: 11 SEQ ID NO: 22 SEQ ID NO: 43 SEQ ID NO: 51 SEQ ID NO: 65 C2 SEQ ID NO: 1 SEQ ID NO: 11 SEQ ID NO: 23 SEQ ID NO: 43 SEQ ID NO: 52 SEQ ID NO: 65 C3 SEQ ID NO: 2 SEQ ID NO: 11 SEQ ID NO: 23 SEQ ID NO: 44 SEQ ID NO: 52 SEQ ID NO: 65 C4 SEQ ID NO: 2 SEQ ID NO: 11 SEQ ID NO: 23 SEQ ID NO: 44 SEQ ID NO: 53 SEQ ID NO: 65 C5 SEQ ID NO: 2 SEQ ID NO: 11 SEQ ID NO: 23 SEQ ID NO: 43 SEQ ID NO: 54 SEQ ID NO: 65 C6 SEQ ID NO: 2 SEQ ID NO: 11 SEQ ID NO: 24 SEQ ID NO: 45 SEQ ID NO: 52 SEQ ID NO: 65 C7 SEQ ID NO: 2 SEQ ID NO: 11 SEQ ID NO: 23 SEQ ID NO: 46 SEQ ID NO: 52 SEQ ID NO: 65 C8 SEQ ID NO: 3 SEQ ID NO: 12 SEQ ID NO: 25 SEQ ID NO: 47 SEQ ID NO: 55 SEQ ID NO: 66 C9 SEQ ID NO: 4 SEQ ID NO: 12 SEQ ID NO: 26 SEQ ID NO: 47 SEQ ID NO: 56 SEQ ID NO: 67 C10 SEQ ID NO: 4 SEQ ID NO: 13 SEQ ID NO: 27 SEQ ID NO: 47 SEQ ID NO: 56 SEQ ID NO: 67 C11 SEQ ID NO: 5 SEQ ID NO: 12 SEQ ID NO: 28 SEQ ID NO: 47 SEQ ID NO: 56 SEQ ID NO: 67 C12 SEQ ID NO: 4 SEQ ID NO: 12 SEQ ID NO: 29 SEQ ID NO: 47 SEQ ID NO: 56 SEQ ID NO: 67 C13 SEQ ID NO: 6 SEQ ID NO: 14 SEQ ID NO: 30 SEQ ID NO: 47 SEQ ID NO: 56 SEQ ID NO: 67 C14 SEQ ID NO: 7 SEQ ID NO: 14 SEQ ID NO: 31 SEQ ID NO: 47 SEQ ID NO: 56 SEQ ID NO: 67 C15 SEQ ID NO: 4 SEQ ID NO: 15 SEQ ID NO: 32 SEQ ID NO: 47 SEQ ID NO: 56 SEQ ID NO: 67 C16 SEQ ID NO: 6 SEQ ID NO: 16 SEQ ID NO: 33 SEQ ID NO: 48 SEQ ID NO: 57 SEQ ID NO: 68 C17 SEQ ID NO: 4 SEQ ID NO: 17 SEQ ID NO: 34 SEQ ID NO: 49 SEQ ID NO: 58 SEQ ID NO: 69 C18 SEQ ID NO: 6 SEQ ID NO: 18 SEQ ID NO: 35 SEQ ID NO: 47 SEQ ID NO: 59 SEQ ID NO: 70 C19 SEQ ID NO: 6 SEQ ID NO: 19 SEQ ID NO: 36 SEQ ID NO: 47 SEQ ID NO: 56 SEQ ID NO: 71 C20 SEQ ID NO: 6 SEQ ID NO: 20 SEQ ID NO: 26 SEQ ID NO: 47 SEQ ID NO: 56 SEQ ID NO: 66 C21 SEQ ID NO: 6 SEQ ID NO: 18 SEQ ID NO: 37 SEQ ID NO: 47 SEQ ID NO: 60 SEQ ID NO: 72 C22 SEQ ID NO: 6 SEQ ID NO: 20 SEQ ID NO: 38 SEQ ID NO: 47 SEQ ID NO: 56 SEQ ID NO: 73 C23 SEQ ID NO: 8 SEQ ID NO: 20 SEQ ID NO: 27 SEQ ID NO: 49 SEQ ID NO: 56 SEQ ID NO: 68 C24 SEQ ID NO: 9 SEQ ID NO: 20 SEQ ID NO: 39 SEQ ID NO: 47 SEQ ID NO: 60 SEQ ID NO: 73 C25 SEQ ID NO: 4 SEQ ID NO: 21 SEQ ID NO: 34 SEQ ID NO: 47 SEQ ID NO: 61 SEQ ID NO: 74 C26 SEQ ID NO: 10 SEQ ID NO: 19 SEQ ID NO: 40 SEQ ID NO: 47 SEQ ID NO: 62 SEQ ID NO: 75 C27 SEQ ID NO: 4 SEQ ID NO: 18 SEQ ID NO: 41 SEQ ID NO: 50 SEQ ID NO: 56 SEQ ID NO: 76 C28 SEQ ID NO: 6 SEQ ID NO: 20 SEQ ID NO: 42 SEQ ID NO: 47 SEQ ID NO: 63 SEQ ID NO: 77 C29 SEQ ID NO: 2 SEQ ID NO: 11 SEQ ID NO: 23 SEQ ID NO: 43 SEQ ID NO: 64 SEQ ID NO: 65 C30 SEQ ID NO: 6 SEQ ID NO: 20 SEQ ID NO: 40 SEQ ID NO: 47 SEQ ID NO: 64 SEQ ID NO: 78

[0054]

[19] The human anti-IL-33 neutralizing monoclonal antibody of item 18, wherein the combination of the amino acid sequences of each of the light chain complementarity determining region 1 (LCDR1), the light chain complementarity determining region 2 (LCDR2), the light chain complementarity determining region 3 (LCDR3), the heavy chain complementarity determining region 1 (HCDR1), the heavy chain complementarity determining region 2 (HCDR2), and the heavy chain complementarity determining region 3 (HCDR3) is selected from the combinations shown in Table 1 as C1 to C28.

[0055]

[20] The human anti-IL-33 neutralizing monoclonal antibody of item 18 or 19, wherein the combination of the amino acid sequences of each of the light chain complementarity determining region 1 (LCDR1), the light chain complementarity determining region 2 (LCDR2), the light chain complementarity determining region 3 (LCDR3), the heavy chain complementarity determining region 1 (HCDR1), the heavy chain complementarity determining region 2 (HCDR2), and the heavy chain complementarity determining region 3 (HCDR3) comprises the amino acid sequences selected from the combinations shown in Table 1 as C1, C8, C15, C17, and C18.

[0056]

[21] The human anti-IL-33 neutralizing monoclonal antibody of any one of items 18 to 20, wherein the amino acid sequences of the antibody framework regions are the amino acid sequences of the germline framework regions or combinations thereof.

[0057]

[22] The human anti-IL-33 neutralizing monoclonal antibody of any one of items 18 to 21, wherein the amino acid sequence of the light chain framework region 1 is residues 1 to 22 of SEQ ID NO: 317 in the Sequence Listing, the amino acid sequence of the light chain framework region 2 is residues 36 to 50 of SEQ ID NO: 317 in the Sequence Listing, the amino acid sequence of the light chain framework region 3 is residues 58 to 89 of SEQ ID NO: 317 in the Sequence Listing, and the amino acid sequence of the light chain framework region 4 is residues 3 to 12 of SEQ ID NO: 401 in the Sequence Listing, and the amino acid sequence of the heavy chain framework region 1 is residues 1 to 30 of SEQ ID NO: 367 in the Sequence Listing or residues 1 to 30 of SEQ ID NO: 368 in the Sequence Listing, the amino acid sequence of the heavy chain framework region 2 is residues 36 to 49 of SEQ ID NO: 367 in the Sequence Listing or residues 36 to 49 of SEQ ID NO: 368 in the Sequence Listing, the amino acid sequence of the heavy chain framework region 3 is residues 67 to 98 of SEQ ID NO: 367 in the Sequence Listing or residues 67 to 98 of SEQ ID NO: 368 in the Sequence Listing, and the amino acid sequence of the heavy chain framework region 4 is residues 5 to 15 of SEQ ID NO: 407 in the Sequence Listing.

[0058]

[23] The human anti-IL-33 neutralizing monoclonal antibody of any one of items 18 to 22, wherein the combination of the amino acid sequences of each of the light chain variable region and the heavy chain variable region is selected from the combinations shown in Table 2 as V1 to V30.

[0059] [Table 2]

[0060] The sequence numbers below refer to the sequence numbers in the sequence listing

[0061] Combination Light chain variable region Heavy chain variable region V1 SEQ ID NO: 79 SEQ ID NO: 105 V2 SEQ ID NO: 80 SEQ ID NO: 106 V3 SEQ ID NO: 81 SEQ ID NO: 107 V4 SEQ ID NO: 81 SEQ ID NO: 108 V5 SEQ ID NO: 81 SEQ ID NO: 109 V6 SEQ ID NO: 82 SEQ ID NO: 110 V7 SEQ ID NO: 81 SEQ ID NO: 111 V8 SEQ ID NO: 83 SEQ ID NO: 112 V9 SEQ ID NO: 84 SEQ ID NO: 113 V10 SEQ ID NO: 85 SEQ ID NO: 113 V11 SEQ ID NO: 86 SEQ ID NO: 113 V12 SEQ ID NO: 87 SEQ ID NO: 113 V13 SEQ ID NO: 88 SEQ ID NO: 113 V14 SEQ ID NO: 89 SEQ ID NO: 113 V15 SEQ ID NO: 90 SEQ ID NO: 113 V16 SEQ ID NO: 91 SEQ ID NO: 114 V17 SEQ ID NO: 92 SEQ ID NO: 115 V18 SEQ ID NO: 93 SEQ ID NO: 116 V19 SEQ ID NO: 94 SEQ ID NO: 117 V20 SEQ ID NO: 95 SEQ ID NO: 118 V21 SEQ ID NO: 96 SEQ ID NO: 119 V22 SEQ ID NO: 97 SEQ ID NO: 120 V23 SEQ ID NO: 98 SEQ ID NO: 121 V24 SEQ ID NO: 99 SEQ ID NO: 122 V25 SEQ ID NO: 100 SEQ ID NO: 123 V26 SEQ ID NO: 101 SEQ ID NO: 124 V27 SEQ ID NO: 102 SEQ ID NO: 125 V28 SEQ ID NO: 103 SEQ ID NO: 126 V29 SEQ ID NO: 81 SEQ ID NO: 127 V30 SEQ ID NO: 104 SEQ ID NO: 128

[0062]

[24] The human anti-IL-33 neutralizing monoclonal antibody of item 23, wherein the combination of the amino acid sequences of the light chain variable region and the heavy chain variable region is selected from the combinations shown in V1 to V28 in Table 2.

[0063]

[25] The human anti-IL-33 neutralizing monoclonal antibody of item 23 or 24, wherein the combination of the amino acid sequences of the light chain variable region and the heavy chain variable region is selected from the combinations shown in V1, V8, V15, V17 and V18 in Table 2.

[0064]

[26] The human anti-IL-33 neutralizing monoclonal antibody of any one of items 18 to 25, wherein the light chain is a lambda chain.

[0065]

[27] The human anti-IL-33 neutralizing monoclonal antibody of any one of items 18 to 26, wherein the human anti-IL-33 neutralizing monoclonal antibody is an IgG.

[0066]

[28] The human anti-IL-33 neutralizing monoclonal antibody of any one of items 18 to 27, wherein the antigen is human IL-33 and monkey IL-33.

[0067]

[29] A nucleic acid molecule encoding a protein portion of the human anti-IL-33 neutralizing monoclonal antibody of any one of items 18 to 28.

[0068]

[30] The nucleic acid molecule of item 29, wherein the combination of the nucleic acid sequences encoding the amino acid sequences of each of the light chain complementarity determining region 1 (LCDR1), the light chain complementarity determining region 2 (LCDR2), the light chain complementarity determining region 3 (LCDR3), the heavy chain complementarity determining region 1 (HCDR1), the heavy chain complementarity determining region 2 (HCDR2) and the heavy chain complementarity determining region 3 (HCDR3) is selected from the combinations shown in CN1 to CN30 in Table 3.

[0069] [Table 3]

[0070] The sequence numbers below refer to the sequence numbers in the sequence listing

[0071] Combination LCDR1 LCDR2 LCDR3 HCDR1 HCDR2 HCDR3 CN1 SEQ ID NO: 129 SEQ ID NO: 140 SEQ ID NO: 156 SEQ ID NO: 181 SEQ ID NO: 191 SEQ ID NO: 209 CN2 SEQ ID NO: 129 SEQ ID NO: 140 SEQ ID NO: 157 SEQ ID NO: 181 SEQ ID NO: 192 SEQ ID NO: 209 CN3 SEQ ID NO: 130 SEQ ID NO: 140 SEQ ID NO: 157 SEQ ID NO: 182 SEQ ID NO: 192 SEQ ID NO: 209 CN4 SEQ ID NO: 130 SEQ ID NO: 140 SEQ ID NO: 157 SEQ ID NO: 182 SEQ ID NO: 193 SEQ ID NO: 209 CN5 SEQ ID NO: 130 SEQ ID NO: 140 SEQ ID NO: 157 SEQ ID NO: 181 SEQ ID NO: 194 SEQ ID NO: 209 CN6 SEQ ID NO: 130 SEQ ID NO: 140 SEQ ID NO: 158 SEQ ID NO: 183 SEQ ID NO: 192 SEQ ID NO: 209 CN7 SEQ ID NO: 130 SEQ ID NO: 140 SEQ ID NO: 157 SEQ ID NO: 184 SEQ ID NO: 192 SEQ ID NO: 209 CN8 SEQ ID NO: 131 SEQ ID NO: 141 SEQ ID NO: 159 SEQ ID NO: 185 SEQ ID NO: 195 SEQ ID NO: 210 CN9 SEQ ID NO: 132 SEQ ID NO: 141 SEQ ID NO: 160 SEQ ID NO: 185 SEQ ID NO: 196 SEQ ID NO: 211 CN10 SEQ ID NO: 132 SEQ ID NO: 142 SEQ ID NO: 161 SEQ ID NO: 185 SEQ ID NO: 196 SEQ ID NO: 211 CN11 SEQ ID NO: 133 SEQ ID NO: 143 SEQ ID NO: 162 SEQ ID NO: 185 SEQ ID NO: 196 SEQ ID NO: 211 CN12 SEQ ID NO: 132 SEQ ID NO: 141 SEQ ID NO: 163 SEQ ID NO: 185 SEQ ID NO: 196 SEQ ID NO: 211 CN13 SEQ ID NO: 134 SEQ ID NO: 144 SEQ ID NO: 164 SEQ ID NO: 185 SEQ ID NO: 196 SEQ ID NO: 211 CN14 SEQ ID NO: 135 SEQ ID NO: 144 SEQ ID NO: 165 SEQ ID NO: 185 SEQ ID NO: 196 SEQ ID NO: 211 CN15 SEQ ID NO: 132 SEQ ID NO: 145 SEQ ID NO: 166 SEQ ID NO: 185 SEQ ID NO: 196 SEQ ID NO: 211 CN16 SEQ ID NO: 134 SEQ ID NO: 146 SEQ ID NO: 167 SEQ ID NO: 186 SEQ ID NO: 197 SEQ ID NO: 212 CN17 SEQ ID NO: 132 SEQ ID NO: 147 SEQ ID NO: 168 SEQ ID NO: 187 SEQ ID NO: 198 SEQ ID NO: 213 CN18 SEQ ID NO: 134 SEQ ID NO: 148 SEQ ID NO: 169 SEQ ID NO: 185 SEQ ID NO: 199 SEQ ID NO: 214 CN19 Serial number 134 Serial number 149 Serial number 170 Serial number 185 Serial number 200 Serial number 215 CN20 Serial number 134 Serial number 150 Serial Number 171 Serial number 185 Serial number 200 Serial number 216 CN21 Serial number 134 Serial Number 148 Serial number 172 Serial number 185 Serial Number 201 Serial number 217 CN22 Serial number 134 Serial number 150 Serial number 173 Serial number 185 Serial number 200 Serial number 218 CN23 Serial number 136 Serial number 151 Serial number 174 Serial Number 188 Serial number 202 Serial number 219 CN24 Serial number 137 Serial number 151 Serial number 175 Serial number 189 Serial number 203 Serial number 220 CN25 Serial number 138 Serial number 152 Serial number 176 Serial number 189 Serial number 204 Serial number 221 CN26 Serial number 139 Serial number 153 Serial number 177 Serial number 189 Serial Number 205 Serial number 222 CN27 Serial number 138 Serial number 154 Serial Number 178 Serial number 190 Serial number 206 Serial number 223 CN28 Serial number 134 Serial number 155 Serial number 179 Serial number 185 Serial number 207 Serial number 224 CN29 Serial number 130 Serial number 140 Serial number 157 Serial Number 181 Serial Number 208 Serial number 209 CN30 Serial number 134 Serial number 155 Serial number 180 Serial number 185 Serial Number 208 Serial number 225

[0072]

[31] A vector comprising the nucleic acid molecule of item 29 or 30.

[0073]

[32] A host cell comprising the vector of item 31.

[0074]

[33] A method of manufacturing the human anti-IL-33 neutralizing monoclonal antibody of any one of items 18 to 28, wherein the method comprises culturing the host cell of item 32 to produce the antibody.

[0075]

[34] A cytokine expression inhibitor comprising the human anti-IL-33 neutralizing monoclonal antibody of any one of items 18 to 28.

[0076]

[35] The inhibitor of item 34, wherein the inhibitor inhibits expression of TNF-a, IFN-g, IL-1b, IL-4, IL-5, IL-6, or IL-13.

[0077]

[36] The inhibitor of item 34 or 35, wherein the inhibitor inhibits expression of IFN-g, IL-5, IL-6, or IL-13.

[0078]

[37] A pharmaceutical composition comprising the human anti-IL-33 neutralizing monoclonal antibody of any one of items 18 to 28.

[0079]

[38] The pharmaceutical composition of item 37, for use in the prevention, treatment, or alleviation of an IL-33 associated disease.

[0080]

[39] The pharmaceutical composition of item 38, wherein the IL-33 associated disease is selected from the group consisting of asthma, atopic dermatitis, hay fever, anaphylactic shock, sinusitis (including eosinophilic sinusitis), Crohn's disease, ulcerative colitis, arthritis, systemic lupus erythematosus, pemphigus, pemphigoid, scleroderma, ankylosing spondylitis, liver fibrosis (including primary biliary cirrhosis), pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), acute kidney injury, vasculitis, and cancer.

[0081]

[40] An anti-IL-33 neutralizing monoclonal antibody that competes for binding to IL-33 with the antibody of item 20 or 25.

[0082]

[41] A method for treating a patient having an IL-33 associated disease, preventing, or alleviating an IL-33 associated disease, the method comprising administering to the subject the antibody of any one of items 1 to 7 and 18 to 28.

[0083]

[42] The method of item 41, wherein the IL-33 associated disease is selected from the group consisting of asthma, atopic dermatitis, hay fever, anaphylactic shock, sinusitis (including eosinophilic sinusitis), Crohn's disease, ulcerative colitis, arthritis, systemic lupus erythematosus, pemphigus, pemphigoid, scleroderma, ankylosing spondylitis, liver fibrosis (including primary biliary cirrhosis), lung fibrosis, chronic obstructive pulmonary disease (COPD), acute kidney injury, vasculitis, and cancer.

[0084]

[43] Use of the antibody of any one of items 1 to 7 and 18 to 28 in the manufacture of a medicament for treating, preventing, or alleviating an IL-33 associated disease.

[0085]

[44] The use of item 43, wherein the IL-33 associated disease is selected from the group consisting of asthma, atopic dermatitis, hay fever, anaphylactic shock, sinusitis (including eosinophilic sinusitis), Crohn's disease, ulcerative colitis, arthritis, systemic lupus erythematosus, pemphigus, pemphigoid, scleroderma, ankylosing spondylitis, liver fibrosis (including primary biliary cirrhosis), lung fibrosis, chronic obstructive pulmonary disease (COPD), acute kidney injury, vasculitis, and cancer.

[0086]

[45] Use of the antibody of any one of items 1 to 7 and 18 to 28 for treating, preventing, or alleviating an IL-33 associated disease.

[0087]

[46] The use of item 45, wherein the IL-33 associated disease is selected from the group consisting of asthma, atopic dermatitis, hay fever, anaphylactic shock, sinusitis (including eosinophilic sinusitis), Crohn's disease, ulcerative colitis, arthritis, systemic lupus erythematosus, pemphigus, pemphigoid, scleroderma, ankylosing spondylitis, liver fibrosis (including primary biliary cirrhosis), lung fibrosis, chronic obstructive pulmonary disease (COPD), acute kidney injury, vasculitis, and cancer.

[0088]

[47] A method of treating a subject in need of inhibition of cytokine expression, preventing, or alleviating an associated disease, the method comprising administering to the subject the antibody of any one of items 1 to 7 and 18 to 28.

[0089]

[48] The method of item 47, wherein the cytokine is TNF-a, IFN-g, IL-1b, IL-4, IL-5, IL-6, or IL-13.

[0090]

[49] Use of the antibody of any one of items 1 to 7 and 18 to 28 for the manufacture of an inhibitor of cytokine expression.

[0091]

[50] The use according to item 49, wherein the cytokine is TNF-a, IFN-g, IL-1b, IL-4, IL-5, IL-6 or IL-13.

[0092] The monoclonal antibody of the present application that binds to the epitope can easily exert neutralization by firmly binding to one continuous amino acid sequence even in the case where IL-33 is decomposed and fragmented, since it binds to the epitope consisting of a continuous amino acid sequence.

[0093] The monoclonal antibody of the present application does not easily induce human anti-human immunoglobulin antibody (HAHA) against the framework region and / or the complementarity determining region of the antibody when administered to a human. The IL-33 neutralization effect is sustained in the organism as long as it is not hindered by HAHA. In addition, it is a safe antibody as long as it does not induce inflammation caused by binding to HAHA. The monoclonal antibody of the present application can be used as a novel diagnostic, prophylactic, therapeutic or alleviating agent for IL-33-related diseases, since it can bind to human IL-33 and neutralize its function. BRIEF DESCRIPTION OF DRAWINGS

[0094] Figure 1 is a diagram illustrating each domain of IL-33 protein and cleavage site.

[0095] Figure 2 is a diagram showing the binding activity of each antibody against human IL-33 protein (residue 112 to residue 270) and each partial peptide fragment (PEP11-26).

[0096] Figure 3 is a diagram showing the three-dimensional structure model of the complex of mature human IL-33 (residue 117 to residue 270) (indicated as "S117-T270" in the figure) and human ST2 (hST2).

[0097] Figure 4 is a diagram showing only a part of PEP12 epitope of human IL-33 (position 117-130 of SEQ ID NO: 226 in the sequence listing) (indicated as "S117-N130" in the figure, and the same hereinafter) and human ST2 in the three-dimensional structure model of Figure 3 .

