Anti-IL-33 neutralizing monoclonal antibody

Abstract

To provide antibodies having an antagonistic action to IL-33, particularly isolated human anti-IL-33 neutralizing monoclonal antibodies or fragments of the antibodies, in which the amino acid sequence in a framework region is an amino acid sequence of a germline or an amino acid sequence of combination thereof.SOLUTION: Epitopes are identified for multiple anti-IL-33 monoclonal antibodies, a human anti-IL-33 neutralizing monoclonal antibody is obtained, further a mutation is introduced into its complementarity determining region, and thereby a complementarity determining region with high binding affinity to IL-33 is identified. By using the identified complementarity determining region, a human anti-IL-33 neutralizing monoclonal antibody in which the amino acid sequence of the framework region is the amino acid sequence of the germline amino acid sequence or a combination thereof is obtained.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a human anti-IL-33 neutralizing monoclonal antibody, an antibody that competes with said antibody, a cytokine expression inhibitor containing these antibodies, and a pharmaceutical composition for the treatment, prevention, or alleviation of IL-33-related diseases. [Background technology]

[0002] Interleukin-33 (IL-33) is a cytokine belonging to the interleukin-1 family, which is thought to play a role in inflammatory states. IL-33 is constitutively expressed in the nuclei of epithelial cells and vascular endothelial cells, and is released along with cell destruction due to infection or tissue damage caused by physical or chemical stress, functioning as an alarm. It is also thought that IL-33 expression can be increased and secreted in response to stimuli such as lipopolysaccharide. Extracellularly released IL-33 can activate intracellular signaling by binding to IL-33 receptors expressed on cells. IL-33 receptors are expressed in various immune system cells and epithelial cells, and IL-33-induced intracellular signaling occurs in these cells.

[0003] IL-33 is thought to induce allergic inflammation (asthma, atopic dermatitis, hay fever, anaphylactic shock, etc.) by inducing the production of Th2 cytokines (IL-4, IL-5, IL-6, IL-13, etc.) from immune system cells that express the IL-33 receptor, such as Th2 cells, mast cells, eosinophils, basophils, NK (natural killer) T cells, and group 2 innate lymphoid cells (Non-patent Literature 1: Tatsukuni Ohno et al., Allergy, 2012, Vol. 67, p1203). Furthermore, among immune system cells that express the IL-33 receptor, mast cells and macrophages are shown to induce the production of IL-1β, IL-6, and TNF (tumor necrosis factor)-α upon IL-33 stimulation, suggesting their involvement in the development of autoantibody-induced arthritis (a model of rheumatoid arthritis) (Non-patent Literature 2: Damo Xu et al., Journal of Immunology, 2010, Vol. 184, p2620). IL-33 antagonists have also been shown to be effective in treating acute kidney injury (Non-patent Literature 3: Ali Akcay et al., Journal of American Society Nephrology, 2011, Vol. 22, p2057).In humans, elevated IL-33 expression has been observed in various inflammatory diseases (rheumatoid arthritis, asthma, systemic sclerosis, hepatic fibrosis, pulmonary fibrosis and other fibrotic diseases, psoriasis, ulcerative colitis, Crohn's disease, multiple sclerosis, ankylosing spondylitis, etc.), and IL-33 is thought to be involved in the onset and maintenance of various diseases (Non-patent Literature 4: Yasushi Matsuyama et al., Journal of Rheumatology, 2010, Vol. 37, p18; Non-patent Literature 5: David Prefontaine et al., Journal of Allergy and Clinical Immunology, 2010, Vol. 125, p752; Non-patent Literature 6: Koichi Yanaba et al., Clinical Rheumatology, 2011, Vol. 30, p825; Non-patent Literature 7: AL Rankin et al., Journal of Immunology, 2010, Vol. 184). p1526;Non-patent document 8: Tamar Mchedlidze et al., Immunity, 2013, Vol. 39, p357;Non-patent document 9: Liang-An Hu et al., Asian Pacific Journal of Cancer Prevention, 2013, Vol. 14, p2563;Non-patent document 10: Luca Pastorelli et al., Proceedings of the National Academy of Sciences of the United States of America, 2010, vol. 107, p8017).

[0004] Given the known involvement of IL-33 in various diseases, particularly inflammatory diseases, the development of IL-33 agonists and antagonists has been underway (Patent Documents 1 to 4). Among these, antibodies against IL-33 have attracted particular attention due to their specificity and potency. However, the antibodies developed so far have been mouse antibodies without identified epitopes (Patent Document 1), or antibodies that identify the caspase-mediated cleavage site of IL-33 and identify that the active form is uncleaved IL-33, using the region containing such cleavage site (residues 155 to 198 of sequence number 226 in the sequence listing) as the epitope (Patent Document 2), or commercially available goat polyclonal antibodies. On January 10, 2014, AnaptysBio announced on its website that it had successfully developed ANB020, a candidate antibody for therapeutic development against IL-33, using its proprietary somatic hypermutation technology (SHM-XEL) ​​platform (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 / > Furthermore, Murphy et al. obtained 20 types of human anti-IL-33 monoclonal antibodies using VelocImmune mice, which are mice into which the variable region gene of a human antibody has been introduced (Patent Document 5). However, the epitopes of these antibodies are not disclosed, and the amino acid sequences of the framework regions of the 20 types of human anti-IL-33 monoclonal antibodies differ from the amino acid sequences of the human germline by two or more amino acid residues. Therefore, when these antibodies are administered to humans, an immune response to these antibodies is induced, leading to the induction of human anti-human immunoglobulin antibodies (HAHA), which can result in reduced efficacy, inflammation, and other side effects. [Prior art documents] [Patent Documents]

[0005]

Patent Document 1

Patent document 2

Patent document 3

Patent document 4

Patent document 5

Non-licensed literature

[0006] [Non-licensed document 1] Tatsukuni Ohno et al., Allergy, 2012, Vol. 67, p120 [Non-licensed document 2] Damo Xu et al., Journal of Immunology, 2010, Vol. 184, p2620 [Non-licensed document 3] Ali Akcay et al., Journal of American Society Nephrology, 2011, Vol.22, p2057

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

[0007] In recent years, the relationship between IL-33 and disease has become clearer, and anti-IL-33 neutralizing monoclonal antibodies that have antagonist activity against IL-33 are desired. The action of anti-IL-33 neutralizing monoclonal antibodies is closely related to the epitope region to which the antibody binds. Since IL-33 is released extracellularly along with cell destruction, it is highly likely to be cleaved by lysosomal proteolytic enzymes, etc., and so-called mature IL-33, and many fragments with IL-33 activity derived from mature IL-33, can be generated. Monoclonal antibodies that bind to epitopes consisting of a continuous amino acid sequence of IL-33 are advantageous because, if the fragment contains such an epitope, they bind more strongly to one of the continuous amino acid sequences of the fragment than monoclonal antibodies that bind to epitopes consisting of discontinuous amino acid sequences, thereby inhibiting the binding of the fragment to the IL-33 receptor. However, identifying epitopes consisting of such consecutive amino acid sequences for generating IL-33 monoclonal antibodies with the desired antagonist activity remained challenging.

[0008] For anti-IL-33 neutralizing monoclonal antibodies that bind to an epitope consisting of a continuous amino acid sequence of IL-33, it is desirable that they exhibit low antigenicity when administered to humans. Ideally, human antibodies should exhibit low antigenicity when administered to humans, and furthermore, their framework region should consist of an amino acid sequence of the human germline or a combination thereof. However, when applying the SHM-XEL platform to human antibodies contained in a human antibody gene library, amino acid sequence mutations occur not only in the complementarity-determining region but also in the framework region. Similarly, when obtaining human anti-IL-33 neutralizing monoclonal antibodies by immunizing mice into which human antibody genes have been introduced with human IL-33 protein, it is impossible to prevent amino acid sequence mutations from occurring in the framework region of the anti-IL-33 neutralizing monoclonal antibody. Therefore, obtaining isolated human monoclonal antibodies against IL-33 in which the amino acid sequence of the framework region consists of an amino acid sequence of the human germline or a combination thereof remains difficult. [Means for solving the problem]

[0009] The inventors of the present invention conducted diligent research to solve the above problems and found that antibodies that strongly bind to epitopes located at positions 155-198, which were previously considered preferred epitopes, have almost no antagonist activity. On the other hand, epitopes consisting of consecutive amino acid sequences located at positions 101-154 or 199-270, particularly those consisting of consecutive amino acid sequences located at positions 111-130, 131-150, 231-250, or 251-270, are important in terms of the antagonist activity of antibodies that bind to these epitopes, leading to the present invention.

[0010] Furthermore, the inventors isolated a human anti-IL-33 neutralizing monoclonal antibody from a human antibody library and, by introducing mutations only in its complementarity-determining region, identified a complementarity-determining region with high binding affinity and good physical properties. This allowed them to obtain a human antibody that does not contain mutations in the amino acid sequence of the germline framework region, yet binds to human IL-33 and neutralizes its function. Therefore, the present invention relates to the following:

[0011] [1] A monoclonal antibody that binds to an epitope consisting of a continuous amino acid sequence located at positions 101-154 or 199-270 of sequence number 226 in the sequence listing. [2] The antibody described in item 1, wherein the epitope consisting of consecutive amino acid sequences located at positions 101-154 or 199-270 of sequence number 226 in the sequence listing is the epitope consisting of consecutive amino acid sequences located at positions 111-130, 131-150, 231-250, or 251-270 of sequence number 226 in the sequence listing. [3] The antibody described in item 1 or 2, wherein the epitope consisting of a continuous amino acid sequence contained in positions 101-154 or 199-270 of sequence number 226 in the sequence listing is an epitope consisting of an amino acid sequence containing an amino acid selected from P118, I119, T120, Y122, L123, R124, S125, L126, S127, Y129, N130, D131, Q132, S133, T135, A137, L138, E139, S142, Y143, E144, I145, Y146, E148, D149, L150, D244, N245, H246, K266, L267, S268 and E269. [4] An antibody as described in any of items 1 to 3, wherein the epitope consisting of a continuous amino acid sequence contained in positions 101 to 154 or 199 to 270 of sequence number 226 in the sequence listing is the epitope consisting of a continuous amino acid sequence located at positions 111 to 130, 131 to 150, 231 to 250, or 251 to 270 of sequence number 226 in the sequence listing. [5] An antibody as described in any of items 1 to 4, wherein the epitope consisting of a continuous amino acid sequence located at positions 101 to 154 or 199 to 270 of sequence number 226 in the sequence listing is the epitope consisting of a continuous amino acid sequence located at positions 138 to 147 or 139 to 147 of sequence number 226 in the sequence listing. [6] A monoclonal antibody that binds to an epitope consisting of a continuous amino acid sequence located at positions 101-154 or 199-270 of sequence number 226 in the sequence listing, and is an IL-33 antagonist, as described in any of items 1-5. [7] A monoclonal antibody that binds to an epitope consisting of a continuous amino acid sequence located at positions 101-154 or 199-270 of sequence number 226 in the sequence listing, thereby inhibiting the binding of IL-33 receptor to IL-33, as described in any of items 1-6. [8] A pharmaceutical composition for the treatment, prevention, or reduction of IL-33-related disease, comprising an antibody described in any of items 1 to 7. [9] A cytokine expression inhibitor containing an antibody described in any of items 1 to 7.

[10] An inhibitor of TNF-α, IFN-γ, IL-1β, IL-4, IL-5, IL-6, or IL-13, as described in item 9.

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

[12] 1) Epitope listed in any of items 1-5, 2) An epitope consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, or added to a sequence of consecutive amino acids of the epitope, and 3) An epitope consisting of an amino acid sequence having at least 90% sequence identity with respect to the sequence of consecutive amino acids of the epitope. An epitope selected from a group consisting of the following.

[13] Antibodies generated or screened using the epitopes described in item 12.

[14] An antibody as described in any of items 1 to 7, wherein the monoclonal antibody that binds to an epitope consisting of a continuous amino acid sequence located at positions 101 to 154 or 199 to 270 of sequence number 226 in the sequence listing is a chimeric antibody, a humanized antibody, or a human antibody.

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

[16] The amino acid sequence of the light chain framework region 1 is from residues 1 to 22 of sequence number 317 in the sequence listing, the amino acid sequence of the light chain framework region 2 is from residues 36 to 50 of sequence number 317 in the sequence listing, the amino acid sequence of the light chain framework region 3 is from residues 58 to 89 of sequence number 317 in the sequence listing, and the amino acid sequence of the light chain framework region 4 is from residues 3 to 12 of sequence number 401 in the sequence listing, and the amino acid sequence of the heavy chain framework region 1 is from residues 1 to 367 in the sequence listing The antibody described in item 15, wherein the amino acid sequence of the heavy chain framework region 2 is residue 30 or residues 1 to 30 of sequence number 368 in the sequence listing, the amino acid sequence of the heavy chain framework region 3 is residues 36 to 49 of sequence number 367 or residues 36 to 49 of sequence number 368 in the sequence listing, the amino acid sequence of the heavy chain framework region 3 is residues 67 to 98 of sequence number 367 or residues 67 to 98 of sequence number 368 in the sequence listing, and the amino acid sequence of the heavy chain framework region 4 is residues 5 to 15 of sequence number 407 in the sequence listing.

[17] The antibody according to item 15 or 16, wherein the amino acid sequence of the light chain framework region 1 is residues 1 to 22 of sequence number 317 in the sequence listing, the amino acid sequence of the light chain framework region 2 is residues 36 to 50 of sequence number 317 in the sequence listing, the amino acid sequence of the light chain framework region 3 is residues 58 to 89 of sequence number 317 in the sequence listing, and the amino acid sequence of the light chain framework region 4 is residues 3 to 12 of sequence number 401 in the sequence listing, and the amino acid sequence of the heavy chain framework region 1 is residues 1 to 30 of sequence number 367 in the sequence listing, the amino acid sequence of the heavy chain framework region 2 is residues 36 to 49 of sequence number 367 in the sequence listing, the amino acid sequence of the heavy chain framework region 3 is residues 67 to 98 of sequence number 368 in the sequence listing, and the amino acid sequence of the heavy chain framework region 4 is residues 5 to 15 of sequence number 407 in the sequence listing.

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

[0012] [Table 1]

[0013]

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

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

[21] A human anti-IL-33 neutralizing monoclonal antibody as described in any of items 18-20, wherein the amino acid sequence of the antibody framework region is the amino acid sequence of the germline framework region or a combination thereof.

[22] The amino acid sequence of the light chain framework region 1 is residues 1 to 22 of sequence number 317 in the sequence listing, the amino acid sequence of the light chain framework region 2 is residues 36 to 50 of sequence number 317 in the sequence listing, the amino acid sequence of the light chain framework region 3 is residues 58 to 89 of sequence number 317 in the sequence listing, and the amino acid sequence of the light chain framework region 4 is residues 3 to 12 of sequence number 401 in the sequence listing, and the amino acid sequence of the heavy chain framework region 1 is residues 1 to 30 of sequence number 367 in the sequence listing or sequence number A human anti-IL-33 neutralizing monoclonal antibody as described in any of items 18-21, wherein the amino acid sequence of heavy chain framework region 2 is residues 36 to 49 of sequence number 367 or sequence number 368 in the sequence listing, the amino acid sequence of heavy chain framework region 3 is residues 67 to 98 of sequence number 367 or sequence number 368 in the sequence listing, and the amino acid sequence of heavy chain framework region 4 is residues 5 to 15 of sequence number 407 in the sequence listing.