[0098] Figure 5 is a diagram showing only PEP14 epitope of human IL-33 and human ST2 in the three-dimensional structure model of Figure 3 .

[0099] Figure 6 is a diagram showing only PEP24 epitope of human IL-33 and human ST2 in the three-dimensional structure model of Figure 3 .

[0100] Figure 7 is Figure 3 In the stereostructure model of

[0101] Figure 8 is a graph showing the effect of human anti-IL-33 neutralizing monoclonal antibody A25-3H04 on inflammation induced by intraperitoneal administration of human IL-33, based on inflammation-related indexes (spleen weight, IL-5 concentration in serum, number of eosinophils in blood, number of basophils in blood, number of neutrophils in blood, IgA concentration in serum, IgE concentration in serum).

[0102] Figure 9 is a graph showing the effect of human anti-IL-33 neutralizing monoclonal antibodies A10-1C04, A23-1A05, A25-2C02 and A26-1F02 on inflammation induced by intraperitoneal administration of human IL-33, based on inflammation-related indexes (spleen weight, IL-5 concentration in serum, number of eosinophils in blood, number of basophils in blood, number of neutrophils in blood, IgA concentration in serum, IgE concentration in serum).

[0103] Figure 10 is a graph showing the concentration of human anti-IL-33 neutralizing monoclonal antibodies (A23-1A05, A25-3H04, A26-1F02, A10-1C04, A25-2C02) in mouse plasma over time.

[0104] Figure 11 is a graph showing the concentration of human anti-IL-33 neutralizing monoclonal antibodies (A10-1C04, A23-1A05) in monkey serum over time. DETAILED DESCRIPTION

[0105] In order to facilitate understanding of the present application, the terms used in the present application are explained below.

[0106] [Epitope]

[0107] In the present application, the term "epitope" refers to a portion of an antigen recognized by an antibody. In the present application, an epitope relates to a sequence consisting of consecutive amino acids necessary for recognition by an antibody.

[0108] [Binding]

[0109] In the present application, the "binding" of the monoclonal antibody to the epitope means that the monoclonal antibody binds to the peptide as the epitope to form a complex. The binding of the monoclonal antibody to the epitope is based on ionic binding, hydrogen bonding, hydrophobic binding, van der Waals force, etc., but is not limited thereto. Whether the monoclonal antibody binds to the epitope can be investigated using, for example, peptide array scanning, KinExA, which are described in the specification.

[0110] [Antibody]

[0111] The term "antibody" in the present application is used in the broadest sense, and includes monoclonal antibodies, polyclonal antibodies, as long as the desired specific binding properties are present. The antibody in the present application can be an antibody derived from any animal, such as a mouse antibody, a human antibody, a rat antibody, a rabbit antibody, a goat antibody, a camel antibody, etc.

[0112] [Monoclonal antibody]

[0113] The monoclonal antibody in the antibody of the present application means an antibody of an antibody group consisting of only a single clone (a single molecular species) in the designed amino acid sequence. The monoclonal antibody includes a chimeric antibody, a humanized antibody, a human antibody, a multispecific antibody, and an artificial antibody, as well as a functionally modified antibody of these antibodies, a conjugated antibody of these antibodies, and a fragment of these antibodies. The monoclonal antibody of the present application can be produced using any known method, such as a hybridoma method, a phage display method, and a genetic engineering method.

[0114] [Chimeric antibody]

[0115] The chimeric antibody means an antibody in which the light chain, the heavy chain, or both are composed of a variable region of a non-human origin and a constant region of a human origin.

[0116] [Humanized antibody]

[0117] The humanized antibody means an antibody in which the variable region is composed of a complementarity determining region of a non-human antibody and a framework region derived from a human antibody, and the constant region is derived from a human antibody.

[0118] [Human antibody]

[0119] The human antibody means an antibody in which both the light chain and the heavy chain are derived from a human. Depending on the difference in the constant region of the heavy chain, the human antibody includes IgG (including IgG1, IgG2, IgG3, and IgG4) having a γ heavy chain, IgM having a μ heavy chain, IgA (including IgA1 and IgA2) having an α heavy chain, IgD having a δ heavy chain, or IgE having an ε heavy chain. In addition, in principle, as the light chain, either of a κ chain and a λ chain is contained.

[0120] [Multi-specific antibody]

[0121] The so-called multispecific antibody is an asymmetric antibody having two or more different antigen specificities and two or more independent antigen recognition sites, and examples include a bispecific antibody having two antigen specificities, a trispecific antibody having three antigen specificities, and the like. The one or more antigens recognized by the multispecific antibody of the present application is an IL-33 molecule.

[0122] [Artificial antibody]

[0123] The so-called artificial antibody is an artificial antibody having the same function as an antibody, such as a protein scaffold, which does not have the structure of an antibody. As the protein scaffold, the Kunitz domain of human serine protease inhibitor, the extracellular domain of human fibronectin, ankyrin, and a lipocalin are used, and if the sequence of the target binding site on the scaffold is changed, a protein scaffold that binds to the epitope of the present application can be generated (Clifford Mintzet. al BioProcess International, 2013, Vol. 11(2), pp40-48).

[0124] [Functionally modified antibody]

[0125] In the present application, the so-called functionally modified antibody is an antibody in which the cytotoxicity, complement activation function, blood half-life, and the like, which are functions other than the antigen binding function possessed by the antibody, are adjusted by mainly modifying the amino acids and sugar chains of the Fc region of the antibody.

[0126] [Coupled antibody]

[0127] In the present application, the so-called coupled antibody is an antibody in which a functional molecule other than an antibody, such as a non-peptide polymer such as polyethylene glycol (PEG), a radioactive substance, a toxin, a low molecular compound, a cytokine, albumin, an enzyme, and the like, is chemically bound or genetically engineered to be bound to the antibody.

[0128] [Fragment]

[0129] In the present application, the so-called fragment of an antibody is a protein containing a part of an antibody, and is capable of binding to an antigen. Examples of the fragment of an antibody include a Fab fragment, a Fv fragment, a F(ab')2 fragment, a Fab' fragment, or an scFv.

[0130] In addition, these fragments of antibodies can be chemically bound or genetically engineered to be bound to a functional molecule other than an antibody, such as a non-peptide polymer such as polyethylene glycol (PEG), a radioactive substance, a toxin, a low molecular compound, a cytokine, albumin, an enzyme, and the like.

[0131] [IL-33]

[0132] IL-33 is a cytokine belonging to the IL-1 family, and human IL-33 consists of 270 amino acids shown by SEQ ID NO: 226 in the sequence listing. IL-33 has a chromatin-binding domain on the N-terminal side, and has an IL-1 -like cytokine domain having 12 β-strands on the C-terminal side, with a molecular weight of 18 kDa, and also has cathepsin G cleavage sites at positions 95 and 109, an elastase cleavage site at position 99, and a caspase cleavage site at position 178. Figure 1 IL-33 is thought to be cleaved by enzymes such as elastase, cathepsin G, or proteinase 3 derived from lysosomes and the like during the process of cell necrosis, to become various fragments including mature IL-33, such as IL-33 (residues 95 to residues 270) (IL-33 represented by the amino acid sequence from position 95 to position 270 of SEQ ID NO: 226 in the sequence listing on the N-terminal side is referred to as "IL-33 (residues 95 to residues 270)". The same is described below), IL-33 (residues 99 to 270), IL-33 (residues 109 to residues 270), IL-33 (residues 112 to residues 270), and the like, and the cytokine functions as an active form. On the other hand, when cell death is apoptosis, caspase activated during the process of apoptosis causes IL-33 to be cleaved at position 178, to become an inactive form of IL-33, such as IL-33 (residues 179 to residues 270).

[0133] IL-33 has a function of being released as a cytokine to the outside of a cell, binding to an IL-33 receptor, and starting intracellular signal transduction in a cell expressing the IL-33 receptor. The signal transduction induced by IL-33 is not limited, and there are NF-κB pathways and MAPKKs pathways, which eventually induce production of various cytokines, chemokines, and inflammatory mediators. As examples of the cytokines induced by IL-33, TNF-α, IL-1β, IFN-γ, IL-3, IL-4, IL-5, IL-6, IL-13, and the like can be given, and in particular, IFN-γ, IL-5, IL-6, and IL-13 are induced. As examples of the chemokines induced by IL-33, CXCL2, CCL2, CCL3, CCL6, CCL17, CCL24, and the like can be given. As examples of the inflammatory mediators induced by IL-33, PGD2, LTB4, and the like can be given. The cytokines, chemokines, and inflammatory mediators induced by IL-33 are associated with migration, cytokine production, and degranulation of immune system cells, and induce inflammation. In the present application, IL-33 refers to any one of full-length IL-33 or an active fragment thereof if IL-33 functions by binding to the IL-33 receptor described later, and can be a derivative or a mutant thereof. In addition, human IL-33 can also be IL-33 derived from other organisms. Among them, human IL-33 represented by the amino acid sequence of SEQ ID NO: 226 in the sequence table is preferred.

[0134] The IL-33 receptor to which IL-33 binds is composed of a heterodimer of ST2 and IL-1RAcP (IL-1 receptor accessory protein). In the IL-33 receptor, a binding site that specifically recognizes IL-33 is present in the extracellular domain of ST2. The receptor for IL-33 is expressed in various immune system cells (Th2 cells, mast cells, eosinophils, basophils, macrophages, dendritic cells, NK cells, NKT cells, type 2 innate lymphoid cells (innate helper cells), nuocytes, Ih2 (innate helper type 2) cells, and the like), epithelial cells, and the like, but is not limited to these cells.

[0135] [IL-33-related diseases]

[0136] In the present application, the so-called IL-33-related disease is a disease induced due to the over-release of IL-33 to the extracellular, and the IL-33-related disease can be prevented, treated or alleviated using an agent capable of hindering the function of IL-33. As the IL-33-related disease, for example, asthma, atopic dermatitis, hay fever, anaphylactic shock, sinusitis (including eosinophilic sinusitis), Crohn's disease, ulcerative colitis, arthritis, systemic lupus erythematosus, pemphigus, pemphigoid, scleroderma, ankylosing spondylitis, liver fibrosis (including primary biliary cirrhosis), pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), acute kidney injury, vasculitis, and cancer can be cited.

[0137] [framework region]

[0138] The so-called framework region is a portion other than the complementarity determining region in the variable region of an immunoglobulin molecule. In the framework region, four framework regions (framework region 1, framework region 2, framework region 3, and framework region 4) are present in the light chain and the heavy chain, respectively. In the present application, the framework region of an immunoglobulin molecule is determined based on the numbering system of Kabat (Kabat et al., 1987, Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA).

[0139] [germ line]

[0140] The so-called germ line is a germ cell such as a sperm or an egg, and unless otherwise specified, it refers to a human germ line. Unlike B lymphocytes that produce antibodies, the immunoglobulin genes of the germ cell are not mutated. Therefore, when described as "the amino acid sequence of the framework region of the germ line", it refers to an amino acid sequence in which no mutation has occurred in the amino acid sequence of the framework region of an immunoglobulin, and when described as "the amino acid sequence of the combination of the amino acid sequences of the framework regions of the germ line", it refers to an amino acid sequence in which the amino acid sequence of one or more of the four framework regions is the amino acid sequence of the framework region of another germ line. In the case of the gene of the light chain variable region of human immunoglobulin, the kappa chain is divided into a VK segment and a JK segment, and the lambda chain is divided into a Vλ segment and a Jλ segment, and the framework region 1 to the framework region 3 are present in the VK segment and the Vλ segment, and the framework region 4 is present in the JK segment and the Jλ segment. The gene of the heavy chain variable region of human immunoglobulin is divided into a VH segment, a DH segment, and a JH segment, and the framework region 1 to the framework region 3 are present in the VH segment, and the framework region 4 is present in the JH segment. The amino acid sequences of the germ line of each segment of Vκ, Vλ, VH, JK, Jλ, and JH of human immunoglobulin are shown in Table 4.

[0141] [table 4-1]

[0142] The sequence numbers below indicate the sequence numbers in the sequence table

[0143] Excerpt Germ cell line name Serial Number Vκ Vκ1-5 Serial Number 278 Vκ Vκ1-6 Serial number 279 Vκ Vκ1-8 Serial number 280 Vκ Vκ1-9 Serial Number 281 Vκ Vκ1-12, Vκ1D-12 Serial number 282 Vκ Vκ1-16 Serial number 283 Vκ Vκ1-17 Serial number 284 Vκ Vκ1-27 Serial number 285 Vκ Vκ1-33, Vκ1D-33 Serial number 286 Vκ Vκ1-39, Vκ1D-39 Serial number 287 Vκ Vκ1D-8 Serial Number 288 Vκ Vκ1D-13 Serial number 289 Vκ Vκ1D-16 Serial number 290 Vκ Vκ1D-17 Serial number 291 Vκ Vκ1D-43 Serial number 292 Vκ Vκ1-NL1 Serial number 293 Vκ Vκ2-24 Serial number 294 Vκ Vκ2-28, Vκ2D-28 Serial number 295 Vκ Vκ2-30 Serial number 296 Vκ Vκ2-40, Vκ2D-40 Serial number 297 Vκ Vκ2D-26 Serial number 298 Vκ Vκ2D-29 Serial number 299 Vκ Vκ2D-30 Serial number 300 Vκ Vκ3-11 Serial Number 301 Vκ Vκ3-15, Vκ3D-15 Serial number 302 Vκ Vκ3-20 Serial number 303 Vκ Vκ3D-7 Serial number 304 Vκ Vκ3D-11 Serial number 305 Vκ Vκ3D-20 Serial number 306 Vκ Vκ3-NL1 Serial number 307 Vκ Vκ3-NL2 SEQ ID NO: 308 Vκ Vκ3-NL3 SEQ ID NO: 309

[0144] [Table 4-2]

[0145] Vκ Vκ3-NL4 SEQ ID NO: 310 Vκ Vκ3-NL5 SEQ ID NO: 311 Vκ Vκ4-1 SEQ ID NO: 312 Vκ Vκ5-2 SEQ ID NO: 313 Vλ Vλ1-36 SEQ ID NO: 314 Vλ Vλ1-40 SEQ ID NO: 315 Vλ Vλ1-44 SEQ ID NO: 316 Vλ Vλ1-47 SEQ ID NO: 317 Vλ Vλ1-51 SEQ ID NO: 318 Vλ Vλ2-8 SEQ ID NO: 319 Vλ Vλ2-11 SEQ ID NO: 320 Vλ Vλ2-14 SEQ ID NO: 321 Vλ Vλ2-18 SEQ ID NO: 322 Vλ Vλ2-23 SEQ ID NO: 323 Vλ Vλ3-1 SEQ ID NO: 324 Vλ Vλ3-9 SEQ ID NO: 325 Vλ Vλ3-10 SEQ ID NO: 326 Vλ Vλ3-12 SEQ ID NO: 327 Vλ Vλ3-16 SEQ ID NO: 328 Vλ Vλ3-19 SEQ ID NO: 329 Vλ Vλ3-21 SEQ ID NO: 330 Vλ Vλ3-22 SEQ ID NO: 331 Vλ Vλ3-25 SEQ ID NO: 332 Vλ Vλ3-27 SEQ ID NO: 333 Vλ Vλ4-3 SEQ ID NO: 334 Vλ Vλ4-60 SEQ ID NO: 335 Vλ Vλ4-69 SEQ ID NO: 336 Vλ Vλ5-37 SEQ ID NO: 337 Vλ Vλ5-39 SEQ ID NO: 338 Vλ Vλ5-45 SEQ ID NO: 339 Vλ Vλ5-52 SEQ ID NO: 340 Vλ Vλ6-57 SEQ ID NO: 341 Vλ Vλ7-43 SEQ ID NO: 342 Vλ Vλ7-46 SEQ ID NO: 343 Vλ Vλ8-61 SEQ ID NO: 344

[0146] [Table 4-3]

[0147] Vλ Vλ9-49 SEQ ID NO: 345 Vλ Vλ10-54 SEQ ID NO: 346 VH VH1-2 SEQ ID NO: 347 VH VH1-3 SEQ ID NO: 348 VH VH1-8 SEQ ID NO: 349 VH VH1-18 SEQ ID NO: 350 VH VH1-24 SEQ ID NO: 351 VH VH1-45 SEQ ID NO: 352 VH VH1-46 SEQ ID NO: 353 VH VH1-58 SEQ ID NO: 354 VH VH1-f SEQ ID NO: 355 VH VH1-69 SEQ ID NO: 356 VH VH2-5 SEQ ID NO: 357 VH VH2-26 SEQ ID NO: 358 VH VH2-70 SEQ ID NO: 359 VH VH3-7 SEQ ID NO: 360 VH VH3-9 SEQ ID NO: 361 VH VH3-11 SEQ ID NO: 362 VH VH3-13 SEQ ID NO: 363 VH VH3-15 SEQ ID NO: 364 VH VH3-20 SEQ ID NO: 365 VH VH3-21 SEQ ID NO: 366 VH VH3-23 SEQ ID NO: 367 VH VH3-30, VH3-30-3 SEQ ID NO: 368 VH VH3-33 SEQ ID NO: 369 VH VH3-43 SEQ ID NO: 370 VH VH3-48 SEQ ID NO: 371 VH VH3-49 SEQ ID NO: 372 VH VH3-53 SEQ ID NO: 373 VH VH3-64 SEQ ID NO: 374 VH VH3-66 SEQ ID NO: 375 VH VH3-72 SEQ ID NO: 376 VH VH3-73 SEQ ID NO: 377 VH VH3-74 SEQ ID NO: 378 VH VH3-d SEQ ID NO: 379

[0148] [Table 4-4]

[0149] VH VH3-NL1 SEQ ID NO: 380 VH VH4-4 SEQ ID NO: 381 VH VH4-28 SEQ ID NO: 382 VH VH4-30-2 SEQ ID NO: 383 VH VH4-30-4 SEQ ID NO: 384 VH VH4-31 SEQ ID NO: 385 VH VH4-34 SEQ ID NO: 386 VH VH4-39 SEQ ID NO: 387 VH VH4-59 SEQ ID NO: 388 VH VH4-b SEQ ID NO: 389 VH VH4-61 SEQ ID NO: 390 VH VH5-a SEQ ID NO: 391 VH VH5-51 SEQ ID NO: 392 VH VH6-1 SEQ ID NO: 393 VH VH7-4-1 SEQ ID NO: 394 JK JK1 SEQ ID NO: 395 JK JK2 SEQ ID NO: 396 JK JK3 SEQ ID NO: 397 JK JK4 SEQ ID NO: 398 JK JK5 SEQ ID NO: 399 JL JL1 SEQ ID NO: 400 JL JL2, JL3 SEQ ID NO: 401 JL JL6 SEQ ID NO: 402 JL JL7 SEQ ID NO: 403 JH JH1 SEQ ID NO: 404 JH JH2 SEQ ID NO: 405 JH JH3 SEQ ID NO: 406 JH JH4 SEQ ID NO: 407 JH JH5 SEQ ID NO: 408 JH JH6 SEQ ID NO: 409

[0150] [Human monoclonal antibody]

[0151] The so-called human monoclonal antibody is a monoclonal antibody having a variable region and a constant region of an immunoglobulin derived from a human germ line. In the present application, the variable region of the human monoclonal antibody can be a recombinant of a part or all of the variable region of another human monoclonal antibody, and from the viewpoint of not impairing the binding property of the antibody, the recombination can occur at the junction of the framework region and the complementarity determining region, and from the viewpoint of not increasing the immunogenicity, each region of the framework region 1 to the framework region 4 can be recombined with each region of the framework region 1 to the framework region 4 of another human monoclonal antibody. In addition, the human monoclonal antibody in the present application can be a mutant of the human monoclonal antibody, and in order to reduce the immunogenicity while maintaining or improving the binding property to the antigen, a human monoclonal antibody containing an amino acid sequence of a complementarity determining region in which a mutation exists in the complementarity determining region of the human monoclonal antibody, and an amino acid sequence of a framework region of the germ line in which no mutation exists in the framework region is preferred.

[0152] [Isolated]

[0153] The "isolation" of the so-called isolated antibody means that it is identified and separated, and / or recovered from components in a natural state. The impurities in the natural state are substances that can hinder the diagnostic or therapeutic use of the antibody, and examples include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In general, in order to isolate the antibody, it is sufficient to purify it by using at least one purification process, and the antibody obtained by purifying it by at least one purification process can be referred to as an "isolated antibody".

[0154] [Neutralization]

[0155] In the present application, the "neutralization" means the ability to bind to the target of interest and the ability to hinder any function of the target. That is, the "anti-IL-33 neutralizing monoclonal antibody" means the monoclonal antibody whose binding to IL-33 results in hindering of the biological activity induced by the IL-33 polypeptide. The hindering of the biological activity of IL-33 includes the hindering of the production of IL-33-induced cytokines such as IL-6, but is not limited thereto. The indicator of the biological activity of IL-33 can be evaluated by one or more of several in vitro or in vivo assays known in the art. Note that the "human anti-IL-33 neutralizing monoclonal antibody" means the human monoclonal antibody that binds to IL-33 and hinders any function of IL-33.

[0156] [Antagonist]

[0157] In the present application, the "antagonist" means the general term of the substance having the neutralizing effect on the target of interest. That is, the "antagonist of IL-33" means the substance that binds to IL-33 and hinders any function of IL-33, including, for example, the anti-IL-33 neutralizing monoclonal antibody.

[0158] [Complementarity-determining region]

[0159] The complementarity-determining region means the region in the variable region of the immunoglobulin molecule that forms the antigen-binding site, also referred to as the hypervariable region, and means the portion in which the amino acid sequence varies particularly greatly in each immunoglobulin molecule. The complementarity-determining region is present in three complementarity-determining regions (complementarity-determining region 1, complementarity-determining region 2, and complementarity-determining region 3) in the light chain and the heavy chain, respectively. In the present application, the complementarity-determining region of the immunoglobulin molecule is determined based on the numbering system of Kabat (Kabat et al., 1987, Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA).

[0160] [Competitive binding]

[0161] In the present application, the "competitively binding monoclonal antibody" means the monoclonal antibody whose binding to IL-33 is significantly reduced in the presence of the monoclonal antibody, as determined using the surface plasmon resonance (SPR) method described in the specification.

[0162] In the present application, the "competitively binding anti-IL-33 neutralizing monoclonal antibody" includes a chimeric antibody, a humanized antibody, a human antibody, a multispecific antibody, and an artificial antibody, as well as a functionally modified antibody of these antibodies, a conjugated antibody of these antibodies, and a fragment of these antibodies.

[0163] Next, the embodiments of the present application will be described. Note that the following embodiments are examples for describing the present application, and the present application is not limited to these embodiments.