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

[0014] [Table 2]

[0015]

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

[25] A human anti-IL-33 neutralizing monoclonal antibody as described in item 23 or 24, wherein the combination of 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.

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

[27] A human anti-IL-33 neutralizing monoclonal antibody described in any of items 18-26, wherein the human anti-IL-33 neutralizing monoclonal antibody is IgG.

[28] A human anti-IL-33 neutralizing monoclonal antibody as described in any of items 18-27, wherein the antigen is human IL-33 or monkey IL-33.

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

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

[0016] [Table 3]

[0017]

[31] A vector containing a nucleic acid molecule as described in item 29 or 30.

[32] Host cells containing the vector described in item 31.

[33] A method for producing a human anti-IL-33 neutralizing monoclonal antibody as described in any of items 18 to 28, wherein the antibody is produced by culturing the host cells described in item 32.

[34] A cytokine expression inhibitor comprising a human anti-IL-33 neutralizing monoclonal antibody as described in any of items 18-28.

[35] An inhibitor of TNF-α, IFN-γ, IL-1β, IL-4, IL-5, IL-6, or IL-13, as described in item 34.

[36] An inhibitor according to item 34 or 35, which suppresses the expression of IFN-γ, IL-5, IL-6, or IL-13.

[37] A pharmaceutical composition comprising a human anti-IL-33 neutralizing monoclonal antibody as described in any of items 18 to 28.

[38] The pharmaceutical composition described in item 37 for the prevention, treatment, or relief of IL-33-related diseases.

[39] The pharmaceutical composition according to item 38, wherein the IL-33-related 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, bullous pemphigoid, scleroderma, ankylosing spondylitis, hepatic fibrosis (including primary biliary cirrhosis), pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), acute kidney injury, vasculitis, and cancer.

[40] An anti-IL-33 neutralizing monoclonal antibody whose binding to IL-33 competes with the antibodies described in item 20 or 25.

[41] A method for treating, preventing or alleviating IL-33-related disease in patients, comprising administering an antibody described in any of items 1-7 and 18-28.

[42] The method according to item 41, wherein IL-33-related diseases are 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, bullous pemphigoid, scleroderma, ankylosing spondylitis, hepatic fibrosis (including primary biliary cirrhosis), pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), acute kidney injury, vasculitis, and cancer.

[43] Use of any of the antibodies described in items 1-7 and 18-28 for the manufacture of a medicine for the treatment, prevention, or reduction of IL-33 related disease.

[44] Use as described in item 43, where IL-33-related diseases are 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, bullous pemphigoid, scleroderma, ankylosing spondylitis, hepatic fibrosis (including primary biliary cirrhosis), pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), acute kidney injury, vasculitis, and cancer.

[45] Use of any of the antibodies described in items 1-7 and 18-28 for the treatment, prevention, or reduction of IL-33 related disease.

[46] Use as described in item 45, where IL-33-related diseases are 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, bullous pemphigoid, scleroderma, ankylosing spondylitis, hepatic fibrosis (including primary biliary cirrhosis), pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), acute kidney injury, vasculitis, and cancer.

[47] A method for treating, preventing or alleviating a patient requiring suppression of cytokine expression, comprising administering to the subject an antibody described in any of items 1-7 and 18-28.

[48] ​​The method according to item 47, wherein the cytokine is TNF-α, IFN-γ, IL-1β, IL-4, IL-5, IL-6, or IL-13.

[49] Use of any of the antibodies described in items 1-7 and 18-28 for the manufacture of cytokine expression inhibitors.

[50] The use described in item 49, wherein the cytokine is TNF-α, IFN-γ, IL-1β, IL-4, IL-5, IL-6, or IL-13. [Effects of the Invention]

[0018] The monoclonal antibody that binds to the epitope of the present invention binds to an epitope consisting of a continuous amino acid sequence. Therefore, even if IL-33 is degraded and fragmented, it can strongly bind to a single continuous amino acid sequence, thus easily exerting its neutralizing effect. The monoclonal antibody of the present invention is less likely to induce human anti-human immunoglobulin antibodies (HAHAs) against the antibody's framework region and / or complementarity-determining region when administered to humans. If not inhibited by HAHAs, its effect of neutralizing IL-33 in vivo persists. Furthermore, it is a safe antibody as long as it does not induce inflammation caused by binding to HAHAs. Because the monoclonal antibody of the present invention can bind to human IL-33 and neutralize its function, it can be used as a novel diagnostic, preventive, therapeutic, or mitigating agent for IL-33-related diseases. [Brief explanation of the drawing]

[0019] [Figure 1] Figure 1 illustrates the various domains and cleavage sites of the IL-33 protein. [Figure 2] Figure 2 shows the binding activity of each antibody to the human IL-33 protein (residues 112 to 270) and each partial peptide fragment (PEP11-26). [Figure 3] Figure 3 shows a model of the three-dimensional structure of the complex between mature human IL-33 (residues 117 to 270) (labeled "S117-T270" in the figure) and human ST2 (hST2). [Figure 4] Figure 4 shows only a portion of the PEP12 epitope of human IL-33 (positions 117-130 of sequence number 226 in the sequence listing) (indicated as "S117-N130" in the figure, and the same notation will be used hereafter) and human ST2 in the three-dimensional structural model of Figure 3. [Figure 5] Figure 5 shows only the PEP14 epitope and human ST2 of human IL-33 in the 3D structural modeling shown in Figure 3. [Figure 6] Figure 6 shows only the PEP24 epitope and human ST2 of human IL-33 in the 3D structural modeling shown in Figure 3. [Figure 7] Figure 7 shows only the PEP26 epitope and human ST2 of human IL-33 in the 3D structural modeling shown in Figure 3. [Figure 8]Figure 8 shows the effect of the human anti-IL-33 neutralizing monoclonal antibody A25-3H04 on inflammation induced by intraperitoneal administration of human IL-33, based on inflammation-related indicators (spleen mass, serum IL-5 concentration, serum eosinophil count, serum basophil count, serum neutrophil count, serum IgA concentration, serum IgE concentration). [Figure 9] Figure 9 shows the effects 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 indicators (spleen mass, serum eosinophil count, serum basophil count, serum neutrophil count, serum IgA concentration, serum IgE concentration). [Figure 10] Figure 10 shows the concentration profiles in mouse plasma of human anti-IL-33 neutralizing monoclonal antibodies (A23-1A05, A25-3H04, A26-1F02, A10-1C04, A25-2C02). [Figure 11] Figure 11 shows the concentration profiles of human anti-IL-33 neutralizing monoclonal antibodies (A10-1C04, A23-1A05) in monkey serum. [Modes for carrying out the invention]

[0020] To facilitate understanding of the present invention, the terms used in this invention are explained below.

[0021] [Epitope] In this invention, an epitope refers to a part of an antigen that is recognized by an antibody. In this invention, an epitope refers to a sequence of consecutive amino acids necessary for antibody recognition.

[0022] [Combine] In this invention, "binding" of a monoclonal antibody to an epitope means that the monoclonal antibody binds to the peptide, which is the epitope, to form a complex. The binding of the monoclonal antibody to the epitope is by ionic bonds, hydrogen bonds, hydrophobic bonds, van der Waals forces, etc., but is not limited to these. Whether a monoclonal antibody binds to an epitope can be investigated, for example, using peptide array scanning or KinExA as described herein.

[0023] [antibody] In this invention, the term "antibody" is used in its broadest sense and includes monoclonal antibodies and polyclonal antibodies as long as the desired specific binding affinity is demonstrated. The antibodies in this invention may be antibodies derived from any animal, such as mouse antibodies, human antibodies, rat antibodies, rabbit antibodies, goat antibodies, or camel antibodies.

[0024] [Monoclonal Antibody] Among the antibodies of the present invention, monoclonal antibodies refer to antibodies that consist of only a single clone (single molecular species) in their designed amino acid sequence. Monoclonal antibodies include chimeric antibodies, humanized antibodies, human antibodies, multispecific antibodies, and artificial antibodies, as well as functionally modified antibodies thereof, as well as their conjugate antibodies and their fragments. Monoclonal antibodies of the present invention can be produced using any known method, such as the hybridoma method, the phage display method, and genetic engineering techniques.

[0025] [Chimera antibody] A chimeric antibody is an antibody in which the light chain, heavy chain, or both are composed of a variable region of non-human origin and a constant region of human origin.

[0026] [Humanized antibodies] Humanized antibodies are antibodies that consist of a variable region comprising a complementarity-determining region of a non-human antibody and a framework region derived from a human antibody, as well as a constant region derived from a human antibody.

[0027] [Human antibodies] Human antibodies are antibodies whose light and heavy chains are derived from humans. Human antibodies include IgG (including IgG1, IgG2, IgG3, and IgG4) with a γ chain heavy chain, IgM with a μ chain heavy chain, IgA (including IgA1 and IgA2) with an α chain heavy chain, IgD with a δ chain heavy chain, or IgE with an ε chain heavy chain, depending on the difference in the constant region of the heavy chain. In principle, the light chain contains either a κ chain or a λ chain.

[0028] [Multispecific antibody] A multispecific antibody is an asymmetric antibody that possesses two or more independent antigen recognition sites, each with two or more different antigen specificities. Examples include a bispecific antibody with two antigen specificities and a trispecific antibody with three antigen specificities. In this invention, one or more antigens recognized by the multispecific antibody are IL-33 molecules.

[0029] [Artificial antibody] An artificial antibody is, for example, a protein scaffold, which does not have the structure of an antibody but has the same function as an antibody. Examples of protein scaffolds include the Kunitz domain of human serine protease inhibitors, the extracellular domain of human fibronectin, ankyrin, and lipocalin. By modifying the sequence of the target binding site on the scaffold, a protein scaffold that binds to the epitope of the present invention can be generated (Clifford Mintz et.al BioProcess International, 2013, Vol.11(2), pp40-48).

[0030] [Modified Antibodies] In this application, a functionally modified antibody refers to an antibody whose cell-killing function, complement activation function, blood half-life, and other functions other than the antigen-binding function are regulated by modifying the amino acids or sugar chains in the Fc region of the antibody.

[0031] [Conjugate antibody] In this application, a conjugated antibody refers to an antibody to which a functional molecule other than the antibody, such as a non-peptide polymer like polyethylene glycol (PEG), a radioactive substance, a toxin, a small molecule compound, a cytokine, albumin, or an enzyme, has been chemically or genetically engineered to it.

[0032] [Fragment] In this application, an antibody fragment refers to a protein containing a portion of an antibody that can bind to an antigen. Examples of antibody fragments include Fab fragments, Fv fragments, F(ab')2 fragments, Fab' fragments, or scFv. Furthermore, these antibody fragments may be chemically or genetically engineered to non-antibody functional molecules such as non-peptide polymers like polyethylene glycol (PEG), radioactive materials, toxins, small molecule compounds, cytokines, albumin, and enzymes.

[0033] [IL-33] IL-33 is a cytokine belonging to the IL-1 family, and human IL-33 consists of 270 amino acids, as shown in sequence number 226 of the sequence listing. IL-33 has a chromatin-binding domain at the N-terminus and an IL-1-like cytokine domain with a molecular weight of 18 kDa and 12 β-strands at the C-terminus. Furthermore, it has cathepsin G cleavage sites at positions 95 and 109, an esterase cleavage site at position 99, and a caspase cleavage site at position 178 (Figure 1). During the process of necrosis in cells, IL-33 is cleaved by enzymes such as esterase, cathepsin G, or proteinase 3, which originate from lysosomes, resulting in various fragments including mature IL-33, such as IL-33(residues 95 to 270) (IL-33 represented by the amino acid sequence from position 95 to 270 from the N-terminus of sequence number 226 in the sequence listing is denoted as "IL-33(residues 95 to 270)"; the same notation will be used hereafter), IL-33(residues 99 to 270), IL-33(residues 109 to 270), and IL-33(residues 112 to 270), which are thought to function as cytokines. On the other hand, when cell death is apoptosis, it is thought that IL-33 is cleaved at position 178 by caspases activated during the apoptosis process, resulting in inactive IL-33, such as IL-33(residues 179 to 270).

[0034] When IL-33 is released extracellularly as a cytokine, it binds to the IL-33 receptor and initiates intracellular signal transduction in cells expressing that receptor. IL-33-induced signal transduction includes, non-limited, the NF-κB pathway and the MAPKKs pathway, ultimately leading to the production of various cytokines, chemokines, and inflammatory mediators. Examples of IL-33-induced cytokines include TNF-α, IL-1β, IFN-γ, IL-3, IL-4, IL-5, IL-6, and IL-13, with IFN-γ, IL-5, IL-6, and IL-13 being particularly induced. Examples of IL-33-induced chemokines include CXCL2, CCL2, CCL3, CCL6, CCL17, and CCL24. Examples of IL-33-induced inflammatory mediators include PGD2 and LTB4. IL-33-induced cytokines, chemokines, and inflammatory mediators are involved in the migration of immune system cells, cytokine production, and degranulation, thereby inducing inflammation. In this invention, IL-33 may refer to full-length IL-33 or its active fragment, or derivatives or mutants thereof, as long as it acts by binding to the IL-33 receptor described later. Furthermore, it may be human IL-33 or IL-33 from another organism. Among these, human IL-33 represented by the amino acid sequence of Sequence ID No. 226 in the sequence listing is preferred.

[0035] 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, the site that specifically recognizes and binds to IL-33 is located in the extracellular domain of ST2. IL-33 receptors are expressed in various immune system cells (Th2 cells, mast cells, eosinophils, basophils, macrophages, dendritic cells, NK cells, NKT cells, group 2 innate lymphoid cells (natural helper cells), nuocytes, Ih2 (innate helper type 2) cells, etc.) and epithelial cells, but are not limited to these cells.

[0036] [IL-33 related diseases] In this application, IL-33-related diseases refer to diseases caused by the excessive extracellular release of IL-33, and IL-33-related diseases can be prevented, treated, or mitigated by drugs that can inhibit the function of IL-33. Examples of IL-33-related diseases include asthma, atopic dermatitis, hay fever, anaphylactic shock, sinusitis (including eosinophilic sinusitis), Crohn's disease, ulcerative colitis, arthritis, systemic lupus erythematosus, pemphigus, bullous pemphigoid, scleroderma, ankylosing spondylitis, hepatic fibrosis (including primary biliary cirrhosis), pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), acute kidney injury, vasculitis, and cancer.

[0037] [Framework domain] The framework region refers to the variable region of an immunoglobulin molecule, excluding the complementarity-determining region. The framework region comprises four areas in both the light and heavy chains (framework region 1, framework region 2, framework region 3, and framework region 4). In this application, the framework region of an immunoglobulin molecule is determined according to Kabat's numbering system (Kabat et al., 1987, Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA).

[0038] [Germ line] The germline refers to a group of germ cells such as sperm and eggs, and unless otherwise specified, it refers to the human germline. Unlike B lymphocytes that produce antibodies, the immunoglobulin genes in germ cells do not undergo mutations. Therefore, when it is written as "amino acid sequence of the germline framework region," it means that the amino acid sequence of the immunoglobulin framework region does not undergo mutations, and when it is written as "amino acid sequence of a combination of germline framework region amino acid sequences," it means that the amino acid sequence of one or more of the four framework regions is the amino acid sequence of a framework region of another germline. The gene for the light chain variable region of human immunoglobulin is divided into the Vκ segment and Jκ segment in the κ chain, and the Vλ segment and Jλ segment in the λ chain. Framework regions 1 to 3 are located in the Vκ segment and Vλ segment, and framework region 4 is located in the Jκ segment and Jλ segment. The gene for the heavy chain variable region of human immunoglobulin is divided into the VH segment, DH segment, and JH segment. Framework regions 1 to 3 are located in the VH segment, and framework region 4 is located in the JH segment. Table 4 shows the germline amino acid sequences of the Vκ, Vλ, VH, Jκ, Jλ, and JH segments of human immunoglobulin.

[0039] [Table 4-1]

[0040] [Table 4-2]

[0041] [Table 4-3]

[0042] [Table 4-4]

[0043] [Human monoclonal antibodies] A human monoclonal antibody refers to a monoclonal antibody having a variable region and a constant region derived from the sequence of human germline immunoglobulins. In this application, the variable region of a human monoclonal antibody may be a recombinant of part or all of the variable region of another human monoclonal antibody. In such recombinants, from the viewpoint of not affecting antibody binding, recombination may occur at the boundary between the framework region and the complementarity-determining region, and from the viewpoint of not increasing immunogenicity, each region of framework region 1 to framework region 4 may be recombined with each region of framework region 1 to framework region 4 of another human monoclonal antibody. Furthermore, in the present invention, a human monoclonal antibody may be a mutant of a human monoclonal antibody. In order to reduce immunogenicity while maintaining or improving antigen binding, a human monoclonal antibody is preferred that includes the amino acid sequence of the complementarity-determining region having a mutation in the complementarity-determining region of the human monoclonal antibody and the amino acid sequence of the germline framework region without a mutation in the framework region.

[0044] [Isolated] The term "isolated" in "isolated antibody" means that it has been identified, separated, and / or recovered from its natural state. Natural impurities are substances that may interfere with the diagnostic or therapeutic use of the antibody, and include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. Generally, an antibody can be isolated by purification through at least one purification step, and an antibody purified through at least one purification step can be called an "isolated antibody."

[0045] [Neutralization] In this application, "neutralization" refers to the action of binding to a target and inhibiting any of the functions of that target. That is, "anti-IL-33 neutralizing monoclonal antibody" means a monoclonal antibody whose binding to IL-33 results in the inhibition of the biological activity induced by the IL-33 polypeptide. Inhibition of IL-33's biological activity includes, but is not limited to, the inhibition of the production of IL-33-inducible cytokines such as IL-6. Indicators of IL-33's biological activity can be evaluated by one or more in vitro or in vivo analyses known in this art. Furthermore, when "human anti-IL-33 neutralizing monoclonal antibody" is used, it means a human monoclonal antibody that binds to IL-33 and inhibits any of its functions.

[0046] [Antagonist] In this application, "antagonist" refers to a general term for substances that have a neutralizing effect on a target. That is, an "IL-33 antagonist" is a substance that can bind to IL-33 and inhibit any of its functions, and includes, for example, an anti-IL-33 neutralizing monoclonal antibody.

[0047] [Complementarity Determination Area] The complementarity-determining region (CRI) is the variable region of an immunoglobulin molecule that forms the antigen-binding site. Also known as the hypervariable region, it refers to the part of the immunoglobulin molecule where the amino acid sequence changes particularly significantly. There are three CRIs in both the light chain and the heavy chain (CRI 1, CRI 2, and CRI 3). In this application, the CRI of an immunoglobulin molecule is determined according to Kabat's numbering system (Kabat et al., 1987, Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA).

[0048] [Competing] In the present invention, "competing" with a monoclonal antibody means that, when measured by the surface plasmon resonance (SPR) method described herein, the presence of the monoclonal antibody significantly reduces the binding to IL-33. In the present invention, "competing anti-IL-33 neutralizing monoclonal antibodies" include chimeric antibodies, humanized antibodies, human antibodies, multispecific antibodies, and artificial antibodies, as well as functionally modified antibodies thereof, as well as conjugate antibodies thereof, and fragments thereof.

[0049] The following describes embodiments of the present invention. Note that the following embodiments are illustrative examples for explaining the present invention, and the present invention is not limited to these embodiments.

[0050] This invention relates to a monoclonal antibody that binds to an epitope of IL-33. Since this monoclonal antibody that binds to the epitope can neutralize the activity of human IL-33, the epitope is preferably an amino acid sequence located at positions 101-154 or 199-270 of human IL-33 as shown in Sequence ID No. 226 of the sequence listing, and more preferably an amino acid sequence located at positions 111-130 (PEP12), 131-150 (PEP14), 231-250 (PEP24), or 251-270 (PEP26) of Sequence ID No. 226 of the sequence listing. IL-33 is often cleaved when released extracellularly. When amino acid residues separated on the primary sequence of IL-33 form an epitope based on protein folding, cleavage of IL-33 can disrupt the folding or cause the separated amino acid residues constituting the epitope to disappear from the fragment, significantly reducing its affinity to the fragment. Therefore, it is preferable that the epitope to which the anti-IL-33 monoclonal antibody binds is a continuous amino acid sequence.

[0051] For a monoclonal antibody that binds to an epitope to exert a neutralizing effect, it is necessary, for example, to prevent IL-33 from binding to the IL-33 receptor. Therefore, in the present invention, it is preferable that a preferred epitope is not only located on the surface of the IL-33 protein, but also in close proximity to the IL-33 receptor. Accordingly, the inventors performed three-dimensional structural modeling based on the crystal structure analysis data shown in Non-Patent Document 11, as described in the examples below, and identified amino acids containing IL-33 atoms (interfacial atoms) with an interatomic distance of 5 Å or less from the nearest atom constituting the IL-33 receptor. Examples of amino acids containing interfacial atoms include P118 (the proline residue at position 118 of sequence number 226 in the sequence listing is denoted as "P118," and this notation will be used hereafter), 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, and K266, L267, S268, E269 of PEP26. A functional epitope to which a monoclonal antibody capable of neutralizing IL-33 specifically binds is preferably one containing an amino acid with an interfacial atom. The neutralizing effect of a monoclonal antibody that specifically binds to a functional epitope is thought to depend on the number of interfacial atoms present in the functional epitope and their positions in the three-dimensional structure, but we do not intend to be bound by such theories.

[0052] A preferred embodiment of the present invention is a monoclonal antibody in which the epitope consisting of a continuous amino acid sequence contained in positions 101-154 or 199-270 of sequence number 226 in the sequence listing is an epitope consisting of a continuous amino acid sequence at positions 111-130 (PEP12), 131-150 (PEP14), 231-250 (PEP24), or 251-270 (PEP26) of sequence number 226 in the sequence listing. A more preferred embodiment of the present invention is a monoclonal antibody in which the epitope consists of a continuous amino acid sequence at positions 138-147 or 139-147 of sequence number 226 in the sequence listing.

[0053] The inventors investigated the minimum amino acid sequence constituting the epitope using two types of monoclonal antibodies that bind to PEP14, and identified the consecutive amino acid sequences at positions 138-147 and 139-147 of sequence number 226 in the sequence listing as the epitope for IL-33. Therefore, the present invention relates to an epitope consisting of the consecutive amino acid sequences at positions 138-147 and 139-147 of sequence number 226 in the sequence listing.

[0054] Whether a monoclonal antibody is a monoclonal antibody that binds to the epitope of the present invention can be determined by methods commonly used in the field of this art, such as ELISA, immunoprecipitation, surface plasmon resonance (SPR), and KinExA. For example, in the peptide array scanning method described in the present example using the SPR method, when tested using the epitope peptide of the present invention, the binding of the monoclonal antibody to the epitope can be measured as a significant increase in the RU value. In addition, the method described in the present example using the KinExA method can measure the dissociation constant (Kd), but a low dissociation constant for the epitope peptide is preferable, for example, preferably 10 μM or less, 1 μM or less, 100 nM or less, 10 nM or less, 100 pM or less, or 10 pM or less.

[0055] Another aspect of the present invention relates to a pharmaceutical composition comprising a monoclonal antibody that binds to an epitope consisting of a continuous amino acid sequence located at positions 101-154 or 199-270 of sequence number 226 in the sequence listing. The present invention also relates to a method for diagnosing, treating, preventing or alleviating IL-33-related disease, comprising administering the monoclonal antibody of the present invention, and to the use of the monoclonal antibody of the present invention for the manufacture of pharmaceuticals for the diagnosis, treatment, prevention or alleviation of IL-33-related disease.

[0056] Examples of IL-33-related diseases include, but are not limited to, asthma, atopic dermatitis, urticaria, hay fever, anaphylactic shock, sinusitis (including eosinophilic sinusitis), allergic encephalomyelitis, eosinophilic syndrome, polymyalgia rheumatica, rheumatic heart disease, multiple sclerosis, arthritis (e.g., rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, osteoarthritis, Reiter's syndrome, etc.), systemic lupus erythematosus (including lupus discoid), pemphigus, bullous pemphigoid, psoriasis, and ankylosing spine. 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.), Sjögren'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 resistance, alopecia areata Greata), antiphospholipid syndrome, autoimmune diseases of the adrenal glands (e.g., autoimmune Addison's disease), celiac pruritus dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), cold agglutinin disease, essential mixed cryoglobulinemia, fibromyalgia-fibromyitis, 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 mellitus, pernicious anemia, polychondritis (pol Polychrondritis, polyglandular syndrome, Stiffman syndrome, vitiligo, sarcoidosis, polyglandular endocrine disorders, other endocrine gland failures, arteriosclerosis, hepatic fibrosis (e.g., primary biliary cirrhosis), pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis), chronic obstructive pulmonary disease (COPD), scleroderma (including CREST syndrome, Raynaud's phenomenon, etc.), tubulointerstitial nephritis, dense deposit disease, acute kidney injury, myocarditis, cardiomyopathy, neuritis (e.g., Guillain-Barré syndrome, etc.), polyarteritis nodosa, cardiotomy syndrome, chronic inflammatory demyelinating polyneuropathy, IgA neuropathy, lichen planus, Meniere's disease, post-myocardial infarction (post-MI), uveitis, uveitis ophthalmitisOpthalmia, vasculitis, primary agammaglobulinemia, cancer (e.g., brain tumor, laryngeal cancer, lip and oral cancer, hypopharyngeal cancer, thyroid cancer, esophageal cancer, breast cancer, lung cancer, gastric cancer, adrenocortical carcinoma, bile duct cancer, gallbladder cancer, liver cancer, pancreatic cancer, bladder cancer, colorectal cancer, uterine cancer, ovarian cancer, prostate cancer, testicular cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, Ewing's tumor, Hodgkin's disease, non-Hodgkin lymphoma, melanoma, mesothelioma, multiple myeloma, etc.), infections resistant to elimination by the immune system (e.g., severe acute respiratory syndrome) Examples include (SARS), lethal cytokine storms associated with highly virulent influenza infections, and sepsis. Preferably, examples include asthma, atopic dermatitis, hay fever, anaphylactic shock, sinusitis (including eosinophilic sinusitis), Crohn's disease, ulcerative colitis, arthritis, systemic lupus erythematosus, pemphigus, bullous pemphigoid, scleroderma, ankylosing spondylitis, hepatic fibrosis (including primary biliary cirrhosis), pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), acute kidney injury, vasculitis, and cancer.

[0057] In yet another aspect of the present invention, the present invention also includes an inhibitor of cytokine, chemokine, or inflammatory mediator expression, which comprises a monoclonal antibody that binds to an epitope consisting of a continuous amino acid sequence located at positions 101-154 or 199-270 of sequence number 226 in the sequence listing.

[0058] The cytokines suppressed by the cytokine, chemokine, or inflammatory mediator expression inhibitor of the present invention are cytokines induced by IL-33, such as TNF-α, IFN-γ, IL-1β, IL-3, IL-4, IL-5, IL-6, and IL-13. The chemokines suppressed by the inhibitor are chemokines induced by IL-33, such as CXCL2, CCL2, CCL3, CCL6, CCL17, and CCL24. The inflammatory mediators suppressed by the inhibitor are inflammatory mediators induced by IL-33, such as PGD2 and LTB4. A particularly preferred embodiment of the present invention is an IFN-γ, IL-5, IL-6, or IL-13 expression inhibitor containing an anti-IL-33 monoclonal antibody, and more preferably an IL-6 production inhibitor.

[0059] In yet another aspect of the present invention, the present invention relates to an epitope to which an anti-IL-33 monoclonal antibody binds. In the present invention, the epitope relates to a sequence of 6 to 20 amino acids necessary for antibody recognition. In another aspect, further epitopes may be formed by including amino acids surrounding the identified sequence or amino acids in the same three-dimensional vicinity, but it is preferable that they be a continuous amino acid sequence that does not include discontinuous amino acid sequences.

[0060] Therefore, the number of amino acid residues in the continuous amino acid sequence constituting the epitope of the present invention is at least 5, preferably at least 6, more preferably at least 7, even more preferably at least 8, and even more preferably at least 9. Furthermore, from the viewpoint of exhibiting more sufficient antigenicity, it is at least 10, more preferably 15, and even more preferably at least 20. On the other hand, if the sequence contained in the epitope becomes long, there is a possibility that it will contain multiple regions that are recognized by the antibody, and in such cases, it may become impossible to produce or screen for antibodies that have the desired neutralizing effect. Therefore, from the viewpoint of ensuring that the antibody that binds to the epitope of the present invention exhibits the desired neutralizing effect, the length of the epitope sequence is preferably 30 or less, more preferably 20 or less, and even more preferably 15 or less. The number of residues in the continuous amino acid sequence contained in the epitope is, for example, a number of residues selected from one of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.

[0061] The epitope may have one or more amino acid mutations, i.e., amino acid substitutions, deletions, or insertions, introduced, as long as its antigenicity is not altered. The number of mutations introduced is preferably 5 or less, more preferably 3 or less, and most preferably 1. Furthermore, the epitope may have modifications, such as modifications to the sugar chains or terminal modifications that were present in the original protein. In another embodiment, the epitope may consist of an amino acid sequence having at least 90%, more preferably at least 95%, even more preferably at least 97%, even more preferably at least 98%, and most preferably 99% sequence identity with respect to the sequence of consecutive amino acids of the epitope identified in the present invention, as long as its antigenicity is not altered. When used as a bait, the epitope peptide may have tags such as histidine or biotin attached, and when used as a vaccine, it may be bound to a carrier protein such as KLH.