[0164] The present application relates to a monoclonal antibody that binds to an epitope of IL-33. The monoclonal antibody that binds to the epitope is capable of neutralizing the activity of human IL-33, and thus the epitope is preferably an amino acid sequence contained in positions 101 to 154 or positions 199 to 270 of human IL-33 shown as SEQ ID NO: 226 in the sequence listing, and more preferably an amino acid sequence contained in positions 111 to 130 (PEP12), positions 131 to 150 (PEP14), positions 231 to 250 (PEP24), or positions 251 to 270 (PEP26) of SEQ ID NO: 226. IL-33 is often cleaved when released to the extracellular space. When the isolated amino acid residues on the primary sequence of IL-33 form an epitope based on the folding of the protein and the like, the folding collapses due to the cleavage of IL-33, or the isolated amino acid residues constituting the epitope disappear from the fragment, which sometimes results in a significant decrease in the affinity to the fragment. Thus, the binding epitope of the anti-IL-33 monoclonal antibody is preferably a continuous amino acid sequence.

[0165] In order for the monoclonal antibody that binds to the epitope to exert a neutralizing effect, for example, it is necessary to block the binding of IL-33 to the IL-33 receptor. Thus, in the present application, it is desirable that the preferred epitope is not only present on the surface of the IL-33 protein, but also present in close proximity to the IL-33 receptor. Thus, the present inventors determined, based on the data of the crystal structure analysis shown in Non-Patent Literature 11, that the preferred epitope is an amino acid sequence contained in positions 111 to 130 (PEP12), positions 131 to 150 (PEP14), positions 231 to 250 (PEP24), or positions 251 to 270 (PEP26) of SEQ ID NO: 226, which is present in close proximity to the IL-33 receptor, by performing a three-dimensional structure modeling as described in the Examples below. amino acids of the atoms (interface atoms) of IL-33 inside. As the amino acid containing the interface atom, P118 of PEP12 (the proline residue at position 118 of SEQ ID NO: 226 in the sequence listing is referred to as "P118". The same is described hereinafter.), 1119, T120, Y122, L123, R124, S125, L126, S127, Y129, N130, D131 of PEP14, Q132, S133, T135, A137, L138, E139, S142, Y143, E144, 1145, Y146, E148, D149, L150, D244 of PEP24, N245, H246, K266 of PEP26, L267, S268, E269 can be mentioned. As the functional epitope specifically binding to the monoclonal antibody capable of neutralizing IL-33, the epitope having the amino acid epitope containing the interface atom is preferred. The neutralization by the monoclonal antibody specifically binding to the functional epitope is considered to depend on the number of the interface atoms present in the functional epitope, the position of the interface atoms in the three-dimensional structure, and the like, but the present application is not limited by this theory.

[0166] As the preferred embodiment of the present application, the monoclonal antibody in which the epitope consisting of the continuous amino acid sequence contained in positions 101 to 154 or positions 199 to 270 of SEQ ID NO: 226 in the sequence listing is the epitope consisting of the continuous amino acid sequence of positions 111 to 130 (PEP12), 131 to 150 (PEP14), 231 to 250 (PEP24), or 251 to 270 (PEP26) of SEQ ID NO: 226 in the sequence listing can be mentioned. As the more preferred embodiment of the present application, the monoclonal antibody in which the epitope is the epitope consisting of the continuous amino acid sequence of positions 138 to 147 or 139 to 147 of SEQ ID NO: 226 in the sequence listing can be mentioned.

[0167] The present inventors have studied the minimum limit of the amino acid sequence constituting the epitope using two monoclonal antibodies binding to PEP14, and as a result, the continuous amino acid sequence of positions 138 to 147 and 139 to 147 of SEQ ID NO: 226 in the sequence listing was determined as the epitope of IL-33. Therefore, the present application relates to the epitope consisting of the continuous amino acid sequence of positions 138 to 147 and 139 to 147 of SEQ ID NO: 226 in the sequence listing.

[0168] Whether the monoclonal antibody is a monoclonal antibody that binds to the epitope of the present application can be investigated by methods commonly practiced in the field such as ELISA method, immunoprecipitation method, surface plasmon resonance (SPR) method, KinExA method, and the like. For example, in the peptide array scanning method described in the present application examples using the SPR method, when the epitope peptide of the present application is used for the test, the binding of the monoclonal antibody to the epitope can be measured as a significant increase in RU value. In addition, although the method described in the present application examples using the KinExA method can measure the dissociation constant (Kd), it is preferred that the dissociation constant for the epitope peptide is low, preferably, for example, 10 μM or less, 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 100 pM or less, 10 pM or less.

[0169] Another aspect of the present application relates to a pharmaceutical composition containing the monoclonal antibody of the present application that binds to an epitope consisting of a consecutive amino acid sequence contained in positions 101 to 154 or positions 199 to 270 of SEQ ID NO: 226 in the sequence listing. In addition, it also relates to a method for the diagnosis, treatment, prevention, or alleviation of an IL-33 related disease, which includes the administration of the monoclonal antibody of the present application, and the use of the monoclonal antibody of the present application for the manufacture of a medicament for the diagnosis, treatment, prevention, or alleviation of an IL-33 related disease.

[0170] Examples of IL-33-related diseases are not limited and can include asthma, atopic dermatitis, urticaria, hay fever, anaphylactic shock, sinusitis (including eosinophilic sinusitis), allergic encephalomyelitis, eosinophilia, polymyalgia rheumatica, rheumatic heart disease, multiple sclerosis, arthritis (e.g., rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, deforming arthropathy, Reiter's syndrome, etc.), systemic lupus erythematosus (including discoid lupus), pemphigus, bullous pemphigoid, psoriasis, ankylosing spondylitis, hepatitis (e.g., autoimmune hepatitis, chronic active hepatitis, etc.), inflammatory bowel disease (e.g., ulcerative colitis, Crohn's disease, gluten-sensitive bowel disease, etc.), and Sjogren's syndrome. Autoimmune hemolytic anemia, autoimmune inflammatory eye disease, autoimmune neonatal thrombocytopenia, autoimmune neutropenia, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, autoimmune thyroiditis, polymyositis, dermatomyositis, myasthenia gravis, adrenergic agonist tolerance, alopecia areata, antiphospholipid syndrome, adrenal autoimmune diseases (e.g., Addison's disease), celiac disease-dermatitis. Sprue-dermatitis, chronic fatigue immune dysfunction (CFIDS), cold agglutinin disease, idiopathic mixed globulinemia, fibromyalgia-fibromyalgia, glomerulonephritis (e.g., IgA nephropathy), Graves' disease, hyperthyroidism (i.e., Hashimoto's thyroiditis), idiopathic thrombocytopenic purpura (ITP), mixed connective tissue disease, type 1 or immune-mediated diabetes, pernicious anemia, polychondritis, autoimmune polyglandular syndrome, and generalized myotonic syndrome (Stiff-Mann syndrome). Syndrome, vitiligo, sarcoidosis, polyendocrinopathy, other endocrine disorders, arteriosclerosis, liver fibrosis (e.g., primary biliary cirrhosis), pulmonary fibrosis (e.g., acute pulmonary fibrosis), chronic obstructive pulmonary disease (COPD), scleroderma (including CREST syndrome, Raynaud's phenomenon), tubulointerstitial nephritis, dense deposit disease, acute kidney injury, myocarditis, cardiomyopathy, neuritis (e.g.,Guillain-Barre syndrome, etc.), polyarteritis nodosa, cardiotomy syndrome, chronic inflammatory demyelinating polyneuropathy, IgA neuropathy, lichen planus, Meniere's syndrome, post-MI, uveitis, uveitis opthalmia, vasculitis, primary agammaglobulinemia, cancer (e.g., brain tumor, laryngeal cancer, oral cancer, hypopharyngeal cancer, thyroid cancer, esophageal cancer, breast cancer, lung cancer, stomach cancer, adrenal cortex cancer, bile duct cancer, gallbladder cancer, liver cancer, pancreatic cancer, bladder cancer, large intestine cancer, uterine cancer, ovarian cancer, prostate cancer, testicular cancer, chronic lymphocytic leukemia, chronic myelocytic leukemia, Ewing's sarcoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, melanoma, mesothelioma, multiple myeloma, etc.), infection showing resistance to rejection from the immune system (e.g., severe acute respiratory syndrome (SARS)), lethal cytokine storm accompanying highly virulent influenza infection, and scurvy, and, among others, asthma, atopic dermatitis, hay fever, anaphylactic shock, sinusitis (including eosinophilic sinusitis), Crohn's disease, ulcerative colitis, arthritis, systemic lupus erythematosus, pemphigus, pemphigoid, scleroderma, ankylosing spondylitis, liver fibrosis (including primary biliary cirrhosis), pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), acute kidney injury, vasculitis, and cancer.

[0171] Another aspect of the present application relates to an expression inhibitor of a cytokine, chemokine, or inflammatory mediator, which contains a monoclonal antibody that binds to an epitope consisting of a continuous amino acid sequence contained in positions 101 to 154 or positions 199 to 270 of SEQ ID NO: 226 in the sequence listing.

[0172] The cytokine inhibited by the expression inhibitor of a cytokine, chemokine, or inflammatory mediator of the present application is a cytokine induced by IL-33, and, for example, TNF-α, IFN-γ, IL-1β, IL-3, IL-4, IL-5, IL-6, IL-13, etc. can be mentioned. In addition, the chemokine inhibited by the inhibitor is a chemokine induced by IL-33, and, for example, CXCL2, CCL2, CCL3, CCL6, CCL17, CCL24, etc. can be mentioned. The inflammatory mediator inhibited by the inhibitor is an inflammatory mediator induced by IL-33, and, for example, PGD2, LTB4, etc. can be mentioned. A particularly preferred aspect of the present application is an expression inhibitor of IFN-γ, IL-5, IL-6, or IL-13 containing an anti-IL-33 monoclonal antibody, and more preferably an inhibitor of production of IL-6.

[0173] Another aspect of the present application relates to an epitope to which the anti-IL-33 monoclonal antibody binds. In the present application, the epitope relates to a sequence consisting of 6 to 20 amino acids necessary for the recognition by the antibody. In other aspects, the epitope can further include amino acids surrounding the recognized sequence or amino acids in the vicinity in the three-dimensional structure, but is preferably a continuous amino acid sequence not including a discontinuous amino acid sequence.

[0174] Therefore, the number of amino acid residues constituting the continuous amino acid sequence of the epitope of the present application is at least 5, preferably at least 6, more preferably at least 7, more preferably at least 8, and more preferably at least 9. Further, from the viewpoint of exerting more sufficient antigenicity, it is at least 10, more preferably 15, and more preferably at least 20. On the other hand, if the sequence contained in the epitope is longer, there is a possibility that a plurality of portions recognized by the antibody are included, and in such a case, it can not be possible to produce or screen an antibody having a desired neutralizing action. Therefore, from the viewpoint of ensuring that the antibody binding to the epitope of the present application exerts a desired neutralizing action, the length of the epitope sequence is preferably 30 or less, more preferably 20 or less, and more preferably 15 or less. As the number of residues of the continuous amino acid sequence contained in the epitope, the number of residues selected from, for example, one of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 is used.

[0175] The epitope can be introduced with one or more amino acid mutations, i.e., amino acid substitution, deletion, or insertion, within a range that does not change its antigenicity. The number of mutations introduced is preferably 5 or less, more preferably 3 or less, and most preferably 1. Further, the epitope can be added with a modification such as a modification such as a sugar chain or the like possessed by the original protein, a terminal modification, or the like. In addition, in other aspects, it can be an epitope consisting of an amino acid sequence having a sequence identity of at least 90%, more preferably at least 95%, more preferably at least 97%, further preferably at least 98%, and most preferably 99% with respect to the sequence consisting of continuous amino acids of the recognized epitope in the present application, within a range that does not change its antigenicity. When the epitope peptide is used as, for example, a bait, a label such as histidine, biotin, or the like can be added, and when it is used as a vaccine, it can be bound to a carrier protein such as KLH or the like.

[0176] Here, the "percent (%) sequence identity" with respect to an identified comparison polypeptide sequence is defined as follows: the percentage of the amino acid residues in the candidate sequence that are identical with the amino acid residues in the identified comparison polypeptide sequence, after aligning the sequences for maximum percentage sequence identity, introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. The comparison for determining percent amino acid sequence identity can be achieved by using various methods known in the art, for example, using BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software, which are available publicly. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Here, however, for purposes of this application, the % amino acid sequence identity values are obtained using the sequence comparison program BLAST in pairwise comparison.

[0177] In the case where BLAST can be used for comparison of amino acid sequences, the % amino acid sequence identity of a provided amino acid sequence A with respect to a provided amino acid sequence B is calculated as follows:

[0178] 100 times the fraction X / Y

[0179] Here, X is the number of amino acid residues determined to be identical in the comparison of sequences A and B by the sequence comparison program BLAST, and Y is the total number of amino acid residues in B. It is known that the % amino acid sequence identity of an amino acid sequence A with respect to an amino acid sequence B is different from the % amino acid sequence identity of B with respect to A, when the length of A is different from the length of B. Unless otherwise specified, all % amino acid sequence identity values herein are obtained using the BLAST computer program, as shown in the immediately preceding figure.

[0180] The epitope discovered in the present application is a functional epitope to which a neutralizing antibody of IL-33 specifically binds. Therefore, by utilizing the functional epitope of the present application, a novel antibody having an IL-33 antagonist effect can be efficiently obtained. That is, by screening a monoclonal antibody population against full-length IL-33 or mature IL-33 for an antibody that binds to the functional epitope of the present application, a monoclonal antibody having an antagonist effect can be obtained. Therefore, in another aspect of the present application, the present application also relates to a screening method for an antibody having an antagonist effect using the functional epitope of IL-33. More specifically, when a clone of an antibody having an IL-33 antagonist effect is enriched from a natural antibody library by a phage display technique or the like, first, the library is screened using full-length or mature IL-33 protein as a bait to enrich antibody clones that bind to various epitopes on the surface of IL-33, and then the library is screened using the functional epitope peptide discovered in the present application as a bait, whereby an antibody having an antagonist effect of IL-33 that specifically binds to the functional epitope can be efficiently screened.

[0181] In the present example, the present inventors investigated IL-33 antagonist activity by changing the antibody concentration with respect to a monoclonal antibody population that was determined to bind to an epitope of 20 residues in length. As a result, an epitope suitable for producing or screening an antibody having an antagonist effect was determined. According to the results, it was confirmed that the antagonist effect of an antibody that binds to an epitope selected from the group consisting of positions 111 to 130 (PEP12), positions 131 to 150 (PEP14), positions 231 to 250 (PEP24), and positions 251 to 271 (PEP26) of SEQ ID NO: 226 in the sequence listing increased in accordance with the antibody concentration, and thus the above epitope is a functional epitope suitable for producing or screening an antibody having an antagonist effect. Therefore, in one aspect of the present application, an epitope comprising a sequence consisting of at least 6, preferably at least 10, more preferably at least 15, consecutive amino acids in a region selected from the group consisting of positions 111 to 130, positions 131 to 150, positions 231 to 250, and positions 251 to 271 of SEQ ID NO: 226 in the sequence listing is involved. In another aspect, the present application relates to an epitope selected from the group consisting of positions 111 to 130, positions 131 to 150, positions 231 to 250, and positions 251 to 270 of SEQ ID NO: 226 in the sequence listing.

[0182] The epitope can be produced using a generally implemented peptide synthesis technique. The produced and purified epitope can be used for immunization of an animal to produce an antibody against the epitope. In addition, in another method, by using the purified epitope in a phage display method, a monoclonal antibody that binds to the epitope can be produced or screened. In addition, the epitope can be used as a vaccine together with an immunological adjuvant.

[0183] The present application relates to a monoclonal antibody that binds to an epitope composed of a continuous amino acid sequence contained in positions 101 to 154 or positions 199 to 270 of SEQ ID NO: 226 in the sequence listing. The monoclonal antibody includes a chimeric antibody, a humanized antibody, a human antibody, a multispecific antibody, and an artificial antibody, as well as a functionally modified antibody of these antibodies, and a conjugated antibody of these antibodies, and a fragment of these antibodies. The monoclonal antibody of the present application can be produced using any of known methods such as a hybridoma method, a phage display method, and a genetic engineering technique.

[0184] In the hybridoma method, a monoclonal antibody can be produced by fusing B cells collected from a spleen or a lymph node of an animal, particularly a rat or a mouse, immunized with an immunogen, and an immortalized cell, such as a myeloma cell, cultivating the hybridoma, screening the hybridoma producing an antibody having a desired binding property, and producing a monoclonal antibody using the screened hybridoma. In addition, a human antibody can be obtained by using a mouse into which a human antibody gene is introduced. In order to obtain a monoclonal antibody from a hybridoma, a method of culturing the hybridoma according to a conventional method and obtaining a culture supernatant thereof, or a method of administering the hybridoma to a suitable mammal to proliferate and obtaining an ascites thereof, or the like can be employed. The former method is suitable for obtaining an antibody with high purity, whereas the latter method is suitable for mass production of an antibody. The technique for preparing a monoclonal antibody can be performed using known techniques, for example, can be produced based on the description of Chapter 2 of Current Protocols in Immunology, Wiley and Sons Inc.

[0185] In the phage display method, phages selected from any phage antibody library are screened using a target immunogen, and phages having a desired binding property to the immunogen are selected. Next, the antibody corresponding sequence contained in the phage is isolated or sequenced, and based on the isolated sequence or the measured sequence information, an expression vector containing a nucleic acid molecule encoding a monoclonal antibody is constructed. Next, by culturing a cell strain transformed with the expression vector, a monoclonal antibody can be produced. As the phage antibody library, by using a human antibody library, a human antibody having a desired binding property can be produced.

[0186] In the genetic engineering method, a mutation is introduced into a sequence corresponding to a complementarity-determining region (CDR) or other sequence in the gene sequence encoding an antibody, the sequence is incorporated into an expression vector, the incorporated expression vector is transformed into a host cell, and a recombinant antibody can be produced (see, for example, Borrebaeck C. A. K. and Larrick J. W. THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990).

[0187] In the present application, a chimeric antibody, a humanized antibody, a multispecific antibody, or an artificial antibody can be used for the purpose of reducing heteroantigenicity against humans or adding other functions, and these antibodies can be produced using known methods.

[0188] A chimeric antibody can be obtained by linking DNA encoding a variable region of a non-human antibody and DNA encoding a constant region of a human antibody, incorporating the linked DNA into an expression vector, and introducing the expression vector into a host to produce the chimeric antibody (see EP 125023, WO 92 / 19759). Using the known methods described above, a chimeric antibody useful in the present application can be obtained.

[0189] A humanized antibody can be obtained by linking a complementarity-determining region (CDR) of a non-human antibody and DNA encoding a region of a human antibody other than the CDR, incorporating the linked DNA into an expression vector, and introducing the expression vector into a host to produce the humanized antibody.

[0190] A multispecific antibody is an asymmetric antibody having two or more different antigen specificities and having two or more independent antigen recognition sites. A multispecific antibody such as a bispecific antibody can be prepared using the antigen binding regions of two or more monoclonal antibodies by a genetic engineering method. The genetic engineering method has been established in the art. For example, a desired bispecific antibody can be obtained using a DVD-Ig technique in which the antigen binding regions of two monoclonal antibodies are linked in series (Wu et al., Nature Biotechnology 25(11), 1290 (2007)), an ART-Ig technique in which the heavy chains of two antibodies that bind to different antigens are combined by changing the Fc region of the antibody (Kitazawa et al., Nature Medicine 18(10), 1570 (2012)).

[0191] The so-called artificial antibody is an artificial antibody such as a protein scaffold which does not have the structure of an antibody but has the same function as an antibody. As the protein scaffold, the Kunitz domain of human serine protease inhibitor, the extracellular domain of human fibronectin, ankyrin, and a lipocalin are used, and if the sequence of the target binding site on the scaffold is changed, a protein scaffold which binds to the epitope of the present application can be produced (Patent Literature 4, Clifford Mintz et. al BioProcess International, 2013, Vol. 11(2), pp40-48).

[0192] By changing the amino acid sequence of the Fc region and the like of the monoclonal antibody of the present application, the sugar chain, the cytotoxicity, the complement activation function, the blood half-life and the like possessed by the antibody can be adjusted (Strohl, Current Opinion in Biotechnology, 2009, vol. 20, p685). Such a functionally modified antibody is produced by, for example, the following method. If a CHO cell in which the a1, 6-fucose transferase (FUT8) gene is disrupted is used as a host cell to produce a monoclonal antibody, the fucose content of the sugar chain is reduced, and an antibody with improved cytotoxicity can be obtained, and if a CHO cell into which the FUT8 gene is introduced is used as a host cell to produce a monoclonal antibody, an antibody with low cytotoxicity can be obtained (International Publication No. 2005 / 035586, International Publication No. 2002 / 31140, International Publication No. 00 / 61739). In addition, by changing the amino acid residues of the Fc region, the complement activation function can be adjusted (U.S. Patent No. 6737056, U.S. Patent No. 7297775, U.S. Patent No. 7317091). Furthermore, by using a mutant of the Fc region with improved binding to FcRn which is one of the Fc receptors, prolongation of the blood half-life can be expected (Hashimoto, M. et al., Shokubutsu Kagaku, 2010, Vol. 82(8), p710). These functionally modified antibodies can be produced by genetic engineering.

[0193] The monoclonal antibody used in the present application can be a conjugated antibody formed by binding various molecules such as a non-peptide polymer such as polyethylene glycol (PEG), a radioactive substance, a toxin, and the like. Such a conjugated antibody can be obtained by performing chemical modification on the obtained antibody. Note that methods of chemical modification have been established in this field. The monoclonal antibody in the present application also includes these conjugated antibodies (D. J. King., Applications and Engineering of Monoclonal antibodies., 1998 T. J. International Ltd, Monoclonal Antibody-Based Therapy of Cancer., 1998 Marcel Dekker Inc; Chari et al., Cancer Res., 1992 Vol 152: 127; Liu et al., Proc Natl Acad Sci USA., 1996 Vol 93: 8681,).

[0194] In the present application, in addition to all the antibodies described above, a fragment of a monoclonal antibody or a modifier of a monoclonal antibody can also be used as long as it has epitope binding and exerts an antagonist activity. For example, as a fragment of an antibody, a Fab fragment, an Fv fragment, an F(ab')2 fragment, a Fab' fragment, or a single chain Fv (scFv) in which Fv of an H chain and an L chain are linked by a suitable linker can be mentioned.

[0195] Furthermore, a fragment of these antibodies can be chemically bound or genetically engineered to bind to a functional molecule other than an antibody, such as a non-peptide polymer such as polyethylene glycol (PEG), a radioactive substance, a toxin, a low-molecular compound, a cytokine, albumin, an enzyme, and the like.