[0062] The "percent (%) sequence identity" of the identified reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of a particular reference polypeptide sequence, after the sequences have been aligned, gaps introduced if necessary to obtain the maximum percentage sequence identity, and any conservative substitutions are not considered part of the sequence identity. Alignment for the purpose of measuring percentage amino acid sequence identity can be achieved by various methods within the scope of the skill of a person skilled in the art, such as using publicly available computer software like BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. A person skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm necessary to achieve the maximum alignment for the full length of the sequences being compared. However, for the purposes of this text, the % amino acid sequence identity value is obtained by using the sequence comparison computer program BLAST in pairwise alignment. When BLAST is used for amino acid sequence comparison, the percentage amino acid sequence identity of a given amino acid sequence A with a given amino acid sequence B is calculated as follows: 100 times the fraction X / Y Here, X is the number of amino acid residues whose scores matched as identical by the program alignment of A and B using the sequence alignment program BLAST, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is different from the length of amino acid sequence B, the % amino acid sequence identity of A to B will be different from the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​here are obtained using the BLAST computer program as shown in the paragraph immediately above.

[0063] The epitope discovered by the present invention is a functional epitope to which an IL-33 neutralizing antibody specifically binds. Therefore, by utilizing the functional epitope of the present invention, it is possible to efficiently obtain, for example, a novel antibody having antagonist activity against IL-33. That is, by screening a group of monoclonal antibodies against full-length IL-33 or mature IL-33 for antibodies that bind to the functional epitope of the present invention, it becomes possible to obtain a monoclonal antibody having antagonist activity. Therefore, in a further aspect of the present invention, the present invention also relates to a method for screening antibodies having antagonist activity using a functional epitope of IL-33. More specifically, when enriching clones of antibodies with IL-33 antagonist activity from a naive antibody library using phage display technology, etc., first, library selection is performed using full-length or mature IL-33 protein as bait to enrich antibody clones that bind to various epitopes on the IL-33 surface. Then, library selection is performed using the functional epitope peptides discovered in this invention as bait, making it possible to efficiently screen for antibodies with IL-33 antagonist activity that specifically bind to functional epitopes.

[0064] In this example, the inventors investigated the IL-33 antagonist activity of a group of monoclonal antibodies whose binding affinity to 20-residue epitopes was identified, using them at varying antibody concentrations. This allowed them to determine epitopes suitable for generating or screening antibodies with antagonistic activity. The results showed that antibodies binding to epitopes selected from the group consisting of positions 111-130 (PEP12), 131-150 (PEP14), 231-250 (PEP24), and 251-271 (PEP26) of sequence number 226 in the sequence listing clearly increased antagonistic activity in proportion to the antibody concentration. Therefore, these were found to be functional epitopes suitable for generating or screening antibodies with antagonistic activity. Accordingly, one aspect of the present invention relates to an epitope comprising a sequence of at least 6, preferably at least 10, more preferably at least 15 consecutive amino acids from a region selected from the group consisting of positions 111-130, 131-150, 231-250, and 251-271 of sequence number 226 in the sequence listing. In another aspect, the present invention relates to an epitope selected from the group consisting of positions 111-130, 131-150, 231-250, and 251-270 of sequence number 226 in the sequence listing.

[0065] Epitopes can be manufactured using standard peptide synthesis techniques. The manufactured and purified epitopes can be used for animal immunization or to produce antibodies against the epitope. Alternatively, purified epitopes can be used in phage display to generate or screen monoclonal antibodies that bind to the epitope. Epitopes can also be used as vaccines in combination with adjuvants.

[0066] The present invention relates to a monoclonal antibody that binds to an epitope consisting of a continuous amino acid sequence located at positions 101-154 or 199-270 of sequence number 226 in the sequence listing. Monoclonal antibodies include chimeric antibodies, humanized antibodies, human antibodies, multispecific antibodies, and artificial antibodies, as well as functionally modified antibodies thereof, as well as their conjugate antibodies and their fragments. The monoclonal antibodies in the present invention may be antibodies derived from any animal, such as mouse antibodies, human antibodies, rat antibodies, rabbit antibodies, goat antibodies, and camel antibodies. The monoclonal antibodies of the present invention can be produced using any known method, such as the hybridoma method, phage display method, and genetic engineering methods.

[0067] In the hybridoma method, B cells collected from the spleen or lymph nodes of animals immunized with an immunogen, particularly rats or mice, are fused with immortalized cells, such as myeloma cells, to create hybridomas. Hybridomas with the desired binding affinity are then screened, and antibodies can be produced using the screened hybridomas. Human antibodies can also be obtained by using mice into which human antibody genes have been introduced. To obtain monoclonal antibodies from hybridomas, methods such as culturing the hybridomas according to conventional methods and obtaining the supernatant, or administering the hybridomas to compatible mammals to proliferate and obtaining the ascites fluid, are employed. The former method is suitable for obtaining high-purity antibodies, while the latter method is suitable for mass production of antibodies. The techniques for producing monoclonal antibodies can be known techniques, for example, by following the description in Chapter 2 of Current Protocols in Immunology, Wiley and Sons Inc.

[0068] In the phage display method, phages selected from a library of phage antibodies are screened using the target immunogen to select phages with the desired binding affinity to the immunogen. Next, the antibody-corresponding sequences contained within the phages are isolated or sequenced, and an expression vector containing a nucleic acid molecule encoding a monoclonal antibody is constructed based on the isolated or sequenced information. Then, by culturing a cell line transfected with this expression vector, monoclonal antibodies can be produced. By using a human antibody library as the phage antibody library, human antibodies with the desired binding affinity can be generated.

[0069] Genetic engineering techniques involve introducing mutations into the complementarity-determining region (CDR) or other sequences in the antibody-coding gene sequence, incorporating these sequences into an expression vector, and transforming host cells with this vector to produce recombinant antibodies (see, for example, Borrebaeck CAK and Larrick JW, THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990).

[0070] In this invention, for purposes such as reducing heterologous antigenicity against humans or adding other functions, for example, chimeric antibodies, humanized antibodies, multispecific antibodies, and artificial antibodies can also be used, and these antibodies can be manufactured using known methods.

[0071] Chimeric antibodies are obtained by ligating DNA encoding the variable region of a non-human antibody with DNA encoding the constant region of a human antibody, incorporating this into an expression vector, and introducing it into a host to induce production (see EP 125023, WO 92 / 19759). Using this known method, chimeric antibodies useful for the present invention can be obtained.

[0072] Humanized antibodies are obtained by ligating the complementarity-determining region (CDR) of a non-human antibody with the DNA encoding the human antibody region of the rest of the antibody, incorporating this into an expression vector, and introducing it into a host to induce production.

[0073] A multispecific antibody is an asymmetric antibody that possesses two or more independent antigen-recognition sites, each with two or more different antigen specificities. Multispecific antibodies, such as bispecific antibodies, can be produced using genetic engineering techniques by utilizing the antigen-binding regions of two or more monoclonal antibodies. These genetic engineering techniques are already well-established in this field. For example, a desired bispecific antibody can be obtained using techniques such as DVD-Ig (Wu et al., Nature Biotechnology 25(11), 1290(2007)), which involves linking the antigen-binding regions of two monoclonal antibodies in series, or ART-Ig (Kitazawa et al., Nature Medicine 18(10), 1570(2012)), which involves modifying the Fc region of an antibody to combine the heavy chains of two antibodies that bind to different antigens.

[0074] An artificial antibody is, for example, a protein scaffold, which does not have the structure of an antibody but has the same function as an antibody. Examples of protein scaffolds include the Kunitz domain of human serine protease inhibitors, the extracellular domain of human fibronectin, ankyrin, and lipocalin. By modifying the sequence of the target binding site on the scaffold, a protein scaffold that binds to the epitope of the present invention can be generated (Patent Document 4, Clifford Mintz et al. BioProcess International, 2013, Vol.11(2), pp40-48).

[0075] By modifying the amino acid sequence, such as the Fc region, or the glycans of the monoclonal antibody of the present invention, it is possible to adjust the cell-killing function, complement activation function, and blood half-life of the antibody, in addition to its antigen-binding function (Strohl, Current Opinion in Biotechnology, 2009, vol.20, p685). Such functionally modified antibodies can be prepared, for example, by the following method. When a monoclonal antibody is produced using CHO cells in which the α1,6-fucosyltransferase (FUT8) gene has been disrupted as the host cell, an antibody with reduced fucose content in the glycans and enhanced cell-killing function is obtained. When the antibody is produced using CHO cells into which the FUT8 gene has been introduced as the host cell, an antibody with low cell-killing function is obtained (International Publication No. 2005 / 035586, International Publication No. 2002 / 31140, International Publication No. 00 / 61739). Furthermore, complement activation function can be regulated by modifying amino acid residues in the Fc region (U.S. Patent Nos. 6,737056, 7,297775, and 7,317091). In addition, the half-life in the blood can be extended by using variants of the Fc region that enhance binding to FcRn, one of the Fc receptors (Shuhei Hashiguchi et al., Biochemistry, 2010, Vol. 82(8), p710). These functionally modified antibodies can be manufactured using genetic engineering.

[0076] The monoclonal antibodies used in the present invention may be conjugated antibodies bound to various molecules, such as non-peptide polymers like polyethylene glycol (PEG), radioactive materials, or toxins. Such conjugated antibodies can be obtained by chemically modifying the obtained antibody. Chemical modification methods have already been established in this field. These conjugated antibodies are also included in the monoclonal antibodies of the present invention (DJ King., Applications and Engineering of Monoclonal antibodies., 1998 TJ International Ltd, Monoclonal Antibody-Based Therapy of Cancer., 1998 Marcel Dekker Inc; Chari et al., Cancer Res., 1992 Vol152:127; Liu et al., Proc Natl Acad Sci USA., 1996 Vol 93:8681).

[0077] In this invention, in addition to the whole antibodies described above, monoclonal antibody fragments or their modifications may be used, as long as they possess epitope binding ability and exhibit antagonist activity. Examples of antibody fragments include Fab fragments, Fv fragments, F(ab')2 fragments, Fab' fragments, or single-chain Fv (scFv) obtained by linking the Fv of the H chain and L chain with an appropriate linker. Furthermore, these antibody fragments may be chemically or genetically engineered to non-antibody functional molecules such as non-peptide polymers like polyethylene glycol (PEG), radioactive materials, toxins, small molecule compounds, cytokines, albumin, and enzymes.

[0078] Production systems for monoclonal antibodies can be either in vitro or in vivo. In vitro production systems include those using eukaryotic cells, such as animal cells, plant cells, or fungal cells, or those using prokaryotic cells, such as bacterial cells like Escherichia coli or Bacillus subtilis. Animal cells used may include mammalian cells, such as commonly used cells like CHO, COS, Myeloma, BHK, HeLa, and Vero, as well as insect cells and plant cells. In vivo production systems include those using animals or plants. When using animals, examples include mammals and insects. Examples of mammals that can be used include goats, pigs, sheep, mice, and cattle (Vicki Glaser, SPECTRUM Biotechnology Applications, 1993). Examples of insects that can be used include silkworms. Examples of plants that can be used include tobacco.

[0079] As described above, when producing monoclonal antibodies in an in vitro or in vivo production system, the DNA encoding the monoclonal antibody heavy chain (H chain) or light chain (L chain) may be incorporated separately into expression vectors to simultaneously transform the host, or the DNA encoding the H chain and L chain may be incorporated into a single expression vector to transform the host (see International Publication No. WO94 / 11523).

[0080] The obtained monoclonal antibody can be purified until homogeneous. The separation and purification of monoclonal antibodies can be performed using the same separation and purification methods used for ordinary proteins. For example, monoclonal antibodies can be separated and purified by appropriately selecting and combining chromatography columns such as affinity chromatography, filters, ultrafiltration, salting out, dialysis, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, etc. (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988), but are not limited to these. Examples of columns used in affinity chromatography include protein A columns and protein G columns. Examples of columns using protein A include Hyper D, POROS, and Sepharose FF (Amersham Biosciences).

[0081] The present invention relates to a monoclonal antibody that binds to an epitope consisting of a continuous amino acid sequence located at positions 101-154 or 199-270 of sequence number 226 in the sequence listing. Because of its low antigenicity when administered to humans, chimeric antibodies, humanized antibodies, and human antibodies are preferred, with human antibodies being the most preferred. Furthermore, among human antibodies, it is preferable that the amino acid sequence of the framework region is the amino acid sequence of the framework region of the human germline or a combination thereof. Therefore, the present invention relates to a human anti-IL-33 neutralizing monoclonal antibody in which the amino acid sequence of the framework region of the antibody is the amino acid sequence of the framework region of the germline or a combination thereof. This human anti-IL-33 neutralizing monoclonal antibody has the characteristic of having no or significantly reduced immunogenicity due to the inclusion of amino acid sequences or combinations of amino acid sequences of the human germline framework region in its variable region framework region, while simultaneously being able to bind to IL-33 and inhibit its function. Therefore, when used as a pharmaceutical, this antibody is less likely to induce human anti-human immunoglobulin antibodies (HAHA), thus not being eliminated in the body, resulting in a long-lasting neutralizing effect on IL-33, and is a safe antibody as long as it does not induce inflammation caused by binding to HAHA.

[0082] Any amino acid sequence of the framework region of the light and heavy chains in the human germline can be used. For example, 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 human antibodies registered in databases such as NCBI (http: / / www.ncbi.nlm.nih.gov / igblast / showGermline.cgi), or amino acid sequences of the germline framework region listed in Table 4 can be used. The light chain variable region may be the variable region of the lambda chain or the variable region of the kappa chain. As the framework region of the light and heavy chains in the human germline, framework regions that appear frequently in vivo and are frequently used are preferred. Examples of such human heavy chain framework regions include framework region 1, framework region 2, and framework region 3 such as VH3-23, VH3-30, VH4-39, and VH4-34, and framework region 4 such as JH4. Furthermore, framework regions 1, 2, and 3 of Vλ1-47, Vλ2-14, Vκ3-20, and Vκ1-39 are examples of human light chain framework regions that frequently appear in vivo, as are framework regions 4 such as Jλ2. Heavy chain framework regions can be used in any combination as long as they are human heavy chain framework regions. For example, framework regions 1 and 2 of VH3-23 and framework region 3 of VH3-30 can be selected and used as a heavy chain framework region. Similarly, light chain framework regions can be used in any combination as long as they are human light chain framework regions.