[0196] The production system for producing the monoclonal antibody can utilize any one of in vitro or in vivo production systems. As the in vitro production system, there can be mentioned a production system using eukaryotic cells such as animal cells, plant cells, or fungal cells, a production system using prokaryotic cells such as bacterial cells of E. coli, Bacillus subtilis, and the like. As the animal cells to be used, there can be mentioned mammalian cells such as commonly used cells of CHO, COS, myeloma, BHK, HeLa, Vero, and the like, insect cells, plant cells, and the like. As the in vivo production system, there can be mentioned a production system using animals, a production system using plants. In the case of using animals, there can be mentioned a production system using, for example, mammals, insects, and the like. As the mammals, there can be mentioned, for example, goats, pigs, sheep, mice, cows, and the like (Vicki Glaser, SPECTRUM Biotechnology Applications, 1993). In addition, as the insects, there can be mentioned, for example, silkworms. In the case of using plants, there can be mentioned, for example, tobacco.

[0197] In the production of the monoclonal antibody using such in vitro or in vivo production system as described above, the DNA encoding the heavy chain (H chain) or the light chain (L chain) of the monoclonal antibody can be separately incorporated into an expression vector and simultaneously transformed into a host, or the DNA encoding the H chain and the L chain can be incorporated into a single expression vector and transformed into a host (see International Publication No. WO94 / 11523).

[0198] The obtained monoclonal antibody can be purified until uniform. The isolation and purification of the monoclonal antibody can be performed using the isolation and purification methods commonly used for proteins. For example, the monoclonal antibody can be isolated and purified by appropriately selecting and combining a column such as affinity chromatography, a filter, ultrafiltration, salting-out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis, and the like (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988), but is not limited to the above-mentioned methods. As the column for affinity chromatography, there can be mentioned a Protein A column, a Protein G column. As the column using, for example, a Protein A column, there can be mentioned Hyper D, POROS, Sepharose F.F. (Amersham Biosciences), and the like.

[0199] The monoclonal antibody of the present invention, which binds to an epitope consisting of a continuous amino acid sequence contained in positions 101-154 or 199-270 of sequence number 226 in the sequence listing, has low antigenicity when administered to humans. Therefore, it is preferably a chimeric antibody, a humanized antibody, a human antibody, and most preferably a human antibody. Furthermore, in the human antibody, the amino acid sequence of the frame region is preferably an amino acid sequence of the frame region of a human germline or a combination thereof. Therefore, the present invention relates to a human anti-IL-33 neutralizing monoclonal antibody, wherein the amino acid sequence of the frame region of the antibody is an amino acid sequence of the frame region of a germline or a combination thereof.

[0200] This human anti-IL-33 neutralizing monoclonal antibody has the following characteristics: because the framework region of the variable region contains the amino acid sequence of the human germline framework region or a combination thereof, these regions are completely non-immunogenic or cause very little immunogenicity. On the other hand, the antibody can bind to IL-33 and inhibit its function. Therefore, when used as a medicine, this antibody is unlikely to induce human anti-human immunoglobulin antibody (HAHA), thus it is not rejected in vivo. As a result, it has a long-lasting IL-33 neutralizing effect and is a safe antibody as long as it does not induce inflammation caused by binding to HAHA.

[0201] Regarding the amino acid sequences of the light and heavy chain framework regions in human germline cells, any sequence can be used as long as it is an amino acid sequence of the framework region of a human germline cell line. For example, sequences registered in databases such as NCBI can be used. http: / / www.ncbi.nlm.nih.gov / igblast / showGermline.cgi The amino acid sequences encoded by the DNA sequences of the heavy chain variable region and the framework region of the light chain variable region of the human antibody in Table 4, and the amino acid sequences of the framework regions of the germline cells listed in Table 4. The light chain variable region can be a variable region of the λ chain or a variable region of the κ chain. As the light chain and heavy chain framework regions in the human germline cells, it is preferred to be framework regions that occur frequently and are commonly used in the organism. Examples of such human heavy chain framework regions include framework regions 1, 2, and 3 of VH3-23, VH3-30, VH4-39, and VH4-34, and framework region 4 of JH4. In addition, examples of human light chain framework regions that occur frequently in the organism include framework regions 1, 2, and 3 of Vλ1-47, Vλ2-14, Vκ3-20, and Vκ1-39, and framework region 4 of Jλ2. For heavy chain framework regions, any human heavy chain framework region can be used in any combination. For example, framework regions 1 and 2 of VH3-23 and framework region 3 of VH3-30 can be selected as heavy chain framework regions. Similarly, for light chain framework regions, any human heavy chain framework region can be used in any combination.

[0202] In the present application, the amino acid sequence of the framework region of the preferred germline is the amino acid sequence of the framework region of VH3-23, VH3-30, JH4, Vl 1-47, Jl 2. Specifically, the following framework region is preferred: in which the amino acid sequence of the light chain framework region 1 is residues 1 to 22 of SEQ ID NO: 317, the amino acid sequence of the light chain framework region 2 is residues 36 to 50 of SEQ ID NO: 317, the amino acid sequence of the light chain framework region 3 is residues 58 to 89 of SEQ ID NO: 317, and the amino acid sequence of the light chain framework region 4 is residues 3 to 12 of SEQ ID NO: 401, and the amino acid sequence of the heavy chain framework region 1 is residues 1 to 30 of SEQ ID NO: 367 or residues 1 to 30 of SEQ ID NO: 368, the amino acid sequence of the heavy chain framework region 2 is residues 36 to 49 of SEQ ID NO: 367 or residues 36 to 49 of SEQ ID NO: 368, the amino acid sequence of the heavy chain framework region 3 is residues 67 to 98 of SEQ ID NO: 367 or residues 67 to 98 of SEQ ID NO: 368, and the amino acid sequence of the heavy chain framework region 4 is residues 5 to 15 of SEQ ID NO: 407, and most preferably the following framework region: in which the amino acid sequence of the light chain framework region 1 is residues 1 to 22 of SEQ ID NO: 317, the amino acid sequence of the light chain framework region 2 is residues 36 to 50 of SEQ ID NO: 317, the amino acid sequence of the light chain framework region 3 is residues 58 to 89 of SEQ ID NO: 317, and the amino acid sequence of the light chain framework region 4 is residues 3 to 12 of SEQ ID NO: 401, and the amino acid sequence of the heavy chain framework region 1 is residues 1 to 30 of SEQ ID NO: 367, the amino acid sequence of the heavy chain framework region 2 is residues 36 to 49 of SEQ ID NO: 367, the amino acid sequence of the heavy chain framework region 3 is residues 67 to 98 of SEQ ID NO: 368, and the amino acid sequence of the heavy chain framework region 4 is residues 5 to 15 of SEQ ID NO: 407.

[0203] As another aspect of the present application, there is provided an isolated human anti-IL-33 neutralizing monoclonal antibody, the amino acid sequence of each of the light chain complementarity determining region 1 (LCDR1), the light chain complementarity determining region 2 (LCDR2), the light chain complementarity determining region 3 (LCDR3), the heavy chain complementarity determining region 1 (HCDR1), the heavy chain complementarity determining region 2 (HCDR2), and the heavy chain complementarity determining region 3 (HCDR3) of the antibody is the amino acid sequence of the complementarity determining region combination shown in Table 1.

[0204] In a more preferable embodiment, the human anti-IL-33 neutralizing monoclonal antibody having a combination of the CDRs of C1 to C30 shown in Table 1 has binding and neutralizing activity against IL-33, particularly mature IL-33 in which binding to the IL-33 receptor and exertion of activity are performed, such as IL-33 (residue 95 to residue 270), IL-33 (residue 99 to residue 270), IL-33 (residue 109 to residue 270), IL-33 (residue 112 to residue 270), and the like. More preferably, the human anti-IL-33 neutralizing monoclonal antibody having a combination of the CDRs of C1 to C30 shown in Table 1 has binding against IL-33 (residue 131 to residue 150).

[0205] In the present application, from the viewpoint of binding and / or property, a combination of improved CDRs is a preferable embodiment. As the human anti-IL-33 neutralizing monoclonal antibody, particularly preferably, the upper limit value of the dissociation rate constant (koff) of the antibody against human IL-33 is about 3.5 x 10 -5 / sec or less, more preferably about 2.0 x 10 -5 / sec or less, further more preferably about 1.5 x 10 -5 / sec or less, further more preferably about 1.0 x 10 -5 / sec or less, and the lower limit value is not particularly limited, for example, 10 -7 / sec or more, more preferably 10 -6 / sec or more, further more preferably about 5 x 10 -6 / sec or more.

[0206] Among the human anti-IL-33 neutralizing monoclonal antibodies, more preferably, an antibody having a low dissociation constant (Kd) against human IL-33 can be cited. As the upper limit value of the dissociation constant of the antibody, for example, 10 -9 M or less, more preferably 10 -10 M or less, further more preferably 10 -12 M or less, and the lower limit value is not particularly limited, for example, 10 -14 M or more, more preferably 10 -13 M or more.

[0207] The human anti-IL-33 neutralizing monoclonal antibody of the present application inhibits the production of IL-6 from HUVEC upon stimulation with IL-33. Among them, an antibody having a strong inhibitory effect is preferred. Specifically, as a preferred embodiment of the present application, a human anti-IL-33 neutralizing monoclonal antibody can be exemplified, which, as described in Example 10 below, inhibits the production of IL-6 from HUVEC upon stimulation with 100 ng / mL of IL-33 by the addition of 1 μg / mL of the human anti-IL-33 neutralizing monoclonal antibody, by a rate (inhibition rate) of about 50% or more, more preferably about 70% or more, and even more preferably about 90% or more.

[0208] The human anti-IL-33 neutralizing monoclonal antibody of the present application inhibits the production of IL-5, IL-6 and / or IL-13 from KU-812 cells upon stimulation with IL-33. Among them, an antibody having a strong inhibitory effect is preferred. Specifically, as a preferred embodiment of the present application, a human anti-IL-33 neutralizing monoclonal antibody can be exemplified, which, as described in Example 11 below, inhibits the production of IL-5, IL-6 and / or IL-13 from KU-812 cells upon stimulation with 100 ng / mL of IL-33 by the addition of 3 μg / mL of the human anti-IL-33 neutralizing monoclonal antibody, by a rate (inhibition rate) of about 30% or more, more preferably about 50% or more, and even more preferably about 70% or more.

[0209] The human anti-IL-33 neutralizing monoclonal antibody of the present application inhibits the production of IFN-γ from human peripheral blood mononuclear cells upon stimulation with IL-33. Among them, an antibody having a strong inhibitory effect is preferred. Specifically, as a preferred embodiment of the present application, a human anti-IL-33 neutralizing monoclonal antibody can be exemplified, which, as described in Example 12 below, inhibits the production of IFN-γ from human peripheral blood mononuclear cells upon stimulation with 10 ng / mL of IL-33 by the addition of 10 μg / mL of the human anti-IL-33 neutralizing monoclonal antibody, by a rate (inhibition rate) of about 80% or more, more preferably about 90% or more, and even more preferably about 95% or more.

[0210] The human anti-IL-33 neutralizing monoclonal antibody of the present application preferably has an excellent anti-inflammatory effect. Specifically, as a preferred embodiment of the present application, there can be mentioned a human anti-IL-33 neutralizing monoclonal antibody in which the increase in the spleen weight, the IgA concentration in serum, the IgE concentration in serum, the number of neutrophils, the number of basophils, the number of eosinophils, and / or the IL-5 concentration in serum caused by the continuous administration of human IL-33 at 0.4 μg / individual for 7 days is inhibited by 30% or more, more preferably 50% or more, and even more preferably 80% or more, as described in Example 13 below.

[0211] Further, the human anti-IL-33 neutralizing monoclonal antibody of the present application preferably has excellent physical properties. Among them, a human anti-IL-33 neutralizing monoclonal antibody in which the shape of the particle size distribution does not show bimodality and the aggregation is extremely low in the evaluation using dynamic light scattering, and a human anti-IL-33 neutralizing monoclonal antibody in which the interaction parameter (kD) as an index of colloidal stability is high, for example, the parameter is preferably -12.4 mL / g or more, more preferably -10 mL / g or more, and even more preferably -8.5 mL / g or more, are preferred.

[0212] The human anti-IL-33 neutralizing monoclonal antibody of the present application preferably has excellent thermodynamic stability. Specifically, a human anti-IL-33 neutralizing monoclonal antibody in which the temperature at which the immunoglobulin domain of the antibody collapses (Tm) is 65°C or more, preferably 68°C or more, more preferably 70°C or more, and even more preferably 73°C or more, is preferred.

[0213] Further, the human anti-IL-33 neutralizing monoclonal antibody of the present application preferably has excellent stability. The stability of the antibody can be determined by a storage stability test, a forced oxidation test, and the like. As a storage stability test, for example, as a preferred embodiment of the present application, the proportion of monomers of the antibody molecule is preferably 90% or more, and more preferably 95% or more, and the binding activity to human IL-33 protein is preferably 95% or more, and more preferably 99% or more, when stored at 40°C for 4 weeks, as described in Example 21 below.

[0214] In addition, when forced oxidation is performed with 1% hydrogen peroxide aqueous solution at 37°C for 24 hours, as described in Example 22 of the present application, it is preferred that the binding activity to human IL-33 protein is maintained at 80% or more, more preferably 85% or more, and even more preferably 90% or more.

[0215] In view of the above, in the present application, a human anti-IL-33 neutralizing monoclonal antibody selected from a combination of the complementarity determining regions of C1 to C28 in Table 1 is a more preferable antibody. In addition, as a more preferable mode of the present application, a human anti-IL-33 neutralizing monoclonal antibody having a combination of the amino acid sequences of specific complementarity determining regions (C1, C8, C15, C17, or C18 in Table 1) can be cited.

[0216] The amino acid sequence of the framework region in the variable region of the human anti-IL-33 neutralizing monoclonal antibody determined by the above combination of the amino acid sequences of the complementarity determining regions can be any framework region as long as antigen-binding property is ensured. From the viewpoint of reducing immunogenicity against humans, the amino acid sequence of the framework region is preferably each amino acid sequence of a human germline framework region or a combination thereof, and more preferably an amino acid sequence of a framework region of a commonly used germline in humans.

[0217] In the present application, the amino acid sequence of the preferred framework region is as follows: the amino acid sequence of the light chain framework region 1 is residues 1 to 22 of SEQ ID NO: 317 in the sequence table, the amino acid sequence of the light chain framework region 2 is residues 36 to 50 of SEQ ID NO: 317 in the sequence table, the amino acid sequence of the light chain framework region 3 is residues 58 to 89 of SEQ ID NO: 317 in the sequence table, and the amino acid sequence of the light chain framework region 4 is residues 3 to 12 of SEQ ID NO: 401 in the sequence table, and the amino acid sequence of the heavy chain framework region 1 is residues 1 to 30 of SEQ ID NO: 367 in the sequence table or residues 1 to 30 of SEQ ID NO: 368 in the sequence table, the amino acid sequence of the heavy chain framework region 2 is residues 36 to 49 of SEQ ID NO: 367 in the sequence table or residues 36 to 49 of SEQ ID NO: 368 in the sequence table, the amino acid sequence of the heavy chain framework region 3 is residues 67 to 98 of SEQ ID NO: 367 in the sequence table or residues 67 to 98 of SEQ ID NO: 368 in the sequence table, and the amino acid sequence of the heavy chain framework region 4 is residues 5 to 15 of SEQ ID NO: 407 in the sequence table. In the present application, the amino acid sequence of the more preferred framework region is as follows: the amino acid sequence of the light chain framework region 1 is residues 1 to 22 of SEQ ID NO: 317 in the sequence table, the amino acid sequence of the light chain framework region 2 is residues 36 to 50 of SEQ ID NO: 317 in the sequence table, the amino acid sequence of the light chain framework region 3 is residues 58 to 89 of SEQ ID NO: 317 in the sequence table, and the amino acid sequence of the light chain framework region 4 is residues 3 to 12 of SEQ ID NO: 401 in the sequence table, and the amino acid sequence of the heavy chain framework region 1 is residues 1 to 30 of SEQ ID NO: 367 in the sequence table, the amino acid sequence of the heavy chain framework region 2 is residues 36 to 49 of SEQ ID NO: 367 in the sequence table, the amino acid sequence of the heavy chain framework region 3 is residues 67 to 98 of SEQ ID NO: 368 in the sequence table, and the amino acid sequence of the heavy chain framework region 4 is residues 5 to 15 of SEQ ID NO: 407 in the sequence table.

[0218] Accordingly, in the present application, preferred combinations of the amino acid sequences of the variable region of the heavy chain and the variable region of the light chain are shown in, for example, Table 2.

[0219] A more preferred embodiment of the present application is a human anti-IL-33 neutralizing monoclonal antibody having a combination of the variable regions of V1 to V28 in Table 2.

[0220] A more preferred embodiment of the present application is a human anti-IL-33 neutralizing monoclonal antibody having a combination of the amino acid sequences of the specific complementarity determining regions (V1, V8, V15, V17, or V18 in Table 2).

[0221] Depending on the difference in the constant region of the heavy chain, there are IgG (including IgG1, IgG2, IgG3, and IgG4), IgM, IgA (including IgA1, IgA2), IgD, or IgE, each of which has a γ chain, a μ chain, an α chain, a δ chain, or an ε heavy chain. As the constant region of the human anti-IL-33 neutralizing monoclonal antibody of the present application, all of the above are included. In addition, depending on the position on the chromosome, there are a κ chain and a λ chain, both of which are included in the present application. In the production of antibody medicines, the κ chain is preferred from the viewpoint of coagulation, but the antibody having a λ light chain is also useful from the viewpoint that the λ chain has a different amino acid sequence from the κ chain and has diversity as well as the κ chain. From the viewpoint of stability in blood, the human anti-IL-33 neutralizing monoclonal antibody of the present application is preferably IgG in which the light chain is a λ chain and the heavy chain is a γ chain, and more preferably IgG1 in which the light chain is a λ chain and the heavy chain is a γ1 chain.

[0222] The amino acid sequence of IL-33 differs depending on the animal species, and thus, there is a difference in the amino acid sequence between human IL-33 shown as SEQ ID NO: 226 in the sequence listing and monkey IL-33 shown as SEQ ID NO: 227 in the sequence listing. In general, monkeys are used as experimental materials in pharmacological tests and safety tests of antibody medicines, and thus, the human anti-IL-33 neutralizing monoclonal antibody of the present application is preferably also bound to monkey IL-33, and more preferably bound to monkey IL-33 with the same degree of affinity as human IL-33. It is particularly preferred that the ratio of koff for monkey IL-33 to koff for human IL-33 with respect to the human anti-IL-33 neutralizing monoclonal antibody be within about 20 times, more preferably within about 10 times, and even more preferably within about 5 times.

[0223] As the fragment of the antibody of the present application, for example, a Fab fragment, an Fv fragment, an F(ab')2 fragment, a Fab' fragment, and an scFv can be given, and these fragments of the antibody can be bound to a functional molecule other than an antibody, such as a non-peptidic polymer such as polyethylene glycol (PEG), a radioactive substance, a toxin, a low-molecular compound, a cytokine, an albumin, an enzyme, and the like.

[0224] For the human anti-IL-33 neutralizing monoclonal antibody of the present application, a multispecific antibody such as a bispecific antibody can be prepared by combining it with another antibody having antigen-binding specificity other than IL-33. As the other antigen other than IL-33, there is no limitation, and TNF-a, IL-6 receptor, CD3, CD20, a4 integrin, BLys, Thymic Stromal Lymphopoietin, IgE, IL-1, IL-2, IL-4, IL-5, IL-6, IL-13, IL-17, IL-23, IL-25, and the like can be mentioned.

[0225] In the human anti-IL-33 neutralizing monoclonal antibody and fragments of the antibody of the present application, by changing the Fc region or the like, a functionally modified antibody that modulates functions such as cytotoxicity, complement activation function, blood half-life, and the like can be obtained (Setoguchi, Yakugaku Zasshi, 2009, Vol. 129(1), p3; Ishii Akiko et al., Nihon Yakurigaku Zasshi, 2010, Vol. 136(5), p280; Hashimoto, Shuhei et al., Shikagaku, 2010, Vol. 82(8), p710; Strohl, Current Opinion in Biotechnology, 2009, vol. 20, p685).

[0226] The human anti-IL-33 neutralizing monoclonal antibody and fragments of the antibody of the present application can bind to other functional molecules to form a conjugated antibody, for example, can bind to a non-peptide polymer such as polyethylene glycol (PEG), a radioactive substance, a toxin, a low molecular compound, albumin, a cytokine, an enzyme, and the like, and add a new function.

[0227] As other aspects of the present application, the following can be mentioned: a nucleic acid molecule encoding a protein portion of a human anti-IL-33 neutralizing monoclonal antibody whose framework region is an amino acid sequence of a germ cell line; a vector containing the nucleic acid molecule, a host cell containing the vector, and a method for producing a human anti-IL-33 neutralizing monoclonal antibody by culturing the host cell.

[0228] Another aspect of the present application includes a composition containing the above-described human anti-IL-33 neutralizing monoclonal antibody. Since IL-33 induces inflammation and the like, the human anti-IL-33 neutralizing monoclonal antibody is expected to have a use for the diagnosis, treatment, prevention, or alleviation of IL-33-related diseases. Thus, one aspect of the present application relates to a pharmaceutical composition containing the human anti-IL-33 neutralizing monoclonal antibody for the diagnosis, treatment, prevention, or alleviation of IL-33-related diseases. Since IL-33 induces cytokines, chemokines, inflammatory mediators, and the like, another aspect of the present application includes an expression inhibitor of cytokines, chemokines, or inflammatory mediators, the inhibitor containing the human anti-IL-33 neutralizing monoclonal antibody.

[0229] The cytokine inhibited by the expression inhibitor of cytokines, chemokines, or inflammatory mediators of the present application is a cytokine induced by IL-33, and examples thereof include TNF-a, IFN-g, IL-1b, IL-3, IL-4, IL-5, IL-6, IL-13, and the like. The chemokine inhibited by the inhibitor is a chemokine induced by IL-33, and examples thereof include CXCL2, CCL2, CCL3, CCL6, CCL17, CCL24, and the like. The inflammatory mediator inhibited by the inhibitor is an inflammatory mediator induced by IL-33, and examples thereof include PGD2, LTB4, and the like. A particularly preferred aspect of the present application is an expression inhibitor of IFN-g, IL-5, IL-6, or IL-13, more preferably an inhibitor of the production of IL-6, containing the human anti-IL-33 neutralizing monoclonal antibody.

[0230] Another aspect of the present application relates to a pharmaceutical composition containing the monoclonal antibody of the present application. In addition, it relates to a method for the diagnosis, treatment, prevention, or alleviation of IL-33-related diseases, which includes the administration of the monoclonal antibody of the present application, and to the use of the monoclonal antibody of the present application for the manufacture of a medicament for the diagnosis, treatment, prevention, or alleviation of IL-33-related diseases.

[0231] As examples of IL-33 related diseases, without limitation, there can be mentioned asthma, atopic dermatitis, urticaria, hay fever, anaphylactic shock, sinusitis (including eosinophilic sinusitis), allergic encephalomyelitis, hypereosinophilic disorders, polymyalgia rheumatica, rheumatic heart disease, multiple sclerosis, arthritis (e.g., rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, Reiter's syndrome, etc.), systemic lupus erythematosus (including discoid lupus), pemphigus, pemphigoid, psoriasis, ankylosing spondylitis, hepatitis (e.g., autoimmune hepatitis, chronic active hepatitis, etc.), inflammatory bowel disease (e.g., ulcerative colitis, Crohn's disease, gluten-sensitive enteropathy, etc.), Sjogren's syndrome, autoimmune hemolytic anemia, autoimmune inflammatory eye disease, autoimmune neonatal thrombocytopenia, autoimmune neutropenia, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, autoimmune thyroiditis, polymyositis, dermatomyositis, myasthenia gravis, adrenalitis (e.g., autoimmune Addison's disease, etc.), alopecia greata, antiphospholipid syndrome, celiac disease-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), cold agglutinin disease, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis (e.g., IgA nephrophathy, etc.), Graves' disease, hyperthyroidism (i.e., Hashimoto's thyroiditis), idiopathic thrombocytopenia purpura (ITP), mixed connective tissue disease, type 1 or immune-mediated diabetes mellitus, pernicious anemia, polychrondritis, autoimmune polyglandular syndrome, stiff-man syndrome, vitiligo, sarcoidosis, polyendocrine autoimmune syndrome, other endocrine deficiencies, arteriosclerosis, liver fibrosis (e.g., primary biliary cirrhosis, etc.), lung fibrosis (e.g., idiopathic pulmonary fibrosis, etc.), chronic obstructive pulmonary disease (COPD), scleroderma (including CREST syndrome, Raynaud phenomenon, etc.), tubulointerstitial nephritis, dense deposit disease, acute kidney injury, myocarditis, cardiomyopathy, neuritis (e.g., Guillain-Barre syndrome, etc.), polyarteritis nodosa, cardiotomy syndrome, chronic inflammatory demyelinating polyneuropathy, IgA neuropathy, lichen planus, Meniere's syndrome, post-MI, uveitis, uveitis opthalmia, vasculitis, primary agammaglobulinemia, cancer (e.g.,Brain tumor, laryngeal cancer, oral and lip cancer, hypopharyngeal cancer, thyroid cancer, esophageal cancer, breast cancer, lung cancer, gastric cancer, adrenal cortex cancer, bile duct cancer, gallbladder cancer, liver cancer, pancreatic cancer, bladder cancer, large intestine cancer, uterine cancer, ovarian cancer, prostate cancer, testicular cancer, chronic lymphocytic leukemia, chronic myelocytic leukemia, Ewing's sarcoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, melanoma, mesothelioma, multiple myeloma, etc.), infection showing resistance to rejection from the immune system (e.g., severe acute respiratory syndrome (SARS)), lethal cytokine storm accompanying highly virulent influenza infection, and scurvy, can be preferably mentioned, asthma, atopic dermatitis, hay fever, anaphylactic shock, sinusitis including eosinophilic sinusitis, Crohn's disease, ulcerative colitis, arthritis, systemic lupus erythematosus, pemphigus, pemphigoid, scleroderma, ankylosing spondylitis, liver fibrosis including primary biliary cirrhosis, pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), acute kidney injury, vasculitis, and cancer, etc.

[0232] In the pharmaceutical composition containing the human anti-IL-33 neutralizing monoclonal antibody of the present application, in addition to the human anti-IL-33 neutralizing monoclonal antibody and salts thereof as active ingredients, pharmacologically allowable carriers, diluents, or excipients can be contained. Further, other active ingredients than the human anti-IL-33 neutralizing monoclonal antibody of the present application, such as anti-inflammatory agents, immunosuppressants, etc. can be contained. Such a composition is provided as a dosage form suitable for non-oral administration or oral administration, and non-oral administration is preferred from the viewpoint of use as an antibody medicine. As non-oral administration, for example, intravenous, intraarterial, subcutaneous, topical, intraperitoneal, intramuscular, transnasal, ophthalmic, transdermal, transmucosal, intrameningeal, transrectal, intramuscular, intracerebral administration, etc. can be mentioned, but are not limited to these administration methods.

[0233] The pharmaceutical composition can be formulated into a suitable dosage form according to the administration route thereof, and can be any dosage form such as an injection, a powder, a transfusion preparation, a granule, a tablet, a suppository, etc., and from the viewpoint of non-oral administration, an injection, a transfusion preparation, a powder for on-the-spot dissolution, etc. are preferred. In addition, these preparations can contain the following components: various auxiliary agents for pharmaceutical use, i.e., carriers, other auxiliary agents, such as stabilizers, preservatives, pain relievers, emulsifiers, and the like.

[0234] The human anti-IL-33 neutralizing monoclonal antibody of the present application can be provided by continuous infusion at intervals of, for example, 1 day, 1 week, 1 month, 1 time, or 1 to 7 times a year, or by administration. The administration can be provided intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracerebroventricularly, or by inhalation. Preferred dosage regimens include the maximum dosage or the highest administration frequency that avoids serious adverse side effects. The total amount per week is usually about 0.05 μg / kg or more of body weight, more usually at least about 0.2 μg / kg, most usually at least about 0.5 μg / kg, typically at least about 1 μg / kg, more typically at least about 10 μg / kg, most typically at least about 100 μg / kg, preferably at least about 0.2 mg / kg, more preferably at least about 1.0 mg / kg, most preferably at least about 2.0 mg / kg, ideally at least about 10 mg / kg, more ideally at least about 25 mg / kg, and most ideally at least about 50 mg / kg.

[0235] The human anti-IL-33 neutralizing monoclonal antibody of the present application is useful for diagnostic tests for detecting the expression of IL-33 in specific cells, tissues, or sera of, for example, patients with IL-33-related diseases. In the diagnostic use, as typical, the human anti-IL-33 neutralizing monoclonal antibody is preferably a conjugated antibody labeled with a detectable moiety.

[0236] As another aspect of the present application, there is provided an anti-IL-33 neutralizing monoclonal antibody that competes with an antibody having an amino acid sequence of a combination of specific complementarity-determining regions (C1, C8, C15, C17, or C18 in Table 1) or a combination of specific variable region amino acid sequences (V1, V8, V15, V17, or V18 in Table 2) for binding to IL-33.

[0237] The anti-IL-33 neutralizing monoclonal antibody that competes with the human anti-IL-33 neutralizing monoclonal antibody having an amino acid sequence of a combination of specific complementarity-determining regions or a combination of specific variable region amino acid sequences described above for binding to IL-33 can be obtained by screening the obtained anti-IL-33 antibodies by, for example, surface plasmon resonance (SPR) method after obtaining the anti-IL-33 antibodies by genetic engineering methods such as phage display, hybridoma methods, and the like.

[0238] By loading biotinylated human IL-33 protein (4 μg / mL) as a ligand on a sensor chip on which avidin was immobilized, 1300 to 1600 RU of human IL-33 protein was immobilized. Next, an arbitrary anti-IL-33 antibody (15 μg / mL) as an analyte was added to bind to the human IL-33 protein immobilized on the sensor chip. By repeating the above operation several times, a state in which all of the molecules of the human IL-33 protein on the sensor chip are bound to the arbitrary anti-IL-33 antibody (saturated state) was achieved, and the binding amount in the saturated state (saturated binding amount 1) was calculated.

[0239] The same experiment was performed using a human anti-IL-33 neutralizing monoclonal antibody of the present application containing an amino acid sequence of a combination of specific complementarity determining regions or an amino acid sequence of a combination of specific variable region amino acid sequences, and the binding amount in the saturated state (saturated binding amount 2) was calculated.

[0240] Next, after saturating the human IL-33 protein on the sensor chip with the human anti-IL-33 neutralizing monoclonal antibody of the present application containing an amino acid sequence of a combination of specific complementarity determining regions or an amino acid sequence of a combination of specific variable region amino acid sequences, an arbitrary anti-IL-33 antibody (15 μg / mL) as an analyte was added, and whether the analyte was bound to the human IL-33 protein to which the human anti-IL-33 neutralizing monoclonal antibody of the present application containing an amino acid sequence of a combination of specific complementarity determining regions or an amino acid sequence of a combination of specific variable region amino acid sequences was already bound in an additional manner was investigated.

[0241] The case where any anti-IL-33 antibody can bind to the human IL-33 protein of the human anti-IL-33 neutralizing monoclonal antibody having the amino acid sequence of the combination of the specific combination of the amino acid sequences containing the specific complementarity-determining region or the specific variable region amino acid sequence in an additional manner while having the saturation binding amount 1 calculated above is judged to be "non-competitive binding". On the other hand, the case where any anti-IL-33 antibody cannot bind to the human IL-33 protein of the human anti-IL-33 neutralizing monoclonal antibody having the amino acid sequence of the combination of the specific combination of the amino acid sequences containing the specific complementarity-determining region or the specific variable region amino acid sequence in an additional manner is judged to be "competitive binding". Alternatively, even in the case where any anti-IL-33 antibody can bind to the human IL-33 protein of the human anti-IL-33 neutralizing monoclonal antibody having the amino acid sequence of the combination of the specific combination of the amino acid sequences containing the specific complementarity-determining region or the specific variable region amino acid sequence in an additional manner, if the additional binding amount is lower than the saturation binding amount 1 and the difference is significant, the antibody is judged to be "competitive binding". The significant difference can be investigated by a usual detection method (e.g., Student's t-test), and the significant criterion is 5% or 1% or less.

[0242] The anti-IL-33 neutralizing monoclonal antibody which competitively binds to IL-33 with the human anti-IL-33 neutralizing monoclonal antibody having the amino acid sequence of the combination of the specific combination of the amino acid sequences containing the specific complementarity-determining region or the specific variable region amino acid sequence described above can be a mouse antibody, a human antibody, a rat antibody, a rabbit antibody, a goat antibody, a camel antibody, or an antibody derived from any animal, a chimeric antibody, a humanized antibody, or a combination of these antibodies.

[0243] The anti-IL-33 neutralizing monoclonal antibody which competitively binds to IL-33 with the human anti-IL-33 neutralizing monoclonal antibody having the amino acid sequence of the combination of the specific combination of the amino acid sequences containing the specific complementarity-determining region or the specific variable region amino acid sequence described above is preferably a chimeric antibody, a humanized antibody, or a human antibody, and most preferably a human antibody.

[0244] The anti-IL-33 neutralizing monoclonal antibody which competitively binds to IL-33 with the human anti-IL-33 neutralizing monoclonal antibody having the amino acid sequence of the combination of the specific combination of the amino acid sequences containing the specific complementarity-determining region or the specific variable region amino acid sequence described above is a fragment of an antibody. As examples of the fragment of an antibody, a Fab fragment, a Fv fragment, a Fab')2 fragment, a Fab' fragment, or an scFv, and preferably a fragment of an antibody to which PEG or the like is bound.

[0245] The following describes the method for producing the anti-IL-33 neutralizing monoclonal antibody or the like of the present application. If a genetic engineering method is used, the human anti-IL-33 neutralizing monoclonal antibody of the present application can be produced by the following method: a combination of a combination of a desired complementarity-determining region and a framework region, and a DNA sequence encoding a light chain variable region and a heavy chain variable region are integrated into an expression vector, the vector is transformed into a host cell, and the host cell is cultured (see, for example, Borrebaeck C.A.K. and Larrick J.W. THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990). In addition, by linking a DNA sequence encoding a constant region of a light chain and a heavy chain to a DNA sequence encoding a heavy chain variable region and a light chain variable region, respectively, a DNA sequence encoding a full length of a heavy chain and a full length of a light chain can be prepared. In the present application, the DNA sequence encoding the full length of the heavy chain and the full length of the light chain of the human anti-IL-33 neutralizing monoclonal antibody is preferably, for example, IgG1 having a lambda chain as a light chain, as shown in Table 5 below. However, when the antibody is produced by a genetic engineering method using an animal cell, the lysine residue at the C-terminal end of the heavy chain is sometimes deleted, and the three nucleotides "aag" at the 3' end of the nucleic acid sequence constituting the heavy chain shown in Table 5 (SEQ ID NOs: 254 to 277 in the sequence listing) can be removed from each of the heavy chain nucleic acid sequences.

[0246] [Table 5]

[0247] The following sequence numbers in Table 5 indicate the sequence numbers in the sequence listing

[0248]

[0249]

[0250] The production system for producing the monoclonal antibody can utilize an in vitro production system. As the in vitro production system, a production system using eukaryotic cells such as animal cells, plant cells, or fungal cells, a production system using prokaryotic cells such as bacterial cells of Escherichia coli, Bacillus subtilis, or the like can be mentioned. As the animal cells to be used, commonly used cells such as CHO, COS, myeloma, BHK, HeLa, Vero, and the like, insect cells, plant cells, and the like can be used, and 293 cells and CHO cells are preferable.

[0251] When producing a monoclonal antibody using the above-described in vitro production system, DNA encoding a heavy chain or a light chain of the monoclonal antibody can be separately incorporated into expression vectors and simultaneously transformed into a host, or DNA encoding the heavy chain and the light chain can be incorporated into a single expression vector and transformed into a host (see International Publication No. 94 / 11523).

[0252] As a vector that can be used in animal cells, pConPlus, pcDM8, pcDNAI / Amp, pcDNA3.1, pREP4, and the like are preferred, but the vectors are not limited to these.

[0253] The obtained monoclonal antibody can be purified until uniform. The isolation and purification of the monoclonal antibody can be performed using an isolation and purification method generally used for proteins. For example, the isolation and purification of the monoclonal antibody can be performed by appropriately selecting and combining a chromatography column such as affinity chromatography, a filter, ultrafiltration, salting-out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis, and the like (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988), but the method is not limited to the above-described methods. As a column for affinity chromatography, a Protein A column, a Protein G column, and the like can be given. As a column such as a Protein A column, Hyper D, POROS, Sepharose F.F. (Amersham Biosciences), and the like can be given.

[0254] A multispecific antibody such as a bispecific antibody can be prepared by combining the human anti-IL-33 neutralizing monoclonal antibody of the present application with another antibody having antigen-binding specificity other than IL-33. As a method for producing a bispecific antibody, a chemical method (Nisonoff, A. et al., Archives of biochemistry and biophysics., 1961, Vol. 90, p. 460-462, Brennan, M. et al., Science, 1985, Vol. 299, p. 81-83) is widely known. In this method, first, two kinds of antibodies are separately hydrolyzed using an enzyme, and then, the disulfide bond of the heavy chain of the antibody is cleaved using a reducing agent, and thereafter, the heterologous antibodies are mixed and reoxidized, thereby obtaining a bivalent reactive antibody. Recently, a production method using a crosslinking agent such as glutaraldehyde, a carbodiimide, and the like has been disclosed (Japanese Patent Application Laid-Open No. 2-1556). A method for producing a multispecific antibody such as a bispecific antibody by genetic engineering has also been established in the art.

[0255] For example, a desired bispecific antibody can be obtained by using a technique of DVD-Ig in which antigen-binding regions of two monoclonal antibodies are linked in series (Wu et al., Nature Biotechnology 25(11), 1290 (2007)), ART-Ig technology in which heavy chains of two antibodies that bind to different antigens are combined by changing the Fc region of the antibody (Kitazawa et al., Nature Medicine 18(10), 1570 (2012)).

[0256] If CHO cells in which a gene of a1, 6-fucosyltransferase (FUT8) is disrupted are used as host cells to produce a monoclonal antibody, the fucose content of sugar chains is reduced, and an antibody with improved cytotoxicity can be obtained, and if CHO cells into which a FUT8 gene is introduced are used as host cells to produce a monoclonal antibody, an antibody with low cytotoxicity can be obtained (International Publication No. 2005 / 035586, International Publication No. 2002 / 31140, International Publication No. 00 / 61739). In addition, by changing the amino acid residues of the Fc region, the complement activation function can be adjusted (U.S. Patent No. 6737056, U.S. Patent No. 7297775, U.S. Patent No. 7317091). Furthermore, by using a mutant of the Fc region with improved binding to FcRn, which is one of Fc receptors, prolongation of the half-life in blood can be expected (Hashimoto, M. et al., Shikagaku, 2010, Vol. 82(8), p710; Strohl, Current Opinion in Biotechnology, 2009, vol. 20, p685). These functionally modified antibodies can be produced by genetic engineering.

[0257] The human anti-IL-33 neutralizing monoclonal antibody of the present application can be conjugated with other functional molecules to prepare a conjugated antibody. For example, when PEG is conjugated to the antibody as a functional molecule, the molecular weight of PEG is not limited, and PEG having a molecular weight of 2,000 to 100,000 Da, more preferably 10,000 to 50,000 Da, can be used, and can be linear or branched. PEG can be conjugated to the N-terminal amino group of the amino acid of the antibody by using, for example, the NHS activity. When a radioactive substance is used as a functional molecule, 131I, 125I, 90Y, 64Cu, 99Tc, 77Lu, or 211At, etc. can be used. The radioactive substance can be directly conjugated to the antibody by the chloroamine T method, etc. When a toxin is used as a functional molecule, bacterial toxins (e.g., diphtheria toxin), plant toxins (e.g., ricin), low-molecular-weight toxins (e.g., gentamicin), maytansinoids, calicheamicin, etc. can be used. When a low-molecular-weight compound is used as a functional molecule, daunomycin, doxorubicin, methotrexate, mitomycin, neocarzinostatin, vindesine, and a fluorescent dye such as FITC, etc. can be mentioned. When an enzyme is used as a functional molecule, luciferase (e.g., firefly luciferase and bacterial luciferase; U.S. Patent No. 4737456), malate dehydrogenase, urease, peroxidase (e.g., horseradish peroxidase (HRPO)), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidase (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidase (e.g., uricase and xanthine oxidase, etc.), lactoperoxidase, microperoxidase, etc. can be used. As a linker used for chemically conjugating a toxin, a low-molecular-weight compound, or an enzyme, a divalent free radical (e.g., alkylene, arylene, heteroarylene), -(CR2) n O(CR2) n -(R is an arbitrary substituent) indicates a linker, a repeating unit of an alkoxy group (e.g., polyethyleneoxy, PEG, polymethyleneoxy, etc.), and an aminoalkyl group (e.g., polyethyleneamino, Jeffamine TM), and divalent acid esters and amides (examples include succinate, succinamide, diglycollate, malonate, and suberate). Methods of chemical modification for binding of functional molecules have been established in the art (D. J. King., Applications and Engineering of Monoclonal antibodies., 1998 T. J. International Ltd, Monoclonal Antibody-Based Therapy of Cancer., 1998 Marcel Dekker Inc; Chari et al., Cancer Res., 1992 Vol 152: 127; Liu et al., Proc Natl Acad Sci USA., 1996 Vol 93: 8681).

[0258] The anti-IL-33 neutralizing monoclonal antibody that competes for binding to IL-33 with the human anti-IL-33 neutralizing monoclonal antibody of the present application containing the amino acid sequence of the specific complementarity determining region (C1, C8, C15, C17, or C18 in Table 1) or the specific combination of the variable region amino acid sequence (V1, V8, V15, V17, or V18 in Table 2) can be a mouse antibody, a human antibody, a rat antibody, a rabbit antibody, a goat antibody, a camel antibody, and the like derived from any animal, or a combination of these antibodies, i.e., a chimeric antibody, a humanized antibody. These anti-IL-33 neutralizing monoclonal antibodies can be obtained using any known method, such as the hybridoma method, the phage display method, and the like genetic engineering methods, and are particularly preferably obtained by genetic engineering methods.

[0259] A chimeric antibody can be obtained by joining the DNA encoding the variable region of the antibody of non-human origin and the DNA encoding the constant region of the antibody of human origin, incorporating it into an expression vector, and introducing it into a host to produce it (see European Publication No. 125023, International Publication No. 92 / 19759).

[0260] A humanized antibody can be obtained by joining the complementarity determining region (CDR) of the antibody of non-human origin and the DNA of the region of the antibody of human origin encoding the other part, incorporating it into an expression vector, and introducing it into a host to produce it.

[0261] Human antibodies can be prepared using, for example, the procedures described in the Examples provided below. In addition, human antibodies can be prepared using the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983 Immunol Today 4: p72), and the EBV- hybridoma technique to produce human monoclonal antibodies (Cole et al., 1985, MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., p. 77) and the like. Furthermore, human antibodies can be produced by immunizing transgenic mice into which human antibody genes have been introduced and cultivating hybridomas by hybridizing the transgenic mice. As the transgenic mice, there can be mentioned HuMab (registered trademark) mice (Medarex), KMTM mice (Kirin Pharma), KM (FCyRIIb-KO) mice, VelocImmune mice (Regeneron), and the like.

[0262] As other modes of the present application, there can be mentioned artificial antibodies which compete with human anti-IL-33 neutralizing monoclonal antibodies of the present application having an amino acid sequence of a combination of specific complementarity determining regions (C1, C8, C15, C17, or C18 in Table 1) or a combination of specific variable region amino acid sequences (V1, V8, V15, V17, or V18 in Table 2) of the present application. As the artificial antibodies, there can be used, for example, the 10th unit of human fibronectin type III domain (FNfn10), and by introducing mutations in the BC, DE, and / or FG loop of the unit, artificial antibodies which bind to a desired target can be obtained. As the artificial antibodies, in addition to the extracellular domain of fibronectin, there can be used the Kunitz domain of serine protease inhibitor, ankyrin, a lipocalin, and the like. These artificial antibodies can be produced by a genetic engineering method in which a vector containing a nucleic acid molecule encoding the peptide is introduced into E. coli, yeast, or animal cells, and the culture supernatant of the host cells is cultivated and purified.

[0263] As artificial antibodies, instead of using the amino acid sequence of the specific protein or the portion thereof as described above, a low-molecular peptide molecule that specifically binds to the epitope of the present application like an antibody can be searched from a random sequence library in which amino acids are randomly combined (for example, Hipolito et al., Current Opinion in Chemical Biology, 2012 Vol 16: 196, Yamagishi et al., Chemistry & Biology, 2011 Vol 18: 1562). For such a peptide, in addition to the genetic engineering method, a chemical synthesis method such as fluorenylmethyloxycarbonyl method (Fmoc), tert-butyloxycarbonyl (tBoc) method, and the like can be used for the production.

[0264] [Combination of sequences of antibodies]

[0265] The combinations of the amino acid sequences of the complementarity-determining regions of the human anti-IL-33 neutralizing monoclonal antibodies described in the present application, i.e., C1 to C30 in Table 1, the combinations of the amino acid sequences of the variable regions, i.e., V1 to V30 in Table 2, the combinations of the nucleic acid sequences of the complementarity-determining regions, i.e., CN1 to CN30 in Table 5, and the combinations of the nucleic acid sequences of the antibodies, i.e., IGN1 to IGN30 in Table 5, each correspond to the sequences of the same clone, and the correspondence is shown in Table 6 below. For example, the combination of the amino acid sequences of the 6 complementarity-determining regions of the complementarity-determining regions of the clone A10-1C04 is C1, and this combination of the amino acid sequences of the complementarity-determining regions can be encoded by the 6 nucleic acid sequences of CN1. In addition, the amino acid sequences of the variable regions of the heavy chain and the light chain of this clone are 2 amino acid sequences of V1, and the amino acid sequences of the lambda light chain and the gamma heavy chain containing the variable region of V1 are encoded by 2 nucleic acid sequences of IGN1.