[0083] In this application, the preferred amino acid sequence of the germline framework region is the amino acid sequence of the framework region VH3-23, VH3-30, JH4, Vλ1-47, and Jλ2. Specifically, the amino acid sequence of the light chain framework region 1 is residues 1 to 22 of sequence number 317 in the sequence listing, the amino acid sequence of the light chain framework region 2 is residues 36 to 50 of sequence number 317 in the sequence listing, the amino acid sequence of the light chain framework region 3 is residues 58 to 89 of sequence number 317 in the sequence listing, and the amino acid sequence of the light chain framework region 4 is residues 3 to 12 of sequence number 401 in the sequence listing, and the amino acid sequence of the heavy chain framework region 1 is residues 1 to 30 of sequence number 367 or sequence number 368 in the sequence listing, the amino acid sequence of the heavy chain framework region 2 is residues 36 to 49 of sequence number 367 or sequence number 368 in the sequence listing, the amino acid sequence of the heavy chain framework region 3 is residues 67 to 98 of sequence number 367 or sequence number 67 to 98 of sequence number 368 in the sequence listing, and the amino acid sequence of the heavy chain framework region 4 is The framework region of the heavy chain is preferably the one consisting of residues 5 to 15 of sequence number 407 in the sequence listing, most preferably the framework region in which the amino acid sequence of the light chain framework region 1 is residues 1 to 22 of sequence number 317 in the sequence listing, the amino acid sequence of the light chain framework region 2 is residues 36 to 50 of sequence number 317 in the sequence listing, the amino acid sequence of the light chain framework region 3 is residues 58 to 89 of sequence number 317 in the sequence listing, and the amino acid sequence of the light chain framework region 4 is residues 3 to 12 of sequence number 401 in the sequence listing, and the amino acid sequence of the heavy chain framework region 1 is residues 1 to 30 of sequence number 367 in the sequence listing, the amino acid sequence of the heavy chain framework region 2 is residues 36 to 49 of sequence number 367 in the sequence listing, the amino acid sequence of the heavy chain framework region 3 is residues 67 to 98 of sequence number 368 in the sequence listing, and the amino acid sequence of the heavy chain framework region 4 is residues 5 to 15 of sequence number 407 in the sequence listing.

[0084] Another aspect of the present invention relates to an isolated human anti-IL-33 neutralizing monoclonal antibody in which the amino acid sequences of each of the light chain complementarity-determining region 1 (LCDR1), light chain complementarity-determining region 2 (LCDR2), light chain complementarity-determining region 3 (LCDR3), heavy chain complementarity-determining region 1 (HCDR1), heavy chain complementarity-determining region 2 (HCDR2), and heavy chain complementarity-determining region 3 (HCDR3) are the amino acid sequences of the complementarity-determining region combinations shown in Table 1.

[0085] In a more preferred embodiment, a human anti-IL-33 neutralizing monoclonal antibody having the combination of complementarity-determining regions C1 to C30 shown in Table 1 has binding and neutralizing activity particularly towards mature IL-33 that binds to the IL-33 receptor and exerts activity, such as IL-33 (residues 95 to 270), IL-33 (residues 99 to 270), IL-33 (residues 109 to 270), and IL-33 (residues 112 to 270). Even more preferably, a human anti-IL-33 neutralizing monoclonal antibody having the combination of complementarity-determining regions C1 to C30 shown in Table 1 has binding affinity to IL-33 (residues 131 to 150).

[0086] In the present invention, a preferred embodiment is a combination of complementary determination regions that are improved in terms of bonding and / or physical properties. Particularly preferred is an upper limit of the dissociation rate constant (koff) for human IL-33 being approximately 3.5 × 10⁻⁶ -5 It is less than or equal to / sec, and more preferably about 2.0 × 10⁻⁶ -5 Less than or equal to / sec, more preferably about 1.5 × 10⁻⁶ -5 Less than or equal to / sec, and more preferably about 1.0 × 10 -5 The lower limit is less than or equal to / sec, and is not particularly limited, but for example, 10 -7 / sec or more, comfort level 10 -6 More than / sec, more preferably about 5 × 10 -6 Examples include human anti-IL-33 neutralizing monoclonal antibodies with a concentration of 1 / sec or higher.

[0087] Among human anti-IL-33 neutralizing monoclonal antibodies, those with a lower dissociation constant (Kd) for human IL-33 are more preferable, and as the upper limit value, for example, 10 -9 M or less, more preferably 10 -10 M or less, even more preferably 10 -12 M or less, and the lower limit value is not particularly limited, but for example, 10 -14 M or more, more preferably 10 -13 or more. Examples of human anti-IL-33 neutralizing monoclonal antibodies include those with such properties.

[0088] The human anti-IL-33 neutralizing monoclonal antibody of the present invention inhibits the production of IL-6 from HUVEC upon IL-33 stimulation, and among them, those with a strong inhibitory effect are preferable. Specifically, as a preferred embodiment of the present invention, as described in Example 10 below, the addition of 1 μg / mL of the human anti-IL-33 neutralizing monoclonal antibody inhibits the production of IL-6 from HUVEC when stimulated with 100 ng / mL of IL-33. The inhibition rate is about 50% or more, more preferably about 70% or more, and even more preferably about 90% or more. Examples of human anti-IL-33 neutralizing monoclonal antibodies include those with such properties.

[0089] The human anti-IL-33 neutralizing monoclonal antibody of the present invention inhibits the production of IL-5, IL-6, and / or IL-13 from KU-812 cells upon IL-33 stimulation. Among them, those with a strong inhibitory effect are preferable. Specifically, as a preferred embodiment of the present invention, as described in Example 11 below, the addition of 3 μg / mL of the human anti-IL-33 neutralizing monoclonal antibody inhibits the production of IL-5, IL-6, and / or IL-13 from KU-812 cells when stimulated with 100 ng / mL of IL-33. The inhibition rate is about 30% or more, more preferably about 50% or more, and even more preferably about 70% or more.

[0090] The human anti-IL-33 neutralizing monoclonal antibody of the present invention inhibits the production of IFN-γ from human peripheral blood mononuclear cells upon IL-33 stimulation. Among these, those with a strong inhibitory effect are preferred. Specifically, a preferred embodiment of the present invention is a human anti-IL-33 neutralizing monoclonal antibody, as described in Example 12 below, in which the addition of 10 μg / mL of the antibody inhibits the production of IFN-γ from human peripheral blood mononuclear cells stimulated with 10 ng / mL of IL-33 by approximately 80% or more, more preferably approximately 90% or more, and even more preferably approximately 95% or more.

[0091] The human anti-IL-33 neutralizing monoclonal antibody of the present invention suppresses inflammation when human IL-33 is administered to mice. Among these, those with a strong anti-inflammatory effect are preferred. Specifically, as described in Example 13 below, a preferred embodiment of the present invention is a human anti-IL-33 neutralizing monoclonal antibody in which, when administered intraperitoneally at a dose of 10 mg / kg daily for 7 days, the increase in spleen weight, serum IgA concentration, IgE concentration, neutrophil count, basophil count, eosinophil count, and / or serum IL-5 concentration compared to 7 days of continuous administration of 0.4 μg / individual human IL-33 is suppressed by approximately 30% or more, more preferably approximately 50% or more, and even more preferably approximately 80% or more.

[0092] Furthermore, the human anti-IL-33 neutralizing monoclonal antibody of the present invention preferably has excellent physical properties. In particular, a human anti-IL-33 neutralizing monoclonal antibody that does not show a bimodal shape in the particle size distribution as evaluated by dynamic light scattering and has extremely low aggregation properties is preferred, and an interaction parameter (kD), which is an indicator of colloidal stability, is preferred to be high, for example, -12.4 mL / g or higher, more preferably -10 mL / g or higher, and even more preferably -8.5 mL / g or higher. The human anti-IL-33 neutralizing monoclonal antibody of the present invention is preferably an antibody with excellent thermodynamic stability. For example, an antibody exhibiting thermodynamic stability such that the temperature (Tm) at which the folding of the immunoglobulin domain collapses is 65°C or higher, preferably 68°C or higher, more preferably 70°C or higher, and even more preferably 73°C or higher.

[0093] Furthermore, the human anti-IL-33 neutralizing monoclonal antibody of the present invention preferably exhibits excellent antibody stability. Antibody stability can be measured by general methods such as storage stability tests and forced oxidation tests. As a storage stability test, for example, in a preferred embodiment of the present invention, as described in Example 21 below, when stored at 40°C for 4 weeks, the monomer content of the antibody molecule is preferably 90% or more, more preferably 95% or more, and the binding activity to the human IL-33 protein is preferably 95% or more, more preferably 99% or more. Furthermore, as described in Example 22 of this application, when forced oxidation is performed with 1% hydrogen peroxide solution at 37°C for 24 hours, it is preferable that the binding activity to human IL-33 protein is retained at 80% or more, more preferably at 85% or more, and even more preferably at 90% or more.

[0094] Considering the above, in the present invention, a human anti-IL-33 neutralizing monoclonal antibody selected from the combinations of complementarity-determining regions C1 to C28 in Table 1 is a more preferred antibody. Furthermore, a more preferred embodiment of the present invention is a human anti-IL-33 neutralizing monoclonal antibody having a specific combination of amino acid sequences of a particular complementarity-determining region (C1, C8, C15, C17, or C18 in Table 1).

[0095] The amino acid sequence of the framework region in the variable region of the human anti-IL-33 neutralizing monoclonal antibody, identified by the combination of amino acid sequences of the complementarity-determining regions described above, may be any framework region as long as antigen binding is ensured. From the viewpoint of reducing immunogenicity in humans, the amino acid sequence of the framework region is preferably an amino acid sequence of each amino acid sequence or a combination thereof of a human germline framework region, but more preferably an amino acid sequence of a germline framework region that is frequently used in the human body.

[0096] In the present invention, the preferred amino acid sequence of the framework region is such that the amino acid sequence of the light chain framework region 1 is residues 1 to 22 of sequence number 317 in the sequence listing, the amino acid sequence of the light chain framework region 2 is residues 36 to 50 of sequence number 317 in the sequence listing, the amino acid sequence of the light chain framework region 3 is residues 58 to 89 of sequence number 317 in the sequence listing, and the amino acid sequence of the light chain framework region 4 is residues 3 to 12 of sequence number 401 in the sequence listing, and the amino acid sequence of the heavy chain framework region 1 is The amino acid sequence of heavy chain framework region 2 is residues 1 to 30 of sequence number 367 in the sequence listing or residues 1 to 30 of sequence number 368 in the sequence listing, the amino acid sequence of heavy chain framework region 3 is residues 36 to 49 of sequence number 367 in the sequence listing or residues 36 to 49 of sequence number 368 in the sequence listing, the amino acid sequence of heavy chain framework region 3 is residues 67 to 98 of sequence number 367 in the sequence listing or residues 67 to 98 of sequence number 368 in the sequence listing, and the amino acid sequence of heavy chain framework region 4 is residues 5 to 15 of sequence number 407 in the sequence listing. A more preferred amino acid sequence for the framework region in the present invention is as follows: the amino acid sequence of the light chain framework region 1 is residues 1 to 22 of sequence number 317 in the sequence listing; the amino acid sequence of the light chain framework region 2 is residues 36 to 50 of sequence number 317 in the sequence listing; the amino acid sequence of the light chain framework region 3 is residues 58 to 89 of sequence number 317 in the sequence listing; and the amino acid sequence of the light chain framework region 4 is residues 3 to 12 of sequence number 401 in the sequence listing; and the amino acid sequence of the heavy chain framework region 1 is residues 1 to 30 of sequence number 367 in the sequence listing; the amino acid sequence of the heavy chain framework region 2 is residues 36 to 49 of sequence number 367 in the sequence listing; the amino acid sequence of the heavy chain framework region 3 is residues 67 to 98 of sequence number 368 in the sequence listing; and the amino acid sequence of the heavy chain framework region 4 is residues 5 to 15 of sequence number 407 in the sequence listing.

[0097] Therefore, preferred combinations of amino acid sequences for the heavy chain variable region and light chain variable region in the present invention are shown, for example, in Table 2.

[0098] A preferred embodiment of the present invention is a human anti-IL-33 neutralizing monoclonal antibody having a combination of the variable regions V1 to 28 in Table 2.

[0099] A more preferred embodiment of the present invention is a human anti-IL-33 neutralizing monoclonal antibody having a specific combination of amino acid sequences of a complementarity-determining region (V1, V8, V15, V17, or V18 in Table 2).

[0100] Human immunoglobulin molecules include IgG (including IgG1, IgG2, IgG3, and IgG4), IgM, IgA (including IgA1 and IgA2), IgD, and IgE, each having a heavy chain of either a γ, μ, α, δ, or ε chain, depending on the constant region of the heavy chain. However, the constant region of the human anti-IL-33 neutralizing monoclonal antibody of the present invention encompasses all of these. Furthermore, the light chain can be either a κ or λ chain, depending on its position on the chromosome, and both are included. When manufacturing antibody drugs, a κ chain is preferred from the viewpoint of aggregation, but antibodies with a λ light chain are also useful because the λ chain has a different amino acid sequence than the κ chain and is just as diverse as the κ chain. From the viewpoint of blood stability, the human anti-IL-33 neutralizing monoclonal antibody of the present invention is preferably IgG with a λ light chain and a γ heavy chain, and more preferably IgG1 with a λ light chain and a γ1 heavy chain.

[0101] Because the amino acid sequence of IL-33 differs depending on the animal species, there are differences in the amino acid sequence between human IL-33, represented by sequence number 226 in the sequence listing, and monkey IL-33, represented by sequence number 227 in the sequence listing. Generally, monkeys are used as experimental material in pharmacological and safety tests of antibody drugs, so it is preferable that the human anti-IL-33 neutralizing monoclonal antibody of the present invention binds to monkey IL-33, and more preferably binds to monkey IL-33 with an affinity similar to that of human IL-33. Particularly preferable is a human anti-IL-33 neutralizing monoclonal antibody in which the ratio of koff to monkey IL-33 to koff to human IL-33 is within approximately 20 times, more preferably within approximately 10 times, and even more preferably within approximately 5 times.

[0102] Examples of antibody fragments of the present invention include Fab fragment, Fv fragment, F(ab')2 fragment, Fab' fragment, and scFv. Furthermore, these antibody fragments may be conjugated with functional molecules other than antibodies, such as non-peptide polymers like polyethylene glycol (PEG), radioactive materials, toxins, small molecule compounds, cytokines, albumin, and enzymes.

[0103] The human anti-IL-33 neutralizing monoclonal antibody of the present invention can be used to produce multispecific antibodies, such as bispecific antibodies, by binding an antibody having antigen-binding specificity other than IL-33 to itself. Other antigens other than IL-33 include, but are not limited to, TNF-α, IL-6 receptor, CD3, CD20, α4 integrin, Blys, thymic stromal lymphopioietin, IgE, IL-1, IL-2, IL-4, IL-5, IL-6, IL-13, IL-17, IL-23, IL-25, and the like.

[0104] The human anti-IL-33 neutralizing monoclonal antibody and its fragments of the present invention can be modified to obtain functionally modified antibodies with altered functions such as cell-killing function, complement activation function, and blood half-life by modifying their Fc region, etc. (Kenya Shitara, Journal of Pharmaceutical Sciences, 2009, Vol.129(1), p3; Akiko Ishii et al., Journal of Japanese Pharmacology, 2010, Vol.136(5), p280; Shuhei Hashiguchi et al., Biochemistry, 2010, Vol.82(8), p710; Strohl, Current Opinion in Biotechnology, 2009, vol.20, p685).

[0105] The human anti-IL-33 neutralizing monoclonal antibody and its antibody fragments of the present invention can be conjugated to other functional molecules to form conjugated antibodies. For example, functional molecules such as non-peptide polymers like polyethylene glycol (PEG), radioactive materials, toxins, low molecular weight compounds, albumin, cytokines, and enzymes can be conjugated to add new functions.

[0106] Another aspect of the present invention includes a nucleic acid molecule encoding the protein portion of a human anti-IL-33 neutralizing monoclonal antibody whose framework region is a germline amino acid sequence, 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.

[0107] In yet another aspect of the present invention, the present invention also exists in a composition containing the above-mentioned human anti-IL-33 neutralizing monoclonal antibody. Since IL-33 induces inflammation, the human anti-IL-33 neutralizing monoclonal antibody is expected to have applications in the diagnosis, treatment, prevention, or mitigation of IL-33-related diseases. Therefore, in one aspect of the present invention, the present invention exists in a pharmaceutical composition for the diagnosis, treatment, prevention, or mitigation of IL-33-related diseases, containing the human anti-IL-33 neutralizing monoclonal antibody. In yet another aspect, since IL-33 induces cytokines, chemokines, inflammatory mediators, etc., the present invention also exists in a cytokine, chemokine, or inflammatory mediator expression inhibitor containing the human anti-IL-33 neutralizing monoclonal antibody.

[0108] The cytokines suppressed by the cytokine, chemokine, or inflammatory mediator expression inhibitor of the present invention are cytokines induced by IL-33, such as TNF-α, IFN-γ, IL-1β, IL-3, IL-4, IL-5, IL-6, and IL-13. The chemokines suppressed by the inhibitor are chemokines induced by IL-33, such as CXCL2, CCL2, CCL3, CCL6, CCL17, and CCL24. The inflammatory mediators suppressed by the inhibitor are inflammatory mediators induced by IL-33, such as PGD2 and LTB4. A particularly preferred embodiment of the present invention is an IFN-γ, IL-5, IL-6, or IL-13 expression inhibitor containing a human anti-IL-33 neutralizing monoclonal antibody, and more preferably an IL-6 production inhibitor.

[0109] Another aspect of the present invention relates to a pharmaceutical composition comprising the monoclonal antibody of the present invention. Furthermore, the present invention relates to a method for diagnosing, treating, preventing or alleviating IL-33-related disease, comprising administering the monoclonal antibody of the present invention, and to the use of the monoclonal antibody of the present invention for the manufacture of pharmaceuticals for the diagnosis, treatment, prevention or alleviation of IL-33-related disease.

[0110] Examples of IL-33-related diseases include, but are not limited to, asthma, atopic dermatitis, urticaria, hay fever, anaphylactic shock, sinusitis (including eosinophilic sinusitis), allergic encephalomyelitis, eosinophilic syndrome, polymyalgia rheumatica, rheumatic heart disease, multiple sclerosis, arthritis (e.g., rheumatoid arthritis, juvenile arthritis, psoriatic arthritis, osteoarthritis, Reiter's syndrome, etc.), systemic lupus erythematosus (including lupus discoid), pemphigus, bullous pemphigoid, psoriasis, and ankylosing spine. 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.), Sjögren'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 resistance, alopecia areata Greata), antiphospholipid syndrome, autoimmune diseases of the adrenal glands (e.g., autoimmune Addison's disease), celiac pruritus dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), cold agglutinin disease, essential mixed cryoglobulinemia, fibromyalgia-fibromyitis, 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 mellitus, pernicious anemia, polychondritis (pol Polychrondritis, polyglandular syndrome, Stiffman syndrome, vitiligo, sarcoidosis, polyglandular endocrine disorders, other endocrine gland failures, arteriosclerosis, hepatic fibrosis (e.g., primary biliary cirrhosis), pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis), chronic obstructive pulmonary disease (COPD), scleroderma (including CREST syndrome, Raynaud's phenomenon, etc.), tubulointerstitial nephritis, dense deposit disease, acute kidney injury, myocarditis, cardiomyopathy, neuritis (e.g., Guillain-Barré syndrome, etc.), polyarteritis nodosa, cardiotomy syndrome, chronic inflammatory demyelinating polyneuropathy, IgA neuropathy, lichen planus, Meniere's disease, post-myocardial infarction (post-MI), uveitis, uveitis ophthalmitisOpthalmia, vasculitis, primary agammaglobulinemia, cancer (e.g., brain tumor, laryngeal cancer, lip and oral cancer, hypopharyngeal cancer, thyroid cancer, esophageal cancer, breast cancer, lung cancer, gastric cancer, adrenocortical carcinoma, bile duct cancer, gallbladder cancer, liver cancer, pancreatic cancer, bladder cancer, colorectal cancer, uterine cancer, ovarian cancer, prostate cancer, testicular cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, Ewing's tumor, Hodgkin's disease, non-Hodgkin lymphoma, melanoma, mesothelioma, multiple myeloma, etc.), infections resistant to elimination by the immune system (e.g., severe acute respiratory syndrome) Examples include (SARS), lethal cytokine storms associated with highly virulent influenza infections, and sepsis. Preferably, examples include asthma, atopic dermatitis, hay fever, anaphylactic shock, sinusitis (including eosinophilic sinusitis), Crohn's disease, ulcerative colitis, arthritis, systemic lupus erythematosus, pemphigus, bullous pemphigoid, scleroderma, ankylosing spondylitis, hepatic fibrosis (including primary biliary cirrhosis), pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), acute kidney injury, vasculitis, and cancer.

[0111] The pharmaceutical composition comprising the human anti-IL-33 neutralizing monoclonal antibody of the present invention may contain, in addition to the active ingredient, the human anti-IL-33 neutralizing monoclonal antibody and its salt, pharmacologically acceptable carriers, diluents, or excipients. Furthermore, it may also contain other active ingredients other than the human anti-IL-33 neutralizing monoclonal antibody of the present invention, such as anti-inflammatory agents or immunosuppressants. Such compositions are provided in dosage forms suitable for parenteral or oral administration, but parenteral administration is preferred from the viewpoint of use as an antibody drug. Examples of parenteral administration include, but are not limited to, intravenous, intra-arterial, subcutaneous, topical, intraperitoneal, intramuscular, nasal, ophthalmic, transdermal, transmucosal, intrameningeal, transrectal, intramuscular, and intracerebral administration.

[0112] The pharmaceutical composition can be appropriately formulated in dosage form depending on the route of administration, and may be any form such as injection, powder, infusion, granules, tablets, or suppositories. However, from the viewpoint of parenteral administration, injection, infusion, or powder that dissolves immediately before use are preferred. Furthermore, these formulations may contain various adjuvants used in pharmaceuticals, i.e., carriers and other auxiliary agents, such as stabilizers, preservatives, analgesics, emulsifiers, and other additives.

[0113] The human anti-IL-33 neutralizing monoclonal antibody of the present invention may be provided, for example, by continuous infusion at intervals of once daily, once weekly, once monthly, or 1 to 7 times per year, or by drug administration. Drug administration may be intravenous, subcutaneous, topical, oral, nasal, rectal, intramuscular, intraventricular, or by inhalation. Preferred dose protocols include the maximum dose or administration frequency to avoid serious undesirable side effects. The total weekly dose is generally at least about 0.05 μg / kg body weight, more generally at least about 0.2 μg / kg, most commonly 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, optimally at least about 10 mg / kg, more optimally at least about 25 mg / kg, and most optimally at least about 50 mg / kg.

[0114] The human anti-IL-33 neutralizing monoclonal antibody of the present invention is useful in diagnostic assays, for example, for detecting IL-33 expression in specific cells, tissues, or serum of patients with IL-33-related diseases. For diagnostic applications, it is typically preferable that the human anti-IL-33 neutralizing monoclonal antibody is a conjugate antibody labeled with a detectable moiety.

[0115] Another aspect of the present invention relates to an anti-IL-33 neutralizing monoclonal antibody that competes for binding to IL-33 with an anti-IL-33 neutralizing monoclonal antibody containing an amino acid sequence of a specific complementarity-determining region combination (C1, C8, C15, C17, or C18 in Table 1) or a specific variable region amino acid sequence combination (V1, V8, V15, V17, or V18 in Table 2).

[0116] Anti-IL-33 neutralizing monoclonal antibodies that compete for binding to IL-33 with human anti-IL-33 neutralizing monoclonal antibodies containing specific amino acid sequence combinations of complementarity-determining regions or specific variable region amino acid sequences, as described above, can be obtained by screening anti-IL-33 antibodies acquired by genetic engineering methods such as phage display or hybridoma methods, for example, using the surface plasmon resonance (SPR) method described below.

[0117] By loading biotinylated human IL-33 protein (4 μg / mL) as a ligand onto an avidin-immobilized sensor chip, approximately 1300 to 1600 RU of human IL-33 protein is immobilized. Next, an arbitrary anti-IL-33 antibody (15 μg / mL) is loaded as an analyte and bound to the human IL-33 protein immobilized on the sensor chip. This process is repeated multiple times to create a state where all molecules of the human IL-33 protein on the sensor chip are bound to the arbitrary anti-IL-33 antibody (saturation state), and the amount of binding in the saturated state (saturation binding amount 1) is determined. Similar experiments were conducted with human anti-IL-33 neutralizing monoclonal antibodies containing amino acid sequences of specific complementarity-determining region amino acid sequences or specific variable region amino acid sequences of the present invention, and the binding amount in a saturated state (saturated binding amount 2) was determined. Next, the human IL-33 protein on the sensor chip is saturated with a human anti-IL-33 neutralizing monoclonal antibody containing an amino acid sequence of a specific complementarity-determining region amino acid sequence combination or a specific variable region amino acid sequence combination according to the present invention. Then, any anti-IL-33 antibody (15 μg / mL) is loaded as an analyte, and it is examined whether it binds to the human IL-33 protein saturated with the human anti-IL-33 neutralizing monoclonal antibody containing an amino acid sequence of a specific complementarity-determining region amino acid sequence combination or a specific variable region amino acid sequence combination according to the present invention. If any anti-IL-33 antibody can bind to a human IL-33 protein saturated with a human anti-IL-33 neutralizing monoclonal antibody containing the amino acid sequence combination of the specific complementarity-determining region of the present invention, or the specific variable region amino acid sequence combination, while exhibiting a saturation binding amount of 1 calculated above, then that antibody is judged to be "non-competitive." On the other hand, if any anti-IL-33 antibody cannot bind to a human IL-33 protein saturated with a human anti-IL-33 neutralizing monoclonal antibody containing the amino acid sequence combination of the specific complementarity-determining region of the present invention, or the specific variable region amino acid sequence combination, then that antibody is judged to be "competitive." Furthermore, even if any anti-IL-33 antibody can bind to a human IL-33 protein saturated with a human anti-IL-33 neutralizing monoclonal antibody containing the amino acid sequence of a specific complementarity-determining region of the present invention, or a specific variable region amino acid sequence, if the amount of additional binding does not reach the saturation binding amount of 1 with a significant difference, the antibody is judged to be "competitive." The significant difference can be examined using a general testing method (e.g., Student's t-test), and the significance level is 5% or 1% or less.

[0118] An anti-IL-33 neutralizing monoclonal antibody that competes for binding to IL-33 with a human anti-IL-33 neutralizing monoclonal antibody containing the amino acid sequence of the specific complementarity-determining region or the specific variable region amino acid sequence may be an antibody derived from any animal, such as a mouse antibody, human antibody, rat antibody, rabbit antibody, goat antibody, or camel antibody, or it may be a chimeric antibody or humanized antibody which is a combination of these antibodies. For a human anti-IL-33 neutralizing monoclonal antibody containing an amino acid sequence of the specific complementarity-determining region or a specific variable region, the anti-IL-33 neutralizing monoclonal antibody that competes for binding to IL-33 is preferably a chimeric antibody, a humanized antibody, or a human antibody, with the human antibody being most preferred.

[0119] Anti-IL-33 neutralizing monoclonal antibodies that compete for binding to IL-33 with human anti-IL-33 neutralizing monoclonal antibodies containing amino acid sequences of specific complementarity-determining regions or specific variable region amino acid sequences include antibody fragments. Examples of antibody fragments include Fab fragments, Fv fragments, F(ab')2 fragments, Fab' fragments, or scFv, but antibody fragments conjugated with PEG are preferred.

[0120] The following describes a method for producing the anti-IL-33 neutralizing monoclonal antibody of the present invention. By using genetic engineering techniques, a DNA sequence containing a desired combination of complementarity-determining regions and framework regions, and including DNA sequences encoding the light chain variable region and the heavy chain variable region, can be incorporated into an expression vector. This vector is then used to transform host cells, which are then cultured to produce the human anti-IL-33 neutralizing monoclonal antibody of the present invention (see, for example, Borrebaeck CAK and Larrick JW THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990). Alternatively, DNA sequences encoding the full length of the heavy chain and the full length of the light chain can be produced by ligating DNA sequences encoding the constant regions of the light chain and the heavy chain to DNA sequences encoding the heavy chain variable region and the light chain variable region, respectively. In the present invention, a preferred DNA sequence encoding the full length of the heavy chain and the full length of the light chain of a human anti-IL-33 neutralizing monoclonal antibody is, for example, IgG1 having a λ chain as the light chain, as shown in Table 5 below. However, when the antibody is produced using animal cells by genetic engineering techniques, the lysine residue at the C-terminus of the heavy chain may be removed, and the three nucleotides "aag" constituting the 3' end of the heavy chain nucleic acid sequences shown in Table 5 (sequence numbers 254-277 in the sequence listing) may be removed from each heavy chain nucleic acid sequence.

[0121] [Table 5]

[0122] For antibody production, in vitro production systems can be used. Examples of in vitro production systems include those using eukaryotic cells, such as animal cells, plant cells, or fungal cells, and those using prokaryotic cells, such as bacterial cells like Escherichia coli and Bacillus subtilis. The animal cells used may include mammalian cells such as CHO, COS, Myeloma, BHK, HeLa, Vero, 293, NS0, Namalwa, and YB2 / 0, as well as insect cells and plant cells, but 293 cells and CHO cells are preferred.

[0123] As described above, when producing antibodies in an in vitro production system, the DNA encoding the heavy chain or light chain of the antibody may be incorporated separately into expression vectors to simultaneously transform the host, or the DNA encoding both the heavy chain and light chain may be incorporated into a single expression vector to transform the host (see International Publication No. 94 / 11523). Preferred vectors for use in animal cells include, but are not limited to, pConPlus, pcDM8, pcDNA I / Amp, pcDNA3.1, and pREP4.

[0124] The obtained antibodies can be purified until homogeneous. Antibodies can be separated and purified using the same methods used for separating and purifying proteins. For example, antibodies can be separated and purified by appropriately selecting and combining chromatography columns such as affinity chromatography, filters, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing, etc. (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988), but are not limited to these. Examples of columns used in affinity chromatography include protein A columns and protein G columns. For example, protein A columns include Hyper D, POROS, and Sepharose FF (Amersham Biosciences).