[0266] [Table 6]

[0267]

[0268] Examples

[0269] Hereinafter, the present application will be described in detail by way of examples, but the present application is not limited to the following examples unless otherwise specified.

[0270] Example 1: Acquisition of Anti-IL-33 Antibodies and Identification of Epitope Peptides

[0271] [Obtaining of antibodies]

[0272] The animals were immunized with human IL-33 protein, and monoclonal antibodies were obtained by preparing hybridomas using the spleen cells of the immunized animals. In addition, antibodies binding to human IL-33 protein were cloned by phage display technique using an antibody library of animals prepared using RNA recovered from the spleen cells of the immunized animals, and a human natural antibody library. In this way, eight (antibodies A to H) anti-IL-33 monoclonal antibodies were obtained.

[0273] [Peptide array scanning]

[0274] To identify the epitopes of the obtained IL-33 antibodies, peptide array scanning was performed to investigate the binding of partial peptides (20 residues in length) of human IL-33 to each antibody. In order to cover the main mature human IL-33 molecule, 16 peptides 20 amino acids in length (PEP11 to PEP26) were synthesized starting at positions 101 valine (V101) to 270 threonine (T270) from the N-terminus, with a 10-amino-acid staggered start position. The sequences and position relationships of these peptides are shown in Table 7.

[0275] [Table 7]

[0276]

[0277] Each peptide biotinylated at the N-terminus was immobilized as a ligand on a neutral avidin sensor chip of a Surface Plasmon Resonance (SPR) device (Bio-Rad, ProteOn XPR36). In addition, the N-terminus of mature human IL-33 (residues 112 to 270) as a positive control was added with an AviTag sequence, and the protein (hIL-33) AviTag sequence was specifically biotinylated using a biotin ligase reaction, and the protein as a ligand was immobilized on the SPR sensor chip. On the sensor chip on which the ligand had been immobilized, the measured antibody as an analyte, human IL-33 receptor protein (recombinant human ST2 Fc chimera) (Enzo Life Science, ALX-201-367-C050) or only buffer (0.05% Tween 20 / PBS) (antibody concentration: 10 μg / mL; flow rate: 100 μl / min) was flowed, and the above measured substance was allowed to bind to the chip, and the amount of the analyte (antibody amount) bound to the ligand on the sensor chip after washing was expressed as an RU value. The results are shown in Table 8. Figure 2 .

[0278] The order from the nearest to the farthest from the N-terminal side of the human IL-33 protein in the antibody epitope is as follows. Antibody A and antibody B bind to PEP12. Antibody C and antibody D bind to PEP14. Antibody E binds to both PEP16 and PEP17. Antibody F binds to PEP24. Antibody G and antibody H bind to PEP26. The commercially available anti-human IL-33 polyclonal antibody (R&D Systems, AF3625) binds to most of the 16 human IL-33 peptides used in the study. On the other hand, the human IL-33 receptor (ST2) binds to the human IL-33 protein but hardly binds to the human IL-33 peptides (PEP11 to PEP26), and in this experiment, it was not possible to know which part of IL-33 is important for binding to ST2. In addition, no binding to the ligand was confirmed in the sample of the buffer alone, mouse IgG (R&D Systems, MAB002). Comparing the binding of the antibodies to hIL-33 (residues 112 to residues 270) among the antibodies used, the result was that the binding to hIL-33 (residues 112 to residues 270) was in the order of antibody G, antibody H, antibody D, antibody E, antibody B, antibody A, antibody C, antibody F from the strongest to the weakest.

[0279] Example 2: Evaluation of IL-33 neutralizing activity of anti-IL-33 monoclonal antibodies - 1

[0280] As an index of the blocking effect against the binding of solid-phase human ST2 and human IL-33, the determination of the IL-33 neutralizing activity of antibody A, antibody B, antibody E, and antibody F was performed. Recombinant human ST2 Fc chimera (Enzo Life Science, ALX-201-367-C050) (1 pg / mL, 50 pL / well) diluted with phosphate buffered saline (PBS) was dispensed in a 96-well microplate (Nunc™, #442404) and left to stand overnight at 4°C. The next day, after the microplate was washed once with PBS containing 1% BSA (PBS-B), the same solution (250 pL / well) was added and left to stand for 2 hours at room temperature. Thereafter, a mixed solution of the test antibody diluted with PBS-B (final concentration: 10 pg / mL) and recombinant human IL-33 protein (ATGen, ILC0701) (final concentration: 1 pg / mL) (50 pL / well) was added and left to stand for 2 hours at room temperature. After the microplate was washed 5 times with PBS containing 0.1% Tween 20 (PBS-T), a goat anti-human IL-33 antibody (R&D Systems: AF3625, final concentration: 1 pg / mL, 50 pL / well) diluted with PBS-B was added and left to stand for 1 hour at room temperature. After the microplate was washed 5 times with PBS-T, a HRP-labeled rabbit anti-goat IgG antibody (In vitrogen: 61-1620, 50 pL / well) diluted 2000-fold with PBS-B was added and left to stand for 1 hour at room temperature. After the microplate was washed 5 times with PBS-T, SureBlue Reserve™ TMB Microwell Peroxidase Substrate (KPL: 5200-0006, 50 pL / well) was added and left to stand for 10 minutes at room temperature. After the reaction was stopped by adding 50 pL / well of 1 M phosphoric acid, the absorbance at 650 nm was measured using a microplate reader (Molecular Devices: SpectraMax® i3x). The absorbance of the sample was corrected by subtracting the absorbance of the blank sample (PBS-B only) and the absorbance of the control sample (PBS-B only). The IL-33 neutralizing activity of the test antibody was calculated by the following equation: IL-33 neutralizing activity (%) = (1 - (absorbance of sample - absorbance of blank sample) / (absorbance of control sample - absorbance of blank sample)) x 100 TMTMB Microwell Peroxidase Substrate (KPL: 52-00-01, 50 μL / well) was allowed to react for 20 minutes at room temperature. The reaction was stopped with TMB Stop Solution (KPL: 50-85-05, 50 μL / well), and the difference in absorbance at wavelengths of 450 nM and 620 nM was measured using a microplate reader (SPECTRA MAX 190, Molecular Devices). The blocking of the binding of the antibodies to ST2 and IL-33 (IL-33 / ST2 binding system competitive binding blocking rate) was calculated by the following method: the blocking rate (%) was calculated with respect to a sample in which only human IL-33 (final concentration: 1 μg / mL) was added, using a sample in which human IL-1β (PeproTech, 200-01B) was added instead of human IL-33 (final concentration: 1 μg / mL) as the background. As a result, antibody A (epitope: PEP12) was 66% blocked, antibody B (epitope: PEP12) was 55% blocked, antibody E (epitope: PEP16-17) was 0% blocked, and antibody F (epitope: PEP24) was 39% blocked, and of the four antibodies studied, all of the antibodies (antibody A, antibody B, and antibody F) except for antibody E exhibited a blocking rate of 30% or more at a final concentration of 10 μg / mL.

[0281] [Table 8]

[0282]

[0283] Example 3: Evaluation of IL-33 neutralizing activity of anti-IL-33 monoclonal antibodies - 2

[0284] Using normal human umbilical cord vein endothelial cells (HUVEC) (LONZA, CLC2517A), a determination of the IL-33 neutralizing activity of the test antibodies (antibodies A to H) was performed using the blocking of IL-6 production induced by human IL-33 as an index. HUVEC (6 x 10 3(0.2 mL / well) for 24 hours at 37°C. After 24 hours, the IL-6 concentration in the culture medium was measured using a commercially available ELISA kit (Thermo Scientific, EH2IL6). In addition, the viability of the cells at the time of collection of the culture medium was measured using a cell counting kit (Dojindo, 345-06463), and it was confirmed that the IL-6 production inhibitory effect did not result in a decrease in the number of viable cells. As the IL-33 neutralizing activity (HUVEC line IL-6 production inhibition rate) of the antibody to be tested, the inhibition rate (%) of IL-6 production relative to the sample treated with recombinant human IL-33 alone was calculated. As a result, antibody A (epitope PEP12) was 51% inhibited, antibody B (epitope PEP12) was 48% inhibited, antibody C (epitope PEP14) was 33% inhibited, antibody D (epitope PEP14) was 38% inhibited, antibody E (epitope PEP16-17) was 0% inhibited, antibody F (epitope PEP24) was 38% inhibited, antibody G (epitope PEP26) was 48% inhibited, and antibody H (epitope PEP26) was 56% inhibited, and all of the eight antibodies except for antibody E exhibited an inhibition rate of 30% or more (Table 9). Among these antibodies, the antibody that binds to an epitope selected from the group consisting of positions 111 to 130, positions 131 to 150, positions 231 to 250, and positions 251 to 270 of SEQ ID NO: 1 exhibited a large increase in neutralizing activity when the antibody concentration was set to 3, 10, and 30 μg / mL (for example, for antibody D, 23, 42, 61% inhibition, respectively), and thus it was found that the above-mentioned epitope is suitable for producing an antibody having an antagonist effect.

[0285] [Table 9]

[0286]

[0287] Antibody E, although it binds to hIL-33 Figure 2However, no neutralizing properties were observed (Tables 8 and 9). Patent Document 2 (WO2008 / 132709) describes three epitopes: epitope 1 (positions 155-198), epitope 2 (positions 165-188), and epitope 3 (positions 175-178). These epitopes have been confirmed to overlap with the epitope peptides (positions 151-180) of antibody E, which was confirmed to lack IL-33 neutralizing activity. Based on these results, it can be concluded that antibodies targeting the epitopes of Patent Document 2 cannot adequately block the binding of IL-33 to ST2 as a receptor, and therefore lack IL-33 neutralizing activity, or if they do, the activity is extremely low.

[0288] Theoretically, besides the quality of the epitopes, insufficient affinity could also be a reason why antibody E did not exhibit IL-33 neutralizing activity. However, there are clones such as antibody D, antibody G, and antibody H that, despite showing a weaker tendency to bind to hIL-33 compared to antibody E, still exhibit clear IL-33 neutralizing activity. Therefore, this possibility is low. Based on the above, it can be considered that when the purpose is to neutralize IL-33 as a cytokine, the epitopes described in Patent Document 2 are epitopes unrelated to IL-33 binding and IL-33 neutralizing activity, while the four epitopes (PEP12, PEP14, PEP24, and PEP26) discovered by the inventors are functional epitopes related to IL-33 binding and IL-33 neutralizing activity. Functional epitopes and their binding antibodies have high antagonistic activity against IL-33, while antibodies binding to non-functional epitopes have low or no antagonistic activity against IL-33.

[0289] Example 4: Mapping of the stereostructure of human IL-33 epitope peptide.

[0290] For the four epitope peptides mentioned above, the ideal epitope interface atoms (distance from the atoms constituting ST2) are specified for the generation of antibodies with antagonistic effects. The epitope peptides were located on the stereostructure of the human IL-33·human ST2 complex, specifically the atoms of IL-33 within the circle. However, the X-ray crystal structure of the human IL-33·human ST2 complex (Research Collaboratory for Structural Bioinformatics: PDB ID 4KC3) lacks a portion of the IL-33 protein structure, thus failing to represent the positions of all identified epitope peptides. Therefore, using the aforementioned X-ray crystal structure (4KC3) as a template, a homology model was constructed. Figure 3, the epitope peptides (PEP12, PEP14, PEP24, PEP26) of the antibodies confirmed to have neutralizing activity this time were positioned (using Accelrys' Discovery Studio 3.5). Figures 4-7 Figures 4-7 In the figure, human IL-33 is indicated in dark gray, and the epitope peptides are indicated in light gray. In order to clearly indicate the position of the contact surface of the receptor on the surface of the IL-33 protein, the interface atoms are indicated by large spheres. The results show that each of these epitope peptides (PEP12, PEP14, PEP24, PEP26) has an amino acid containing an interface atom. As the amino acid containing an interface atom, P118, I119, T120, Y122, L123, R124, S125, L126, S127, Y129, N130 of PEP12, D131, Q132, S133, T135, A137, L138, E139, S142, Y143, E144, I145, Y146, E148, D149, L150 of PEP14, D244, N245, H246 of PEP24, K266, L267, S268, E269 of PEP26 can be mentioned. It can be considered that, as the epitope to which an antibody having an antagonist action specifically binds, an epitope having an amino acid containing an interface atom is preferable.

[0291] Example 5: Acquisition of human anti-IL-33 antibodies (parental clones)

[0292] Using a phage display library of human scFv (BioInvent, n-CoDeR) (Soderlind et al., Nature biotechnology, 2000 Vol. 18(8), p852), two parental clones (scFv) (indicated as the molecular type scFv, the same is described below) (clone name: A00-0070, A00-0036) were acquired, which bind to mature IL-33 (residue 112 to residue 270), hinder the binding of IL-33 to ST2, and hinder the activity of IL-33 with the IL-33-induced IL-6 production of normal human umbilical vein endothelial cells (HUVEC) as an index. The base sequences of these antibodies were determined, and the amino acid sequences of the variable regions of the light chains and the heavy chains were determined. The combination of the amino acid sequences of the light chain and the heavy chain variable regions of A00-0070 and A00-0036 are V29 and V30 in Table 2, respectively.

[0293] Example 6: Determination of amino acid substitutions for improved complementarity determining regions

[0294] ​To improve the affinity to IL-33 and the physical properties (reduction of aggregation due to reduction of surface hydrophobicity and improvement of solubility) of the two parent clones, Fab ribosome display and Fab phage display were used to improve the complementarity determining regions. The improvement of the complementarity determining regions was performed in two stages. In the first stage, single amino acid substitutions were determined with the aim of improving the affinity to IL-33 and the physical properties. In the second stage, combinations of these single amino acid substitutions were determined (Fujino et al., Biochem. Biophys. Res. Commun., 2012 Vol. 428(3), p395).

[0295] Fab ribosome display vectors were constructed using the light chain and heavy chain variable regions of the two parent clones. Using these as templates, gene site-directed mutagenesis PCR and overlap extension PCR were used to perform multistage PCR to substitute each of the total of 20 natural amino acids for all of the amino acid residues of the six complementarity determining regions (LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, HCDR3) that constitute the antibody, thereby constructing a comprehensive single amino acid substitution mutant library. Using the Fab ribosome display method (Fujino et al., Biochem. Biophys. Res. Commun., 2012 Vol. 428(3), p395) using a reconstituted cell-free translation system, PURE system (Genefrontier, PUREfrex) (Shimizu et al., Nature Biotechnology, 2001 Vol. 19(8), p751), the comprehensive single amino acid substitution mutant library was repeatedly enriched several times using recombinant human IL-33 protein (ATGen, ILC0701) as bait. The base sequences of the light chain and heavy chain variable regions of each clone (Fab) (indicates that the molecular form is Fab, and the same is described below) contained in the library before enrichment (initial state after construction) and the library after enrichment were determined using a second-generation sequencer (Roche, 454). Sequence data of several thousand reads were obtained from each of the libraries before and after enrichment, and the presence frequencies of all of the single amino acid substitution mutants in the complementarity determining regions were calculated. Next, the change ratios (enrichment ratios) of the presence frequencies of all of the single amino acid substitution mutants in the library before enrichment and the library after enrichment were calculated, and the size of the enrichment ratio of the library enrichment was used as an index to determine the single amino acid substitutions useful for improving the affinity to human IL-33 protein. Further, the positions at which the amino acid substitutions were introduced in the custom library constructed in the second stage were determined based on the total number of these single amino acid substitutions and the distribution state in the amino acid sequence.

[0296] In the parent clone A00-0070, it was determined that the amino acid substitution was introduced into the 12th asparagine of LCDR1 (SEQ ID NO: 2 in the sequence table), the 4th glutamine of LCDR2 (SEQ ID NO: 11 in the sequence table), the 2nd serine, the 3rd tyrosine, the 6th serine of LCDR3 (SEQ ID NO: 23 in the sequence table), the 1st aspartic acid, the 5th asparagine of HCDR1 (SEQ ID NO: 43 in the sequence table), the 4th serine, the 5th serine, the 7th serine, the 9th isoleucine of HCDR2 (SEQ ID NO: 64 in the sequence table). In the parent clone A00-0036, it was determined that the amino acid substitution was introduced into the 9th asparagine, the 13th asparagine of LCDR1 (SEQ ID NO: 6 in the sequence table), the 6th arginine, the 7th leucine of LCDR2 (SEQ ID NO: 20 in the sequence table), the 1st alanine, the 9th alanine, the 10th valine of LCDR3 (SEQ ID NO: 40 in the sequence table), the 1st asparagine of HCDR1 (SEQ ID NO: 47 in the sequence table), the 4th serine, the 5th serine, the 6th serine, the 7th serine, the 8th tyrosine, the 9th isoleucine, the 10th tyrosine, the 11th tyrosine, the 13th aspartic acid, the 16th lysine of HCDR2 (SEQ ID NO: 64 in the sequence table), the 2nd glycine, the 5th histidine, the 6th aspartic acid of HCDR3 (SEQ ID NO: 78 in the sequence table).

[0297] In order to improve the physical properties, an identity model of the two parent clones was created using a protein structure analysis program (Accelrys, Discovery Studio), and the region in which the surface hydrophobicity was high in the complementarity determining region was predicted. Then, in order to reduce the surface hydrophobicity of such a region, in the parent clone A00-0070, it was determined that the amino acid substitution was introduced into the 3rd tyrosine of LCDR3 (SEQ ID NO: 23 in the sequence table), the 7th serine, the 9th isoleucine of HCDR2 (SEQ ID NO: 64 in the sequence table), and in the parent clone A00-0036, it was determined that the amino acid substitution was introduced into the 6th arginine, the 7th leucine of LCDR2 (SEQ ID NO: 20 in the sequence table), the 7th serine, the 8th tyrosine, the 9th isoleucine of HCDR2 (SEQ ID NO: 64 in the sequence table). From the data of the enrichment ratio in the mutation analysis using a comprehensive single amino acid substitution mutant library, the amino acid substitution of the above-mentioned site useful for maintaining the binding performance to the human IL-33 protein and reducing the surface hydrophobicity was determined.

[0298] Example 7: Preparation of a human anti-IL-33 antibody in which the complementarity determining region was improved

[0299] By combining a plurality of the above-mentioned useful amino acid substitutions aimed at improving affinity and improving physical properties, a comprehensive custom library for improvement of the complementarity-determining region was designed. Vectors for Fab ribosome display and Fab phage display were constructed, and using the Fab ribosome display vector as a template, a multi-stage PCR reaction was performed using gene site-directed mutation PCR and overlap extension PCR, and using the Fab phage display vector as a template, site-specific mutation introduction by the Kunkel method (Fellouse et al., J. Mol. Biol. 2007 Vol. 373, p924) was performed. Thus, the complementarity-determining region was randomized based on the above design, and a custom library for improvement of the complementarity-determining region was constructed. The Fab ribosome display library and the Fab phage display library were repeatedly enriched several times using human IL-33 protein and cynomolgus monkey (cynomolgus monkey) IL-33 protein (GenBank: EHH57404, residues 112 to 269 of SEQ ID NO: 227 in the sequence listing) as bait. In the latter stage of enrichment, negative selection was performed using a hydrophobic column carrier such as Octyl Sepharose (GE Healthcare) or Phenyl Sepharose (GE Healthcare) before binding to the IL-33 protein, and Fabs with high affinity to IL-33 protein and low surface hydrophobicity were enriched.

[0300] The recombinant protein used as bait was prepared by the following method. Mature human IL-33 (residues 112 to 270) and mature cynomolgus monkey IL-33 (residues 112 to 269 of SEQ ID NO: 227 in the sequence listing) having a 6His tag-AviTag added to the N-terminal side were inserted into pET30a(-), and an expression vector was constructed, and a recombinant protein was prepared. After pre-culturing Escherichia coli BL21 (DE3) strain containing the expression vector in 5 mL LB medium, 1 mL of the pre-culture was inoculated into 50 mL of expression medium (Merck, Overnight Express; kanamycin was added), and the expression culture was performed at 30°C / 200 rpm for about 18 hours. After washing and recovering the bacterial cells, the cells were lysed in BagBuster (Novagen), and the supernatant was recovered. The 6His tag-AviTag-cynomolgus monkey IL33 (residues 112 to 269) contained in the supernatant was purified using Ni-NTA Agarose (QIAGEN), and biotin modification specific to AviTag was introduced using a commercially available biotin ligase (Avidity, BirA).

[0301] Using the enriched library, a library of E. coli expressing Fabs by secretion was constructed, and using the culture supernatant of several hundred clones of E. coli, determination of the dissociation rate constant (koff) was performed using surface plasmon resonance (SPR) (Bio-Rad, ProteOn XPR36). As a ligand, the above-described human IL-33 protein (4 μg / mL) and the above-described cynomolgus IL-33 protein (4 μg / mL) were added to a sensor chip (Bio-Rad, NLC sensor chip), and 1300 to 1600 RU of the human IL-33 protein and 1100 to 1500 RU of the cynomolgus IL-33 protein were immobilized. Subsequently, as an analyte, the culture supernatant of E. coli was added, and a sensorgram was obtained with an association phase of 1 minute and a dissociation phase of 10 to 30 minutes. Using a SPR data analysis program (Bio-Rad, ProteOn Manager v3.1.0), interspot correction and blank correction of the sensorgram were performed, and koff was calculated by Langmuir off-rate analysis.

[0302] From the clones (Fabs) in which the complementarity determining regions were improved, 28 clones in which the affinity for the human IL-33 protein was improved and which had binding to the cynomolgus IL-33 protein were identified, and were used for advanced evaluation in Example 8 and thereafter (V1 to V28 in Table 2). As shown in Table 10, these clones (Fabs) exhibited high affinity (low Koff value) for the human and cynomolgus IL-33 proteins compared to the parent clone (Fab). There were no amino acid substitutions in the framework regions in the variable regions of these clones. Even if the same single amino acid substitution is present in the complementarity determining region, the effect of the increase in affinity differs between the single amino acid substitution mutant and the multiple amino acid substitution mutant, and thus, there are amino acid substitutions that, although the enrichment ratio in the comprehensive single amino acid substitution mutant library in the first stage was low, were present at a high frequency in the sequences of the 28 clones used for advanced evaluation, and conversely, there are amino acid substitutions that, although the enrichment ratio in the first stage was high, were present at a low frequency in the sequences of the 28 clones used for advanced evaluation.

[0303] [Table 10]

[0304]

[0305] Example 8: Preparation of IgG antibody

[0306] For 7 clones of human anti-IL-33 antibodies (A10-1C04, A23-1A05, A25-2C02, A25-3H04, A26-1F02, A00-0070, A00-0036), expression vectors for IgG expression mammalian cells were constructed by inserting DNA encoding the amino acid sequences of light and heavy chains downstream of the CMV promoter. The DNA sequences of the light chains of each clone were SEQ ID NOs: 228, 232, 239, 241, 242, 230, and 253, and the DNA sequences of the heavy chains were SEQ ID NOs: 254, 261, 262, 264, 265, 276, and 277, respectively, in the sequence listing. The above expression vectors were introduced into FreeStyle 293-F cells (Life Technologies) using a gene introduction reagent NeoFection-293-1 (Astec). After the introduction of the genes, the cells were cultured for 5 days, after which the culture supernatant was obtained. Stable expression strains of CHO cells were established by using the GS system (Lonza) using a pConPlus vector and CHOK1SV cells. The CHO cell stable expression strains were cultured from a concentration of 0.3 x 10 6 cells / mL using a WAVE Bioreactor SYSTEM 20 / 50EHT (GE Healthcare), and the culture solution containing the secreted IgG was recovered. The IgG was purified from the culture supernatant by affinity chromatography using an AKTA explorer 100 (GE Healthcare) and Protein A resin (GE Healthcare, HiTrap MabSelect SuRe). The IgG bound to the Protein A resin was eluted in an elution buffer at pH 3.2, and immediately after the pH was neutralized to about neutral, it was dialyzed against PBS (pH 7.2). For the purpose of improving the purity of the purification, the IgG after Protein A column purification was purified using CHT (ceramic hydroxyapatite Type I resin) (BIORAD). The IgG bound to the CHT was eluted in a gradient of NaCl concentration, and after the target fraction was recovered, it was dialyzed against PBS (pH 7.2). The antibody obtained by this purification method was used as the "neutralized purified antibody".

[0307] The purification method described above was carried out with the addition of a step of washing with 6 Column Volumes of 100 mM sodium carbonate buffer (pH 11.0) for 6 minutes before the step of elution from the Protein A resin. The antibody obtained by the purification method was taken as "alkali-purified antibody". The recovery rate in each step of the alkali-purified antibody is shown in Table 11. The alkali-purified antibody after purification was concentrated by centrifugation with VIVASPIN Turbo 15 30000 MWCO (Sartorius).

[0308] [Table 11]

[0309]

[0310] Example 9: Affinity for IL-33 protein

[0311] The dissociation constant (Kd) of the test antibody (IgG) (indicates the molecular type is IgG, described below the same) in PBS was measured by a kinetic exclusion assay (KinExA) to determine the affinity of the antibody against human IL-33 protein (Sapidyne, KinExA3200). The concentration of human IL-33 protein (ATGen, ILC0701) was titrated to a wide range (2-fold dilution by 12 stages, resulting in a concentration range of 1 to 2048 times, so that the upper limit of the final concentration is several nM to several tens of nM) against a certain concentration (final concentration of several tens of pM to several hundred pM) of the test antibody, a mixed sample of the test antibody and human IL-33 protein was prepared, and incubated at room temperature until the antigen-antibody reaction reached equilibrium. After reaching equilibrium, the presence of free anti-IL-33 antibody was measured using KinExA3200. Using the KinExA data analysis program (Sapidyne, KinExA Pro Software v3.5.3), the presence of anti-IL-33 antibody not bound to human IL-33 protein (vertical axis) and antigen concentration (horizontal axis) was plotted to fit the theoretical equation, and the Kd was calculated. 50 mg of Azlactone beads (Sapidyne) was suspended in 1 mL of coating solution (10 μg / mL human IL-33 protein (ATGen, ILC0701), 50 mM sodium carbonate, pH 9.6) and incubated at room temperature for 1 hour to prepare anti-IL-33 antibody-captured glass beads. The detection antibody used anti-human F(ab)'2-DyLight649 (Jackson, 309-495-006). As shown in Table 12, when using a neutral purified antibody, the affinity of A23-1A05 against human IL-33 protein was weakest (Kd = 231 pM) and A25-2C02 was strongest (Kd = 720 fM) among the antibodies (A10-1C04, A23-1A05, A25-2C02, A25-3H04, A26-1F02) in which the complementarity determining region was improved.

[0312] Similarly, the affinities of the base purified antibodies against human IL-33 protein (residue 112 - residue 270) (ATGen, ILC0701) or full length human IL-33 protein were measured by KinExA assay (Table 12). The affinities against human IL-33 protein (residue 112 - residue 270) were as follows: A10-1C04 was Kd = 100.3 pM, A23-1A05 was Kd = 195.3 pM, A25-2C02 was Kd = 700 fM, A25-3H04 was Kd = 7.7 pM, A26-1F02 was Kd = 5.3 pM. The affinities against full length human IL-33 protein were as follows: A10-1C04 was Kd = 179.8 pM, A26-1F02 was Kd = 10.4 pM.

[0313] The recombinant protein used as a ligand was prepared by the following method. The full length human IL-33 protein to which NusA tag-6 His tag-TEV protease cleavage sequence was added at the N-terminal side was inserted into pET30a(+) to construct an expression vector, and a recombinant protein was prepared. After the E. coli BL21(DE3) strain containing the expression vector was pre-cultured in 50 mL of LB medium, it was inoculated at a density of OD = 0.5 and cultured at 37°C for 4 hours with shaking. After 4 hours, the culture temperature was changed to 13°C and cultured for 30 minutes with shaking, and then IPTG was added to a final concentration of 0.1 mM, and the culture was continued at 13°C for 72 hours with shaking to obtain full length IL-33 expressing E. coli. After the full length IL-33 expressing E. coli was lysed with BugBuster Master Mix (Novagen), centrifugal separation was performed to obtain a supernatant fraction. The recovered supernatant was subjected to IMAC purification using a HisTrap FF Crude column (GE Healthcare) and anion exchange purification using a CaptoQ Impress column (GE Healthcare) to improve the purity of the protein. The sample after anion exchange was concentrated by centrifugation using VIVASPIN6 (5,000 MWCO). In 1750 μL of the concentrated solution, 100 μL of Turbo TEV protease (Nacalai Tesque, Inc.) and 4.5 μL of 1M DTT were added and incubated at 4°C to remove the NusTag and HisTag. To remove the NusTag and Turbo TEV protease (HisTag fusion) contained in the sample after the removal of the label, it was subjected to a Ni Sepharose Excel column (GE Healthcare) and the effluent fraction was recovered. DTT was added to the effluent fraction to a final concentration of 3.3 mM, and it was used as a full length human IL-33 protein for the assay using KinExA.

[0314] [Table 12]

[0315]

[0316] *: Values in the table are dissociation constants of anti-IL-33 monoclonal antibody (IgG) against various ligands purified by a specific method

[0317] Example 10: Evaluation of in vitro human IL-33 neutralizing activity using HUVEC

[0318] The in vitro human IL-33 neutralizing activity of the test antibody (IgG) was evaluated using IL-33-induced IL-6 production by HUVEC as an index. As a positive control, a commercially available polyclonal anti-IL-33 antibody (R&D Systems, AF3625) was used. HUVEC (LONZA, CLC2517A) were suspended in EGM-2 medium (LONZA, CLCC-3156, CLCC-4176) and seeded at 6 x 10 3 / well) in a 96-well microplate (IWAKI) and confirmed that the cells became confluent. A mixed solution of anti-IL-33 antibody (final concentration of 1 μg / mL (about 6.7 nM)) and recombinant human IL-33 (ATGen, ILC0701) (final concentration of 100 ng / mL (about 5 nM)) was added to the medium and incubated at 37°C for 24 hours. The medium was collected and the IL-6 concentration in the culture supernatant was measured using a commercially available ELISA kit (Thermo Scientific, EH2IL6). In addition, the viability of the cells at the time of collection of the medium was measured using a cell counting kit (Dojindo, 345-06463) and it was confirmed that the IL-6 production inhibitory effect did not cause a decrease in the number of viable cells. As the IL-33 neutralizing activity of the test antibody, the inhibition rate (%) with respect to the IL-6 production induced by treatment with IL-33 alone was calculated. When a neutralized antibody was used, A10-1C04 was 67% inhibited, A23-1A05 was 74% inhibited, A25-2C02 was 96% inhibited, A25-3H04 was 97% inhibited, and A26-1F02 was 96% inhibited, all of which exhibited strong neutralizing activity, while A00-0070 as a parent clone was 4% inhibited and A00-0036 was -2% inhibited, both of which exhibited very weak neutralizing activity. By increasing the concentration to 10 μg / mL, A00-0070 was 42% inhibited and A00-0036 was 38% inhibited, both of which exhibited moderate neutralizing activity. On the other hand, when the commercially available polyclonal antibody (R&D Systems, AF3625) was added at a final concentration of 1 μg / mL, it exhibited moderate neutralizing activity of 30% inhibition.

[0319] ​Similarly, a mixed solution of the test antibody (final concentration: 0.1 to 10 μg / mL (about 0.67 to 67 nM)) and recombinant human IL-33 (ATGen, ILC0701) (final concentration: 100 ng / mL (about 5 nM)) was added to HUVEC as a neutralizing activity of the antibody, and the inhibitory effect (IC50 value) on IL-6 production induced by IL-33 alone was calculated. A10-1C04 was IC50 = 0.35 μg / mL, A23-1A05 was IC50 = 0.27 μg / mL, A25-2C02 was IC50 = 0.19 μg / mL, A25-3H04 was IC50 = 0.21 μg / mL, and A26-1F02 was IC50 = 0.23 μg / mL.

[0320] In addition, a mixed solution of the test antibody (final concentration: 0.1 to 3 μg / mL) and recombinant cynomolgus IL-33 (using the protein prepared by the method described in Example 7 without biotinylation) (final concentration: 100 ng / mL) was added to HUVEC as a neutralizing activity of the antibody, and the inhibitory effect (IC50 value) on IL-6 production induced by IL-33 alone was calculated. The IC50 of A10-1C04 was 0.43 μg / mL, and it was confirmed that A10-1C04 neutralized human IL-33 and cynomolgus IL-33 with the same strength.

[0321] Example 11: Evaluation of in vitro human IL-33 neutralizing activity using KU-812 cells

[0322] The in vitro human IL-33 neutralizing activity of the test antibody (IgG) was evaluated using IL-33-induced IL-5, IL-6, and IL-13 production in KU-812 cells as an index. As a positive control, a commercially available polyclonal anti-IL-33 antibody (R&D Systems, AF3625) was used. Human basophil cell line KU-812 cells (ECACC, EC90071807) were seeded in a 96-well microplate (Falcon) at 1 x 105 cells / well and incubated at 37°C for 24 hours in 5% CO2. 4IL-5, IL-6, IL-13 in RPMI-1640 medium containing 10% FBS. In addition, the survival rate of cells at the time of collecting the culture medium was measured using a cell counting kit (Dojindo, 345-06463), and it was confirmed that the production inhibitory effect of IL-5, IL-6, IL-13 did not cause a decrease in the number of surviving cells. In this evaluation system, the neutralizing activity of A26-1F02 against the production of IL-5, IL-6, IL-13 was 70%, 82%, 72%, respectively, and it was confirmed that A26-1F02 showed strong neutralizing activity against the production of any of the cytokines, compared to the commercially available polyclonal antibody (47%, 51%, 41% inhibition, respectively).

[0323] Similarly, a mixed solution of the test antibody (final concentration: 100 to 0.01 μg / mL (about 667 to 0.067 nM)) and recombinant human IL-33 (ATGen, ILC0701) (final concentration: 3 ng / mL (about 0.15 nM)), human IL-3 (PeproTech, 200-03) (final concentration: 10 ng / mL (about 0.67 nM)), and human complement C5a (Sigma-Aldrich, C5788) (final concentration: 1 nM) was added to KU-812 cells, and incubated at 37°C for 24 hours. The concentrations of IL-5 and IL-13 in RPMI-1640 medium containing 10% FBS were measured. In addition, the survival rate of cells at the time of collecting the culture medium was measured using a cell counting kit, and it was confirmed that the production inhibitory effect of IL-5 and IL-13 did not cause a decrease in the number of surviving cells. In this evaluation system, the test antibody (A10-1C04, A23-1A05, A25-2C02, A25-3H04, A26-1F02) showed more than 50% inhibition of the production of IL-5 and IL-13 at a final concentration of 1 μg / mL.

[0324] Example 12: Evaluation of in vitro human IL-33 neutralizing activity using human peripheral blood mononuclear cells

[0325] The in vitro human IL-33 neutralizing activity of the test antibody (IgG) was evaluated using the IL-33-induced IFN-γ production from human peripheral blood mononuclear cells (PBMC) as an index. As a positive control, a commercially available polyclonal anti-IL-33 antibody (R&D Systems, AF3625) was used. PBMC were prepared and seeded in a 96-well microplate (2 x 10 5 cells / well), and recombinant human IL-12 (and light pharmaceutical industry) was added (final concentration: 10 ng / mL). A mixture of the test antibody and recombinant human IL-33 protein (10 ng / mL) was added, and incubation was performed at 37°C for 48 hours. Thereafter, the culture supernatant was collected, and the amount of IFN-γ produced in the culture medium was determined using an AlfaLISA™ human IFN-γ immune assay kit (PerkinElmer) to evaluate the IL-33 neutralizing activity. The inhibition rate when the alkali-purified antibody was allowed to act at a final concentration of 10 μg / mL in this evaluation system was as follows: 96.9% inhibition for A10-1C04, 97.5% inhibition for A23-1A05, 98.75% inhibition for A25-2C02, 97.9% inhibition for A25-3H04, and 98.25% inhibition for A26-1F02.

[0326] Example 13: Evaluation of the effect on inflammation induced by intraperitoneal administration of human IL-33

[0327] By intraperitoneal administration of human IL-33 to mice, various inflammatory changes were induced. That is, an increase in IgE, IgA, IL-5 in blood, an increase in neutrophils, eosinophils, basophils, an increase in splenocytes (an increase in the weight of the spleen), and pathological changes in various mucosal organs were produced. Using these changes as an index, the anti-inflammatory effect in vivo of the test antibody (IgG) was evaluated.

[0328] To male C57BL6 (6-8 weeks old) (Charles River Japan), human IL-33 protein (R&D Systems, 3625-IL-010) was intraperitoneally administered at 0.4 μg / individual for 7 days (Day 0-Day 6). Subsequently, the test antibody (IgG) was intraperitoneally administered for 7 days (Day 0-Day 6). Seven days after the start of administration (Day 7), the spleen weight of the group to which PBS instead of human IL-33 protein (indicated as "vehicle" in the figure) was administered was 76 ± 4 mg on average, compared with 90 ± 7 mg on average for the group to which IL-33 protein was administered. In addition, the spleen weight of the group to which human control IgG (MP Biomedicals, 55908) (indicated as "mpk" in the figure) was intraperitoneally administered at 10 mg / kg in addition to IL-33 protein was 93 ± 4 mg on average, compared with 66 ± 3 mg on average for the group to which A26-1F02, a neutral purified antibody, was intraperitoneally administered at 10 mg / kg in addition to IL-33 protein.

[0329] Next, on the day before administration of human IL-33 protein (Day -1), the basic purified antibody was administered subcutaneously only once (sc, one shot) and evaluated. Seven days after the start of administration (Day 7), the spleen weight of the group to which PBS instead of human IL-33 protein was administered was 70 mg on average, compared with 152 mg on average for the group to which the above human control IgG (10 mg / kg) was subcutaneously administered in addition to IL-33 protein. In this regard, Figure 8 As shown, the spleen weight of the group to which A25-3H04 (1, 3, 5, 10 mg / kg) was subcutaneously administered in addition to IL-33 protein was 143, 106, 109, 78 mg, respectively, and the increase in spleen weight due to inflammation was inhibited in a dose-dependent manner by A25-3H04. As with the above anti-inflammatory effect on spleen weight, the increased IgA concentration in serum, IgE concentration in serum, number of neutrophils, number of basophils, number of eosinophils in blood, and IL-5 concentration in serum due to administration of human IL-33 were also inhibited by A25-3H04 Figure 8 ). From the above results, it was confirmed that A25-3H04 exhibited an inhibitory effect on IL-33-induced inflammatory reactions in vivo. In addition, the concentration of A25-3H04 in the blood of mice 7 days after the start of administration (Day 7) was measured, and as a result, the concentrations were 0.6, 3.7, 6.5, 20.3 μg / mL, respectively, corresponding to administration of 1, 3, 5, 10 mg / kg.

[0330] The in vivo anti-inflammatory effects of the other tested antibodies (IgG) were also evaluated by subcutaneous administration (10 mg / kg) in the same protocol. The results are shown in Figure 9 As shown, the spleen weight of the group to which human control IgG was subcutaneously administered was 181 mg on average, and in comparison, the spleen weight of the group to which each of the alkali-purified antibodies (A10-1C04, A23-1A05, A25-2C02, A26-1F02) was subcutaneously administered in addition to the administration of IL-33 protein was 82 mg, 92 mg, 100 mg, and 77 mg, respectively, and the increase in the spleen weight due to inflammation was inhibited. As with the above-mentioned anti-inflammatory effects on the spleen weight, the increased IgA concentration in the serum, IgE concentration in the serum, and the number of neutrophils, basophils, and eosinophils in the blood due to the administration of human IL-33 were also inhibited by the alkali-purified antibodies (A10-1C04, A23-1A05, A25-2C02, A26-1F02) (Fig. 6). Figure 9 Based on the above results, it was confirmed that A25-3H04 exhibited an inhibitory effect on IL-33-induced in vivo inflammation, and likewise, the other tested antibodies (A10-1C04, A23-1A05, A25-2C02, A26-1F02) also exhibited anti-inflammatory effects.

[0331] Example 14: Evaluation of the effects on lung disorders induced by intratracheal administration of human IL-33

[0332] IL-33 protein was intratracheally administered to mice, after which bronchoalveolar lavage fluid (BALF) was collected, and the total number of cells, the number of eosinophils, and the number of neutrophils in the BALF increased, and mucus hyperplasia of the tracheal epithelium was observed. In addition, cytokines such as IL-4, 5, 6, and 13 in the BALF were also produced. By intraperitoneally, subcutaneously, or intravenously administering the tested antibodies (IgG) to this system, the effects of the tested antibodies on lung disorders can be evaluated.

[0333] Example 15: Evaluation of the effects on airway hypersensitivity induced by intranasal administration of human IL-33

[0334] IL-33 protein was intranasally administered to mice, after which airway hypersensitivity was induced by inhaling acetylcholine. By intraperitoneally, subcutaneously, or intravenously administering the tested antibodies (IgG) to this evaluation system, the effects of the tested antibodies on airway hypersensitivity can be evaluated.

[0335] Example 16: Evaluation of the effects on IL-33 using human IL-33 knock-in mice

[0336] When the mite antigen or papain is administered intranasally or intratracheally to a mouse into which human IL-33 is embedded, tracheal inflammation is induced, and BALF is recovered from the mouse. The total number of cells in the BALF increases. With regard to tracheal inflammation caused by the mite antigen or papain, it is known that the protease activity of the mite antigen or papain causes IL-33 to be released from tracheal epithelial cells (Oboki et al., Proceedings of the National Academy of Sciences of the United States of America, 2010, vol. 107, p 18581). By administering the test antibody (IgG) intraperitoneally, subcutaneously, or intravenously to the evaluation system, the effect of the test antibody on tracheal inflammation caused by the protease and on in vivo-induced IL-33 can be evaluated.

[0337] Example 17: Evaluation of the effect on inflammation in a sepsis model caused by intraperitoneal administration of LPS

[0338] By administering LPS intraperitoneally to a mouse into which human IL-33 is embedded, sepsis is induced (Oboki et al., Proceedings of the National Academy of Sciences of the United States of America, 2010, vol. 107, p 18581), and the test antibody (IgG) is administered intraperitoneally, subcutaneously, or intravenously before the administration of LPS, and the effect of the test antibody on the mortality rate after the administration of LPS can be evaluated. In addition, inflammatory cytokines such as IL-6, TNF-a, and the like are detected at high concentrations in the blood within several hours after the administration of LPS, and by measuring these concentrations, the anti-inflammatory effect of the test antibody can be evaluated.

[0339] Example 18: Evaluation of the in vivo effect on cancer using a cancer-bearing mouse

[0340] A mouse cancer cell line, a human cancer cell line, is implanted into a mouse by the subcutaneous or intravenous route at a cell number appropriate for each cancer cell line, and human IL-33 is administered. The test antibody (IgG) is administered intraperitoneally, subcutaneously, or intravenously to the mouse, and after the implantation of the cancer cell line, the number of cancer cells in the organs as the primary cancer site and metastatic lesions is evaluated in terms of volume and cell number, and the effect of the test antibody on cancer can be evaluated.

[0341] Example 19: Evaluation of the colloidal stability of an antibody

[0342] The presence or absence of aggregates was analyzed by dynamic light scattering to evaluate the colloidal stability of the antibody (IgG) to be measured. Each of the alkali-purified antibodies was concentrated to about 50 mg / mL using VIVASPIN or VIVASPIN TURBO (Sartorius, 10,000 to 50,000 MWCO). Centrifugation was performed at 4°C, and the number of revolutions and the time were changed as appropriate. The antibody solution to be measured was sequentially diluted, and the particle diameter was measured using a dynamic light scattering device (Nanotrac UPA UT-151) using a sample of 200 to 250 μL, thereby obtaining data in a concentration range of about 1 mg / mL to about 50 mg / mL. The particle diameter distribution of the antibody protein was calculated from the cumulative data for 200 seconds, and the presence or absence of aggregates was evaluated. For the antibodies to be measured (A10-1C04, A23-1A05, A25-2C02, A25-3H04, and A26-1F02), the peak in the particle diameter distribution around 10 nM shifted to the high-particle-diameter side to a very slight extent as the antibody concentration increased, and in addition, there was no peak of several tens of nM or more in the particle diameter that was not dependent on the antibody concentration and derived from irreversible aggregates. Based on the above results, the good colloidal stability of the antibodies to be measured was confirmed.

[0343] To quantitatively evaluate the colloidal stability, the calculation of the interaction parameter (k D ) was performed. The interaction parameter, which indicates the concentration dependence of the diffusion coefficient (inversely proportional to the particle diameter), is an important index also used for the formulation design of high-concentration preparations of proteins such as antibodies. It has been reported that if the value of the interaction parameter is higher than -12.4 mL / g, the colloidal stability is excellent due to the repulsive interaction, and the self-association is low (Saito et al., Pharm. Res., 2013. Vol. 30 p1263). The antibody solution to be measured dissolved in PBS (pH 7.2) was concentrated to several 10 mg / mL using an ultrafiltration membrane, and the resulting sample was sequentially diluted 2-fold with the same solvent, and the particle diameter of the sample was measured using a dynamic light scattering measuring device (Nanotrac UPA UT151, Nikkiso). The diffusion coefficient was calculated from the obtained particle diameter by the following Stokes-Einstein equation.