[0125] By conjugating the human anti-IL-33 neutralizing monoclonal antibody according to this application with an antibody having antigen-binding specificity other than IL-33, multispecific antibodies such as bispecific antibodies can be produced. Methods for producing bispecific antibodies are already well known, including chemical methods (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). These methods involve first hydrolyzing two types of antibodies enzymatically, then cleaving the disulfide bonds in the heavy chains of the antibodies with a reducing agent, and subsequently mixing the different antibodies and re-oxidizing them to obtain a bivalent reactive antibody. Recently, preparation methods using crosslinking agents such as glutaraldehyde and carbodiimide have also been disclosed (Japanese Patent Publication No. 2-1556). Methods for producing multispecific antibodies, such as bispecific antibodies, through genetic engineering are already established in this field. For example, the DVD-Ig technique (Wu et al., Nature Biotechnology 25(11), 1290(2007)), which involves linking the antigen-binding regions of two monoclonal antibodies in series, or the ART-Ig technique (Kitazawa et al., Nature Medicine 18(10), 1570(2012)), which involves modifying the Fc region of an antibody to combine the heavy chains of two antibodies that bind to different antigens, can be used to obtain the desired bispecific antibody.

[0126] Functionally modified or conjugated human anti-IL-33 neutralizing monoclonal antibodies are prepared by the following methods. For example, when the human anti-IL-33 neutralizing monoclonal antibody of this invention is produced using CHO cells in which the α1,6-fucosyltransferase (FUT8) gene has been disrupted as the host cell, an antibody with reduced fucose content in the sugar chain and enhanced cell-killing function is obtained. When the antibody is produced using CHO cells into which the FUT8 gene has been introduced as the host cell, an antibody with low cell-killing function is obtained (International Publication No. 2005 / 035586, International Publication No. 2002 / 31140, International Publication No. 00 / 61739). Furthermore, complement activation function can be regulated by modifying the amino acid residues in the Fc region (U.S. Patent No. 6,737056, U.S. Patent No. 7,297775, U.S. Patent No. 7,317091). Furthermore, by using mutants of the Fc region that enhance binding to FcRn, one of the Fc receptors, the half-life in the blood can be extended (Shuhei Hashiguchi et al., Biochemistry, 2010, Vol.82(8), p710; Strohl, Current Opinion in Biotechnology, 2009, vol.20, p685). These functionally modified antibodies can be manufactured using genetic engineering.

[0127] The human anti-IL-33 neutralizing monoclonal antibody of the present invention can be conjugated to other functional molecules to produce a conjugated antibody. For example, when PEG is conjugated to the antibody as a functional molecule, PEG with a molecular weight of 2,000 to 100,000 Da, more preferably 10,000 to 50,000 Da, can be used without limitation, and may be linear or branched. PEG can be conjugated to the N-terminal amino group of an amino acid in the antibody, for example, by using an NHS active group. When using a radioactive substance as a functional molecule, 131 I, 125 I, 90 Y, 64 Cu, 99 Tc, 77 Lu or 211At and other methods are used. Radioactive materials can be directly bound to antibodies by methods such as the chloramine T method. When toxins are used as functional molecules, bacterial toxins (e.g., diphtheria toxin), plant toxins (e.g., lysine), low molecular weight toxins (e.g., geldanamycin), meitansinoids, and calicheamicin are used. When low molecular weight compounds are used as functional molecules, examples include daunomycin, doxorubicin, metrolexate, mitomycin, neocarutinostatin, vindesine, and fluorescent dyes such as FITC. When enzymes are used as functional molecules, luciferases (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, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., uricase and xanthine oxidase, etc.), lactoperoxidase, microperoxidase, etc. are used. Divalent radicals (e.g., alkylene, arylene, heteroarylene), -(CR2) are used as linkers when chemically binding toxins, low molecular weight compounds, or enzymes. n O(CR2) nLinkers represented by -(R is any substituent), repeating alkoxy units (e.g., polyethyleneoxy, PEG, polymethyleneoxy, etc.) and alkylaminos (e.g., polyethyleneamino, Jeffamine™), as well as diacitates and amides (e.g., succinate, succinamide, diglycolate, malonate, and caproamide, etc.) are examples. Chemical modification methods for attaching functional molecules are already established in this field (DJKing., Applications and Engineering of Monoclonal antibodies., 1998 TJ International Ltd, Monoclonal Antibody-Based Therapy of Cancer., 1998 Marcel Dekker Inc; Chari et al., Cancer Res., 1992 Vol152:127; Liu et al., Proc Natl Acad Sci USA., 1996 Vol 93:8681).

[0128] The human anti-IL-33 neutralizing monoclonal antibody, which contains an amino acid sequence of a specific complementarity-determining region (C1, C8, C15, C17, or C18 in Table 1) or a specific variable region (V1, V8, V15, V17, or V18 in Table 2), competes for binding to IL-33 with the anti-IL-33 neutralizing monoclonal antibody that competes for binding to IL-33. This anti-IL-33 neutralizing monoclonal antibody may be derived from any animal, such as a mouse antibody, human antibody, rat antibody, rabbit antibody, goat antibody, or camel antibody, or it may be a chimeric antibody or humanized antibody, which is a combination of these antibodies. These anti-IL-33 neutralizing monoclonal antibodies can be obtained using any known method, such as a genetic engineering method like the hybridoma method or phage display method, but are particularly preferably obtained by a genetic engineering method.

[0129] Chimeric antibodies are obtained by ligating DNA encoding the variable region of a non-human antibody with DNA encoding the constant region of a human antibody, incorporating this into an expression vector, and transforming host cells to produce the antibodies (see European Publication No. 125023, International Publication No. 92 / 19759).

[0130] Humanized antibodies are obtained by ligating the complementarity-determining region of a non-human antibody with the DNA encoding the human antibody region of the rest of the antibody, incorporating this into an expression vector, and introducing it into a host to induce production.

[0131] Human antibodies can be prepared, for example, using the procedures described in the examples provided below. Human antibodies can also be prepared using trioma technology, human B-cell hybridoma technology (Kozbor et al., 1983 Immunol Today 4: p72), and EBV hybridoma technology for generating human monoclonal antibodies (Cole et al., 1985, MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., p. 77). Furthermore, human antibodies can also be produced by immunizing transgenic mice into which human antibody genes have been introduced with antigen proteins to create hybridomas. Examples of transgenic mice include HuMab® mice (Medarex), KMTM mice (Kirin Pharma), KM (FCγRIIb-KO) mice, and VelocImmune mice (Regeneron).

[0132] Another aspect of the present invention includes a human anti-IL-33 neutralizing monoclonal antibody containing an amino acid sequence of a specific complementarity-determining region (C1, C8, C15, C17, or C18 in Table 1) or a specific variable region (V1, V8, V15, V17, or V18 in Table 2), and an artificial antibody that competes for binding to IL-33. For example, the 10th unit (FNfn10) of the human fibronectin type III domain can be used as an artificial antibody, and an artificial antibody that binds to a desired target can be obtained by introducing mutations into the BC, DE, and / or FG loops of this unit. In addition to the extracellular domain of fibronectin, peptides such as the Kunitz domain of serine protease inhibitors, ankyrin, and lipocalin can also be used as artificial antibodies. These artificial antibodies can be genetically engineered by introducing a vector containing a nucleic acid molecule encoding the peptide into E. coli, yeast, or animal cells, and purifying the culture supernatant obtained by culturing the host cells.

[0133] As artificial antibodies, instead of using specific proteins or parts of amino acid sequences as described above, it is also possible to search for small peptide molecules that specifically bind to the epitopes of the present invention, like antibodies, from a random sequence library of randomly combined amino acids (e.g., Hipolito et al., Current Opinion in Chemical Biology, 2012 Vol 16: 196, Yamagishi et al., Chemistry & Biology, 2011 Vol 18: 1562). Such peptides can be produced not only by genetic engineering methods but also by chemical synthesis methods such as the fluorenylmethyloxycarbonyl method and the t-butyloxycarbonyl method.

[0134] [Combinations of antibody sequences] Table 1 shows C1 to C30, which are combinations of amino acid sequences in the complementarity-determining region of the human anti-IL-33 neutralizing monoclonal antibody described in this application; Table 2 shows V1 to V30, which are combinations of amino acid sequences in the variable region; Table 5 shows CN1 to CN30, which are combinations of nucleic acid sequences in the complementarity-determining region; and Table 5 shows IGN1 to IGN30, which are combinations of nucleic acid sequences in the antibody. Each of these corresponds to the sequence of the same clone, and the correspondence is shown in Table 6 below. For example, the amino acid sequence of the complementarity-determining region of clone A10-1C04 ​​is a combination of the six amino acid sequences of the complementarity-determining region in C1, and this combination of amino acid sequences of the complementarity-determining region can be encoded by the six nucleic acid sequences in CN1. Furthermore, the amino acid sequences of the variable regions of the heavy and light chains of the clone are the two amino acid sequences in V1, and the amino acid sequences of the light chain of the λ chain and the heavy chain of the γ chain, which include the variable region of V1, are encoded by the two nucleic acid sequences in IGN1.

[0135] [Table 6] [Examples]

[0136] The present invention will be described in detail below with reference to examples, but unless otherwise specified, the present invention is not limited to the following.

[0137] Example 1: Acquisition of anti-IL-33 antibody and identification of epitope peptide [Acquisition of antibodies] Monoclonal antibodies were obtained by immunizing animals with human IL-33 protein and creating hybridomas from the splenocytes of the immunized animals. Furthermore, antibodies that bind to human IL-33 protein were cloned using phage display technology from an animal antibody library created using RNA recovered from the splenocytes of the immunized animals, and from a human naive antibody library. In this way, eight types (antibodies A-H) of anti-IL-33 monoclonal antibodies were obtained.

[0138] [Peptide array scan] To identify the epitopes of the acquired IL-33 antibodies, a peptide array scan was performed to examine the binding affinity of partial peptides (20 residues long) of human IL-33 to each antibody. To cover the main mature human IL-33 molecules, a total of 16 20-amino acid peptides (PEP11 to PEP26) were synthesized, shifting the starting position by 10 amino acids from valine at position 101 (V101) to threonine at position 270 (T270) from the N-terminus. The sequences and positional relationships of these peptides are shown in Table 7.

[0139] [Table 7]

[0140] Each peptide with its N-terminus biotinylated was immobilized as a ligand on a Neutroavidin sensor chip of a Surface Plasmon Resonance (SPR) device (Bio-Rad, ProteOn XPR36). As a positive control, an avidag sequence was added to the N-terminus of mature human IL-33 (residues 112 to 270), and the resulting biotinylated protein (hIL-33) was immobilized as a ligand on the SPR sensor chip via a biotin ligase reaction, specifically targeting the avidag sequence. A ligand-immobilized sensor chip was subjected to binding using either the test antibody, human IL-33 receptor white matter (recombinant human ST2 Fc chimera) (Enzo Life Science, ALX-201-367-C050), or buffer (0.05% Tween20 / PBS) as the analyte (antibody concentration: 10 μg / ml; flow rate: 100 μl / min). After washing, the amount of analyte (antibody amount) bound to the ligand on the sensor chip was expressed as the RU value. The results are shown in Figure 2.

[0141] Antibodies A and B bound to PEP12, starting with those possessing an epitope at the N-terminus of the human IL-33 protein. Antibodies C and D bound to PEP14. Antibody E bound to both PEP16 and PEP17. Antibody F bound to PEP24. Antibodies G and H bound to PEP26. A commercially available anti-human IL-33 polyclonal antibody (R&D Systems, AF3625) bound to most of the 16 human IL-33 peptides used in the study. On the other hand, the human IL-33 receptor (ST2) bound to the human IL-33 protein but hardly bound to the human IL-33 peptides (PEP11-PEP26), and this study did not determine which part of IL-33 is important for binding to ST2. Furthermore, no ligand binding was observed with buffer alone or with mouse IgG (R&D Systems, MAB002). When comparing the binding affinity to hIL-33 (residues 112 to 270) among the antibodies used, the order of binding strength to hIL-33 (residues 112 to 270) was antibody G, antibody H, antibody D, antibody E, antibody B, antibody A, antibody C, and antibody F.

[0142] Example 2: Evaluation of IL-33 neutralizing activity of anti-IL-33 monoclonal antibody - 1 The IL-33 neutralizing activity of antibodies A, B, E, and F was measured using their inhibitory effect on the binding of immobilized human ST2 to human IL-33 as an indicator. Recombinant human ST2 Fc chimera (Enzo Life Science, ALX-201-367-C050) (1 μg / mL, 50 μL / well), diluted in phosphate-buffered saline (PBS), was dispensed into a 96-well microplate (Nunc™, #442404) and left to stand overnight at 4°C. The following day, the plate was washed once with PBS-B containing 1% BSA, and the same solution (250 μL / well) was added and blocked at room temperature for 2 hours. Subsequently, a mixed solution (50 μL / well) of the test antibody (final concentration 10 μg / mL) and recombinant human IL-33 protein (ATGen, ILC0701) (final concentration 1 μg / mL), diluted in PBS-B, was added and incubated at room temperature for 2 hours. After washing the microplate five times with PBS containing 0.1% Tween 20 (PBS-T), goat anti-human IL-33 antibody (R&D Systems: AF3625, final concentration 1 μg / mL, 50 μL / well) diluted with PBS-B was added and incubated at room temperature for 1 hour. After washing the microplate five times with PBS-T, HRP-labeled rabbit anti-goat IgG antibody (Invitrogen: 61-1620, 50 μL / well) diluted 2000-fold with PBS-B was added and incubated at room temperature for 1 hour. After washing the microplate five times with PBS-T, SureBlue™ TMB Microwell Peroxidase Substrate (KPL: 52-00-01, 50 μL / well) was added and reacted at room temperature for 20 minutes. 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 plate reader (SPECTRA MAX 190, Molecular Devices). The inhibitory effect of the antibody on the binding of ST2 to IL-33 (IL-33 / ST2 binding system competitive inhibition rate) was determined by using a sample to which human IL-1β (PeproTech, 200-01B) (final concentration 1 μg / mL) was added instead of human IL-33 as the background, and calculating the inhibition rate (%) compared to a sample to which human IL-33 (final concentration 1 μg / mL) was added alone.As a result, antibody A (epitope PEP12) inhibited 66%, antibody B (epitope PEP12) inhibited 55%, antibody E (epitope PEP16-17) inhibited 0%, and antibody F (epitope PEP24) inhibited 39%. Of the four antibodies examined, all antibodies except antibody E (antibody A, antibody B, antibody F) showed an inhibition rate of 30% or more at a final concentration of 10 μg / mL.