[0344]

[0345] In the above equation, D is the diffusion coefficient (cm 2 / sec), KB is the Boltzmann constant (J / K), T is the thermodynamic temperature (K), π is the circular constant, η is the viscosity of the diluent (poise), and d is the particle diameter (nM). The concentration dependence of the diffusion coefficient was plotted, and the interaction parameter was calculated by fitting to the following calculation equation.

[0346] D = D0(1 + k D c)

[0347] D is a diffusion coefficient obtained by the Stokes-Einstein equation, D0is a diffusion coefficient at infinite dilution, and c is a concentration of the antibody to be measured at the time of measurement (g / mL). The slope of the fitting line, i.e., the interaction parameter (k D ) is calculated according to this equation. The results are as follows: A10-1C04 is k D = -8.1 mL / g (analytical range: 0.41-63.7 mg / mL), A23-1A05 is k D = -5.6 mL / g (analytical range: 0.40-61.8 mg / mL), A25-2C02 is k D = -6.2 mL / g (analytical range: 0.43-66.3 mg / mL), A25-3H04 is k D = -7.5 mL / g (analytical range: 0.34-56.5 mg / mL), A26-1F02 is k D = -6.7 mL / g (analytical range: 0.35-62.7 mg / mL), and the interaction parameter of any of the antibodies is higher than -12.4 mL / g, and the colloidal stability is excellent.

[0348] Example 20: Evaluation of the thermodynamic stability of the antibodies

[0349] The thermal stability of the test antibody (IgG) was evaluated at the temperature at which the folding of the immunoglobulin domain collapses (Tm). According to the attached file, Protein Thermal Shift Dye (Life Technologies) was added to a solution of the test antibody at about 10 μg / mL, the temperature was raised at a rate of about 1°C / min using Real-time PCR 7500 Fast (Life Technologies), and the fluorescence intensity was measured simultaneously. The Tm was determined by analyzing the data obtained with Protein Thermal Shift (Life Technologies). Note that when multiple Tm were confirmed, they were recorded in order of temperature from low to high as Tm1, Tm2. The results were as follows: when the neutral purified antibody was used, A10-1C04 was Tm = 73.9°C, A23-1A05 was Tm1 = 69.3°C, Tm2 = 77.6°C, A25-2C02 was Tm1 = 69.3°C, Tm2 = 80.3°C, A25-3H04 was Tm1 = 70.0°C, Tm2 = 76.4°C, and A26-1F02 was Tm = 74.5°C. In addition, when the alkali purified antibody was used, A10-1C04 was Tm = 73.7°C, A23-1A05 was Tm1 = 69.5°C, Tm2 = 77.5°C, A25-2C02 was Tm1 = 69.5°C, Tm2 = 80.4°C, A25-3H04 was Tm1 = 70.1°C, Tm2 = 76.4°C, and A26-1F02 was Tm = 74.4°C. The Tm of any of the antibodies was 65°C or higher, and good thermal stability was exhibited.

[0350] Example 21: Evaluation of storage stability of antibodies

[0351] To evaluate the storage stability of the test antibody (IgG), each of the alkali purified antibodies was dissolved in a citric acid buffer (50 mM citric acid, 150 mM NaCl (pH 6.3)) at a concentration of about 10 mg / mL, and stored at 40°C for 4 weeks. To evaluate the monomer purity of the antibody after storage, the monomer purity was measured by gel filtration analysis (SEC) and microchip capillary SDS electrophoresis (mCE-SDS), and the antigen binding activity was measured using surface plasmon resonance.

[0352] A column composed of two TSKgel G3000SWXL (Tosoh) connected in series was installed in an HPLC device (Beckman System Gold, 126 solvent manager, 166 detector, 508 auto sampler), and gel filtration analysis was performed. As a mobile phase solvent, 0.1 M phosphate buffer (pH 6.7) containing 0.1 M sodium sulfate was used, and separation was performed at a flow rate of 0.5 mL / min, and detection was performed at UV 215 nM. An antibody storage solution of about 10 mg / mL was diluted 100-fold, and 50 μL of this was injected as a sample for analysis. The monomer purity obtained by gel filtration analysis is shown in Table 13. Each of the antibodies tested (A10-1C04, A23-1A05, A25-2C02, A25-3H04, A26-1F02) maintained a monomer purity of 90% or more after storage at 40°C for 4 weeks, and exhibited good storage stability.

[0353] Capillary SDS electrophoresis was performed using Lab Chip GX II (PerkinElmer). Reduction was performed under denaturing conditions using the reagent kit HT Protein Express Reagent (PerkinElmer) dedicated to this device, according to the standard protocol of the manufacturer. As a sample for analysis, 2 μL of an antibody storage solution of about 10 mg / mL was added. The reagent for electrophoresis was taken from the above-mentioned kit, and measurement was performed according to the built-in protocol HT Antibody 200 for antibody analysis using the dedicated chip HT Protein Express Lab Chip, version 2 (PerkinElmer). As shown in Table 13, each of the antibodies tested (A10-1C04, A23-1A05, A25-2C02, A25-3H04, A26-1F02) maintained a monomer purity of 90% or more after storage at 40°C for 4 weeks even under denaturing and reducing conditions, and exhibited good storage stability.

[0354] To investigate whether irreversible aggregates were formed independent of the antibody concentration after storage, particle size measurement was performed. The antibody storage solution was diluted 10-fold (final concentration: about 1 mg / mL) with a citric acid buffer (50 mM citric acid, 150 mM NaCl (pH 6.3)), and particle size measurement was performed by dynamic light scattering method (Nanotrac UP AUT-151, Nikkiso) for the resulting analysis sample. Measurement was performed for 200 seconds of cumulative time. No aggregates were detected for any of the antibodies (A10-1C04, A23-1A05, A25-2C02, A25-3H04, A26-1F02, A00-0070, A00-0036) after storage at 40°C for 4 weeks, and antibodies with excellent storage stability were obtained.

[0355] To investigate the presence or absence of antigen-binding ability after storage, antigen-binding activity assay was performed using a surface plasmon resonance device Biacore T200 (GE Healthcare). Human IL-33 protein (ATGen, ILC0701) was immobilized on a Sensor Chip CM5 (GE Healthcare) using an amine coupling kit (GE Healthcare) (immobilization amount: about 3000 to 6000 RU). Next, the antibody storage solution was diluted 10-fold with citrate buffer (50 mM citric acid, 150 mM NaCl (pH 6.3)), and the total protein concentration in the solution was measured using a microabsorptiometer Astragene II (Astranet) (protein concentration: about 1 mg / mL). The antibody solution having the measured total protein concentration was diluted 1000-fold with HBS-EP buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% (v / v) Surfactant P20 (pH 7.4)) as an analyte solution. The assay was performed at 25°C. Each analyte solution was added for 36 seconds to obtain a sensorgram of the binding phase. The assay was performed at a flow rate of 5 μL / min, 100 μL / min, and the concentration of the antibody having antigen-binding activity was calculated from the sensorgrams obtained at the two flow rates by using Calibration Free Concentration Analysis using a data analysis program (GE Healthcare, Biacore T200 Evaluation Software v1.0). As a control, the antigen-binding activity of each of the measured antibodies after storage at 4°C for 4 weeks was also measured, and the proportion of the antigen-binding activity of the measured antibodies after storage at 40°C for 4 weeks was calculated. As shown in Table 13, each of the measured antibodies (A10-1C04, A23-1A05, A25-2C02, A25-3H04, A26-1F02) maintained a monomer purity of 90% or more after storage at 40°C for 4 weeks, and exhibited good storage stability.

[0356] Table

[13]

[0357]

[0358] Example 22: Evaluation of antibody stability using forced oxidation

[0359] The effect of oxidation on the antigen-binding activity of the test antibodies (IgG) was investigated. An aqueous hydrogen peroxide solution (final concentration 1%) was added to each of the purified antibodies in a final concentration of about 1 mg / mL, and allowed to oxidize at 37°C for 24 hours. After that, the oxidation was terminated by adding an 80 mM methionine solution. Next, the test antibody solution was exchanged to PBS using a desalting column Zeba spin (Thermo Scientific). The antigen-binding activity of the test antibodies subjected to the oxidation treatment was investigated using a surface plasmon resonance device Biacore T200 (GE Healthcare) in the same manner as in Example 21 described above. The proportion of the antigen-binding activity after the oxidation treatment relative to the antigen-binding activity of each of the test antibodies without the oxidation treatment was calculated, and as a result, in terms of the proportion of the retention of the binding activity, A10-1C04 was 83%, A23-1A05 was 95%, A25-2C02 was 100.5%, A25-3H04 was 98.7%, and A26-1F02 was 89.5%. According to these results, each of the test antibodies (A10-1C04, A23-1A05, A25-2C02, A25-3H04, A26-1F02) exhibited stability in which 80% or more of the antigen-binding activity was retained in the forced oxidation treatment with 1% aqueous hydrogen peroxide solution.

[0360] Example 23: Evaluation of aggregate formation caused by physical stress (agitation)

[0361] The test antibodies (IgG) were diluted to 0.2 mg / mL with PBS, and physical stress was applied by agitation in a batch cell installed in an Aggregates Sizer (Shimadzu Corporation). After the stirrer was moved up and down at room temperature for 30 minutes (190 times / minute), the aggregate concentration of 40 nM to 20 μΜ was measured with the Aggregates Sizer. When the purified antibodies were used, the aggregate concentration caused by agitation was as follows: A10-1C04 was 17.2 μg / mL, A23-1A05 was 16.4 μg / mL, A25-2C02 was 13.3 μg / mL, A25-3H04 was 23.4 μg / mL, and A26-1F02 was 17.0 μg / mL. The aggregate formation induced by physical stress was 15% or less for each of the antibodies, and each of the test antibodies was stable to physical stress.

[0362] Example 24: Evaluation of the time course of the concentration of antibodies in mouse blood

[0363] After the intravenous administration (3 mg / kg) of the test antibodies (IgG) labeled with fluorescence to male C57BL6 mice (8 to 10 weeks old) (Charles River Japan), the concentration of the test antibodies was measured by detecting the fluorescence in the plasma. As shown inFigure 10 As shown, the elimination half-life of any of the tested antibodies (A10-1C04, A23-1A05, A25-2C02, A25-3H04, A26-1F02) was 100 hours or more when the antibody was purified using a base, showing good stability in blood.

[0364] Example 25: Evaluation of concentration transition in monkey blood

[0365] After intravenous administration (1 mg / kg) of the tested antibody (IgG) to a male cynomolgus monkey (2-3 years old) (Hamri Co., Ltd.), the concentration of the tested antibody in serum was measured using a Human Therapeutic IgG1 EIA Kit (Cayman Chemical, 500910). The base-purified antibody A10-1C04 was administered to two cynomolgus monkeys (No. 201, 202), and the base-purified anti-A23-1A05 was administered to one cynomolgus monkey (No. 301). As shown in Figure 11 As shown, the elimination half-life of A10-1C04 was 16.56 days (No. 201) and 11.40 days (No. 202), and the clearance was 3.598 mL / day / kg (No. 201) and 5.451 mL / day / kg (No. 202). In addition, the elimination half-life of A23-1A05 was 10.87 days, and the clearance was 10.07 mL / day / kg. Any of the tested antibodies showed good stability in blood in cynomolgus monkeys.

[0366] Example 26: Evaluation of immunogenicity of antibodies

[0367] To evaluate the immunogenicity of the tested antibodies (IgG), an in vitro T cell assay (Lonza) was performed. To represent a target population, the number of providers was set to 50, and 50 μg / mL of each of the base-purified antibodies was added to the dendritic cells derived from human peripheral blood collected from the providers, so that the dendritic cells took up the antibodies. On the other hand, CD4-positive T cells derived from human peripheral blood collected from the same providers were isolated. Thereafter, the two, the dendritic cells that had taken up the tested antibodies and the CD4-positive T cells, were co-cultured, and the reaction (proliferation) of the CD4-positive T cells was measured. As a negative control, the same was performed using a buffer (PBS) containing no tested antibody, and the reaction of the CD4-positive T cells was obtained, and by comparing the two, the risk of immunogenicity when the antibody is administered to humans was evaluated. As a result, no difference in the reaction of T cells between any of the tested antibodies (A10-1C04, A25-2C02, A25-3H04, A26-1F02) and the negative control was confirmed.

[0368] Example 27: Evaluation of human tissue cross-reactivity

[0369] The cross-reactivity of the test antibodies (IgG) with human tissues (frozen sections of 35 tissues from the same provider, meeting the standards of FDA and EMA guidelines) was evaluated by immunohistochemical staining method (Covance Laboratories Ltd.). The 35 tissues included adrenal gland, bladder, blood cells, bone marrow, breast, cerebellum, cerebral cortex, colon, endothelial cells (blood vessels), eyeball, fallopian tube, gastrointestinal tract (including smooth muscle), heart, kidney (glomerulus, renal tubule), liver, lung, lymph node, ovary, pancreas, parathyroid gland, parotid gland, peripheral nerve, pituitary gland, placenta, prostate, skin, spinal cord, spleen, striated muscle, testis, thymus, thyroid gland, tonsil, ureter, uterus (cervix, endometrium). As a result, when using alkali-purified antibodies, strong staining was confirmed in the vascular endothelial cells (positive control) in which IL-33 was widely expressed, for any of the test antibodies (A10-1C04, A23-1A05, A26-1F02, A25-2C02). In addition, in various tissues such as epithelial cells, interstitial cells, nerve tissues, muscle tissues, and blood cells, cross-reactivity with the cytoplasm or nucleus was confirmed, but cross-reactivity with the cell membrane was not confirmed in any of the tissues. According to the ICH S6(R1) guidelines and other papers (Toxicologic Pathology 2010, 38(7): 1138-1166), cross-reactivity with the cytoplasm and nucleus, to which antibodies are difficult to reach in vivo, is not significantly meaningful in toxicology. Therefore, no toxicity concerns were found for any of the test antibodies (A10-1C04, A23-1A05, A26-1F02, A25-2C02).

[0370] Example 28: Screening of epitope region of A10-1C04 and A25-3H04

[0371] The anti-IL-33 monoclonal antibodies A10-1C04 and A25-3H04 bind to the PEP14 epitope described in Example 1 above. For shorter continuous amino acid sequences contained in the PEP14 consisting of 20 amino acid residues, two epitopes (LEDESYEIYV (SEQ ID NO: 426 in the Sequence Listing) and EDESYEIYV (SEQ ID NO: 427 in the Sequence Listing)) were found by conducting experiments using a phage display library exhibiting such amino acid sequences. The peptide LEDESYEIYV corresponds to residues 138 to 147 of human IL-33 shown in SEQ ID NO: 226 in the Sequence Listing, and the peptide EDESYEIYV corresponds to residues 139 to 147 of IL-33 shown in SEQ ID NO: 226 in the Sequence Listing. These peptides were synthesized, and Kd was calculated as the affinity to the base purified antibody by KinExA experiments identical to those in Example 9 (Table 14).

[0372] [Table 14]

[0373]

[0374] Industrial applicability

[0375] The antibody having a neutralizing action of the present application can be used as a pharmaceutical composition for the diagnosis, treatment, prevention, or alleviation of IL-33-related diseases.

Claims

1. An anti-IL-33 neutralizing monoclonal antibody, which is the following antibody (1) to antibody (10), Antibody (1): A human anti-IL-33 neutralizing monoclonal antibody A10-1C04, wherein, the amino acid sequence of the light chain complementarity determining region 1 (LCDR1) is SEQ ID NO: 1 of the sequence listing, the amino acid sequence of the light chain complementarity determining region 2 (LCDR2) is SEQ ID NO: 11 of the sequence listing, the amino acid sequence of the light chain complementarity determining region 3 (LCDR3) is SEQ ID NO: 22 of the sequence listing, and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 105 of the sequence listing; Antibody (2): the human anti-IL-33 neutralizing monoclonal antibody A23-1A05, wherein the amino acid sequence of the light chain complementarity determining region 1 (LCDR1) is SEQ ID NO: 3 of the sequence listing, the amino acid sequence of the light chain complementarity determining region 2 (LCDR2) is SEQ ID NO: 12 of the sequence listing, the amino acid sequence of the light chain complementarity determining region 3 (LCDR3) is SEQ ID NO: 25 of the sequence listing, and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 112 of the sequence listing; Antibody (3): the human anti-IL-33 neutralizing monoclonal antibody A25-3H04, wherein the amino acid sequence of the light chain complementarity determining region 1 (LCDR1) is SEQ ID NO: 4 of the sequence listing, the amino acid sequence of the light chain complementarity determining region 2 (LCDR2) is SEQ ID NO: 15 of the sequence listing, the amino acid sequence of the light chain complementarity determining region 3 (LCDR3) is SEQ ID NO: 32 of the sequence listing, and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 113 of the sequence listing; Antibody (4): the human anti-IL-33 neutralizing monoclonal antibody A25-2C02, wherein the amino acid sequence of the light chain complementarity determining region 1 (LCDR1) is SEQ ID NO: 4 of the sequence listing, the amino acid sequence of the light chain complementarity determining region 2 (LCDR2) is SEQ ID NO: 17 of the sequence listing, the amino acid sequence of the light chain complementarity determining region 3 (LCDR3) is SEQ ID NO: 34 of the sequence listing, and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 115 of the sequence listing; Antibody (5): the human anti-IL-33 neutralizing monoclonal antibody A26-1F02, wherein the amino acid sequence of the light chain complementarity determining region 1 (LCDR1) is SEQ ID NO: 6 of the sequence listing, the amino acid sequence of the light chain complementarity determining region 2 (LCDR2) is SEQ ID NO: 18 of the sequence listing, the amino acid sequence of the light chain complementarity determining region 3 (LCDR3) is SEQ ID NO: 35 of the sequence listing, and the amino acid sequence of the heavy chain variable region is SEQ ID NO: 116 of the sequence listing; Antibody (6): the human anti-IL-33 neutralizing monoclonal antibody A10-1C04, wherein the amino acid sequence of the heavy chain complementarity determining region 1 (HCDR1) is SEQ ID NO: 43 of the sequence listing, the amino acid sequence of the heavy chain complementarity determining region 2 (HCDR2) is SEQ ID NO: 51 of the sequence listing, the amino acid sequence of the heavy chain complementarity determining region 3 (HCDR3) is SEQ ID NO: 65 of the sequence listing, and the amino acid sequence of the light chain variable region is SEQ ID NO: 79 of the sequence listing; Antibody (7): the human anti-IL-33 neutralizing monoclonal antibody A10-1C04, wherein the amino acid sequence of the heavy chain complementarity determining region 1 (HCDR1) is SEQ ID NO: 43 of the sequence listing, the amino acid sequence of the heavy chain complementarity determining region 2 (HCDR2) is SEQ ID NO: 51 of the sequence listing, the amino acid sequence of the heavy chain complementarity determining region 3 (HCDR3) is SEQ ID NO: 65 of the sequence listing, and the amino acid sequence of the light chain variable region is SEQ ID NO: 79 of the sequence listing; A human anti-IL-33 neutralizing monoclonal antibody A23-1A05, wherein the amino acid sequence of the heavy chain complementarity determining region 1 (HCDR1) is SEQ ID NO: 47 of the Sequence Listing, the amino acid sequence of the heavy chain complementarity determining region 2 (HCDR2) is SEQ ID NO: 55 of the Sequence Listing, the amino acid sequence of the heavy chain complementarity determining region 3 (HCDR3) is SEQ ID NO: 66 of the Sequence Listing, and the amino acid sequence of the light chain variable region is SEQ ID NO: 83 of the Sequence Listing; Antibody (8): A human anti-IL-33 neutralizing monoclonal antibody A25-3H04, wherein the amino acid sequence of the heavy chain complementarity determining region 1 (HCDR1) is SEQ ID NO: 47 of the Sequence Listing, the amino acid sequence of the heavy chain complementarity determining region 2 (HCDR2) is SEQ ID NO: 56 of the Sequence Listing, the amino acid sequence of the heavy chain complementarity determining region 3 (HCDR3) is SEQ ID NO: 67 of the Sequence Listing, and the amino acid sequence of the light chain variable region is SEQ ID NO: 90 of the Sequence Listing; Antibody (9): A human anti-IL-33 neutralizing monoclonal antibody A25-2C02, wherein the amino acid sequence of the heavy chain complementarity determining region 1 (HCDR1) is SEQ ID NO: 49 of the Sequence Listing, the amino acid sequence of the heavy chain complementarity determining region 2 (HCDR2) is SEQ ID NO: 58 of the Sequence Listing, the amino acid sequence of the heavy chain complementarity determining region 3 (HCDR3) is SEQ ID NO: 69 of the Sequence Listing, and the amino acid sequence of the light chain variable region is SEQ ID NO: 92 of the Sequence Listing; Antibody (10): A human anti-IL-33 neutralizing monoclonal antibody A26-1F02, wherein the amino acid sequence of the heavy chain complementarity determining region 1 (HCDR1) is SEQ ID NO: 47 of the Sequence Listing, the amino acid sequence of the heavy chain complementarity determining region 2 (HCDR2) is SEQ ID NO: 59 of the Sequence Listing, the amino acid sequence of the heavy chain complementarity determining region 3 (HCDR3) is SEQ ID NO: 70 of the Sequence Listing, and the amino acid sequence of the light chain variable region is SEQ ID NO: 93 of the Sequence Listing; The sequence numbers below in Table 1 indicate the sequence numbers in the Sequence Listing The sequence numbers below in Table 2 indicate the sequence numbers in the Sequence Listing 。 2. The anti-IL-33 neutralizing monoclonal antibody of claim 1, wherein, The anti-IL-33 neutralizing monoclonal antibody is an antagonist of IL-33.

3. The anti-IL-33 neutralizing monoclonal antibody of claim 1, wherein, The anti-IL-33 neutralizing monoclonal antibody hinders the binding of an IL-33 receptor to IL-33.

4. A pharmaceutical composition for treating, preventing, or alleviating an IL-33- related disease, the composition containing the anti-IL-33 neutralizing monoclonal antibody according to any one of claims 1 to 3.

5. A cytokine expression inhibitor, the inhibitor containing the anti-IL-33 neutralizing monoclonal antibody according to any one of claims 1 to 3.

6. The inhibitor according to claim 5, which inhibits the expression of TNF-α, IFN-γ, IL-1β, IL-4, IL-5, IL-6, or IL-13.

7. The inhibitor according to claim 6, which inhibits the expression of IFN-γ, IL-5, IL-6, or IL-13.

8. The anti-IL-33 neutralizing monoclonal antibody of any one of claims 1-3, wherein, The anti-IL-33 neutralizing monoclonal antibody is a chimeric antibody, a humanized antibody, or a human antibody.

9. The anti-IL-33 neutralizing monoclonal antibody of claim 8, wherein, The amino acid sequence of the framework regions is that of the amino acid sequence of the framework regions of a human germ line or a combination thereof. The amino acid sequence of the framework regions is that of the amino acid sequence of the framework regions of a human germ line or a combination thereof.

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