[0143] [Table 8]

[0144] Example 3: Evaluation of IL-33 neutralizing activity of anti-IL-33 monoclonal antibody - 2 The IL-33 neutralizing activity of test antibodies (antibodies A-H) was measured using normal human umbilical vein endothelial cells (HUVECs) (LONZA, CLC2517A) as an indicator of their inhibitory effect on human IL-33-induced IL-6 production. HUVECs were seeded in 96-well microplates (IWAKI, MT4940-010) (6 × 10⁶). 3The cells were confirmed to be confluent after adding 0.1 mL / well. Anti-IL-33 antibody (final concentration 10 μg / mL) and recombinant human IL-33 (ATGen, ILC0701, final concentration 100 ng / mL) were added to the culture medium (EGM-2 medium (LONZA, CLCC-3156, CLCC-4176)) (0.2 mL / well), and incubated at 37°C for 24 hours. After 24 hours, the IL-6 concentration in the culture medium was measured using a commercially available ELISA kit (Thermo Scientific, EH2IL6). Cell viability at the time of culture medium collection was also measured using a cell counting kit (Dojindo, 345-06463) to confirm that the IL-6 production inhibitory effect was not due to a decrease in the number of viable cells. The IL-33 neutralizing activity of the test antibody (HUVEC system IL-6 production inhibition rate) was calculated as the inhibition rate (%) of IL-6 production by recombinant human IL-33 monotherapy. As a result, antibody A (epitope PEP12) inhibited 51%, antibody B (epitope PEP12) inhibited 48%, antibody C (epitope PEP14) inhibited 33%, antibody D (epitope PEP14) inhibited 38%, antibody E (epitope PEP16-17) inhibited 0%, antibody F (epitope PEP24) inhibited 38%, antibody G (epitope PEP26) inhibited 48%, and antibody H (epitope PEP26) inhibited 56%. Of the eight antibodies, all except antibody E showed an inhibition rate of 30% or more (Table 9). Among these antibodies, antibodies that bind to epitopes selected from the groups consisting of positions 111-130, 131-150, 231-250, and 251-270 of SEQ ID NO: 1 showed a significant increase in neutralization activity at antibody concentrations of 3, 10, and 30 μg / mL (for example, antibody D showed 23%, 42%, and 61% inhibition, respectively), indicating that these epitopes are suitable for generating antibodies with antagonist activity.

[0145] [Table 9]

[0146] Despite binding to hIL-33 (Figure 2), antibody E did not exhibit functional neutralizing activity (Tables 8 and 9). Patent document 2 (WO2008 / 132709) describes three types of epitopes: epitope 1 (positions 155-198), epitope 2 (positions 165-188), and epitope 3 (positions 175-178). It became clear that these epitopes overlap with the epitope peptide (positions 151-180) of antibody E, which was confirmed to lack IL-33 neutralizing activity. From these results, it was concluded that antibodies against the epitopes in Patent document 2 could not sufficiently inhibit the binding of IL-33 to its receptor, ST2, and therefore either had no IL-33 neutralizing activity, or had very low activity.

[0147] Theoretically, besides the superiority or inferiority of the epitope, another possible reason why antibody E did not show neutralizing activity against IL-33 is insufficient affinity. However, given the existence of clones such as antibody D, antibody G, and antibody H, which tend to bind to hIL-33 more weakly than antibody E, yet still showed clear neutralizing activity against IL-33, this possibility is considered unlikely. From these findings, it can be concluded that when the objective is to neutralize the cytokine IL-33, the epitopes described in Patent Document 2 are epitopes in which binding to IL-33 and neutralizing activity against IL-33 are unrelated, whereas the four epitopes we have discovered (PEP12, PEP14, PEP24, PEP26) are considered to be functional epitopes in which binding to IL-33 and neutralizing activity against IL-33 are related. Antibodies that bind to functional epitopes are thought to have high antagonist activity against IL-33, while antibodies that bind to non-functional epitopes are thought to have low or no antagonist activity against IL-33.

[0148] Example 4: Mapping of epitope peptides to the human IL-33 structure To further identify the interfacial atoms (IL-33 atoms within a minimum of 5 Å of the atoms constituting ST2) that are favorable epitopes for the generation of antibodies with antagonist activity for the four epitope peptides mentioned above, the epitope peptides were mapped onto the three-dimensional structure of the human IL-33·human ST2 complex. Because the X-ray crystal structure of the human IL-33·human ST2 complex (Research Collaboratory for Structural Bioinformatics: PDB ID 4KC3) lacked some structural elements of the IL-33 protein, it was not possible to show the positions of all the epitope peptides identified. Therefore, a homology model was constructed using the above X-ray crystal structure (4KC3) as a template (Figure 3, using Accelrys Discovery Studio 3.5), and the epitope peptides of the antibodies that showed neutralizing activity (PEP12, PEP14, PEP24, PEP26) were mapped (Figures 4-7). In Figures 4-7, human IL-33 and epitope peptides are shown in dark gray, while ST2, which binds to them, is shown in light gray. To clearly indicate the location of the contact surface with the receptor on the IL-33 protein surface, interface atoms are highlighted with larger spheres. As a result, it was found that these epitope peptides (PEP12, PEP14, PEP24, and PEP26) each contain amino acids that include interface atoms. Examples of amino acids containing interfacial atoms include P118, I119, T120, Y122, L123, R124, S125, L126, S127, Y129, and N130 of PEP12; D131, Q132, S133, T135, A137, L138, E139, S142, Y143, E144, I145, Y146, E148, D149, and L150 of PEP14; D244, N245, and H246 of PEP24; and K266, L267, S268, and E269 of PEP26. Epitopes containing interfacial atoms are considered preferable as epitopes to which antibodies with antagonist activity specifically bind.

[0149] Example 5: Acquisition of human anti-IL-33 antibody (parent clone) Using a human scFv phage display library (BioInvent, n-CoDeR) (Soderlind et al., Nature biotechnology, 2000 Vol.18(8), p852), we obtained two parental clones (scFv) (meaning the molecular form is scFv; the same notation applies hereafter) (clone names: A00-0070, A00-0036) that bind to mature IL-33 (residues 112 to 270), inhibit the binding of IL-33 to ST2, and inhibit IL-33 activity using IL-33-dependent IL-6 production in normal human umbilical vein endothelial cells (HUVECs), as described later, as an indicator. The nucleotide sequences of these antibodies were determined, and the amino acid sequences of the light chain and heavy chain variable regions were determined. The amino acid sequence combinations of the light chain and heavy chain variable regions of A00-0070 and A00-0036 were V29 and V30, respectively, in Table 2.

[0150] Example 6: Determination of amino acid substitutions to improve the complementarity determination region To improve the affinity for IL-33 and enhance the physical properties (reduction of aggregation by reducing surface hydrophobicity and improvement of solubility) of two parent clones, the complementarity determination regions were improved using Fab ribosome display and Fab phage display. The improvement of the complementarity determination regions was carried out in two stages: in the first stage, a single amino acid substitution that could improve the affinity for IL-33 and enhance the physical properties was determined, and in the second stage, multiple combinations of these single amino acid substitutions were determined (Fujino et al., Biochem. Biophys. Res. Commun., 2012 Vol. 428(3), p395).

[0151] A Fab ribosome display vector was constructed using the light and heavy chain variable regions of two parental clones. Using this as a template, a comprehensive single-amino acid substitution mutant library was constructed by performing multi-step PCR reactions using site-directed mutagenesis PCR and overlap extension PCR, in which all amino acid residues constituting the six complementarity-determining regions of the antibody (LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, HCDR3) were replaced with one of each of the 20 native amino acids. Using the Fab ribosome display method (Fujino et al., Biochem. Biophys. Res. Commun., 2012 Vol. 428(3), p395) with the reconstituted cell-free translation system PURE system (Genefrontier, PUREfrex) (Shimizu et al., Nature Biotechnology, , 2001 Vol. 19(8), p751), a comprehensive library of single-amino acid substitution mutants was enriched several times using recombinant human IL-33 protein (ATGen, ILC0701) as bait. The nucleotide sequences of the light chain and heavy chain variable regions of each clone (Fab) (meaning the molecular form is Fab, the same notation applies below) in the pre-enrichment (immediately after construction) and post-enrichment libraries were determined using a next-generation sequencer (Roche, 454). Thousands of reads of sequence data were obtained from each library before and after enrichment, and the frequency of all single-amino acid substitution mutants in the complementarity-determining region was calculated. Next, the ratio of change in the frequency of all single-amino acid substitution mutants (enrichment ratio) between the pre-enrichment and post-enrichment libraries was calculated. Using the magnitude of the enrichment ratio due to library enrichment as an indicator, single-amino acid substitutions considered useful for improving affinity to human IL-33 protein were determined. Furthermore, considering the total number of these single-amino acid substitutions and their distribution on the amino acid sequence, the positions in which amino acid substitutions would be introduced in the custom library constructed in the second stage were determined.

[0152] In the parent clone A00-0070, we decided to introduce amino acid substitutions at the following positions: asparagine position 12 in LCDR1 (sequence number 2 in the sequence listing), glutamine position 4 in LCDR2 (sequence number 11 in the sequence listing), serine position 2, tyrosine position 3, and serine position 6 in LCDR3 (sequence number 23 in the sequence listing), aspartic acid position 1 and asparagine position 5 in HCDR1 (sequence number 43 in the sequence listing), and serine position 4, 5, 7, and isoleucine position 9 in HCDR2 (sequence number 64 in the sequence listing). In the parent clone A00-0036, we decided to introduce amino acid substitutions at the following positions: asparagine 9 and 13 in LCDR1 (sequence number 6 in the sequence listing); arginine 6 and leucine 7 in LCDR2 (sequence number 20 in the sequence listing); alanine 1, 9, and 10 in LCDR3 (sequence number 40 in the sequence listing); asparagine 1 in HCDR1 (sequence number 47 in the sequence listing); serine 4, 5, 6, 7, 8, tyrosine, 9, isoleucine, 10, 11, 13, aspartic acid, and 16th lysine in HCDR2 (sequence number 64 in the sequence listing); and glycine 2, histidine 5, and 6 aspartic acid in HCDR3 (sequence number 78 in the sequence listing).

[0153] To improve the physical properties, homology models of two parent clones were created using protein structure analysis programs (Accelrys, Discovery Studio), and regions with high surface hydrophobicity were predicted in the complementarity-determining regions. Next, to reduce the surface hydrophobicity of these regions, it was decided to introduce amino acid substitutions at the 3rd tyrosine position of LCDR3 (sequence number 23 in the sequence listing) and the 7th serine and 9th isoleucine positions of HCDR2 (sequence number 64 in the sequence listing) in parent clones A00-0070, and at the 6th arginine and 7th leucine positions of LCDR2 (sequence number 20 in the sequence listing) and the 7th serine, 8th tyrosine, and 9th isoleucine positions of HCDR2 (sequence number 64 in the sequence listing) in parent clone A00-0036. Amino acid substitutions that are considered useful for reducing surface hydrophobicity while maintaining binding ability to human IL-33 protein at these sites were determined by considering enrichment ratio data from mutation analysis using a comprehensive single-amino acid substitution mutant library.

[0154] Example 7: Creation of a human anti-IL-33 antibody with an improved complementarity-determining region. A custom library for improving complementarity-determining regions was designed by combining multiple useful amino acid substitutions mentioned above, with the aim of improving affinity and physical properties. Vectors for Fab ribosome display and Fab phage display were constructed, and a custom library for improving complementarity-determining regions was constructed by performing multi-step PCR reactions using site-directed mutagenesis PCR and overlap extension PCR with the Fab ribosome display vector as a template, and site-directed mutagenesis induction by Kunkel's method using the Fab phage display vector as a template (Fellouse et al., J. Mol. Biol. 2007 Vol. 373, p924), thereby randomizing the complementarity-determining regions based on the above design. Human IL-33 protein and cynomolgus monkey IL-33 protein (GenBank: EHH57404, from residue 112 Ser to residue 269 Glu of sequence number 227 in the sequence listing) were used as bait, and library enrichment using Fab ribosome display and Fab phage display was repeated for several rounds. In the later rounds, negative selection was performed using hydrophobic column supports such as Octyl Sepharose (GE Healthcare) or Phenyl Sepharose (GE Healthcare) before binding to the IL-33 protein, thereby enriching Fabs that had high affinity for the IL-33 protein and low surface hydrophobicity. Recombinant proteins used as bait were prepared as follows: Mature human IL-33 (residues 112 to 270) and mature cynomolgus monkey IL-33 (residues 112 to 269 of sequence number 227 in the sequence listing) were modified by adding a 6His tag-Avi tag to the N-terminus and inserting it into pET30a(-) to construct expression vectors, thereby preparing recombinant proteins. Escherichia coli BL21(DE3) strains containing the expression vectors were pre-cultured in 5 mL of LB medium, and then 1 mL of the pre-culture solution was inoculated into 50 mL of expression medium (Merck, Overnight Express; with kanamycin added), and expression culture was performed at 30°C / 200 rpm for approximately 18 hours. After washing the collected cells, they were lysed with BagBuster (Novagen), and the supernatant was collected. The 6His-tagged-Avi-tagged cynomolgus monkey IL33 (residues 112 to 269) contained in the supernatant was purified using Ni-NTA agarose (QIAGEN), and biotin modification was introduced specifically to the Avi-tagged portion using commercially available biotin ligases (Avidity, BirA).

[0155] A Fab-secreting E. coli library was constructed using the concentrated library, and the dissociation rate constant (koff) was measured by surface plasmon resonance (SPR) using the culture supernatant of several hundred E. coli clones (Bio-Rad, ProteOn XPR36). Biotinylated human IL-33 protein (4 μg / mL) and cynomolgus monkey IL-33 protein (4 μg / mL) were loaded as ligands onto a sensor chip (Bio-Rad, NLC sensor chip), and 1300 to 1600 RU equivalent of human IL-33 protein and 1100 to 1500 RU equivalent of cynomolgus monkey IL-33 protein were immobilized. Next, the E. coli culture supernatant was loaded as an analyte, and sensorgrams were obtained for 1 minute of the bound phase and 10 to 30 minutes of the dissociation phase. Interspot correction and blank correction were performed on the sensorgram using the SPR data analysis program (Bio-Rad, ProteOn Manager v3.1.0), and koff was determined by Langmuir's off-rate analysis.

[0156] From among the clones (Fab) with improved complementarity-determining regions, 28 clones were selected to proceed to higher-level evaluations from Example 8 onward, which showed improved affinity for human IL-33 protein and binding affinity for cynomolgus monkey IL-33 protein (V1 to V28 in Table 2). As shown in Table 10, these clones (Fab) showed higher affinity (lower Koff values) for human and cynomolgus monkey IL-33 proteins compared to the parent clone (Fab). There were no amino acid substitutions in the framework region within the variable region of these clones. Even with the same single amino acid substitution in the complementarity-determining region, the affinity-improving effect differs between single-amino acid substitution mutants and multi-amino acid substitution mutants. Therefore, there were amino acid substitutions that were frequently found in the sequences of the 28 clones for higher-level evaluation despite having a small enrichment ratio from the comprehensive single-amino acid substitution mutant library in the first stage, and conversely, amino acid substitutions that were frequently found in the sequences of the 28 clones for higher-level evaluation despite having a large enrichment ratio in the first stage.

[0157] [Table 10]

[0158] Example 8: Preparation of IgG antibody For seven clones of human anti-IL-33 antibody obtained (A10-1C04, A23-1A05, A25-2C02, A25-3H04, A26-1F02, A00-0070, A00-0036), expression vectors for mammalian cells expressing IgG were constructed by inserting DNA encoding the light chain and heavy chain amino acid sequences downstream of the CMV promoter. For the light chain DNA sequences of each clone, sequence numbers 228, 232, 239, 241, 242, 230, and 253 from the sequence listing were used, and for the heavy chain DNA sequences, sequence numbers 254, 261, 262, 264, 265, 276, and 277 from the sequence listing were used, respectively. The above expression vectors were introduced into FreeStyle 293-F cells (Life Technologies) using the gene transfer reagent NeoFection-293-1 (Astec). After gene transfer, the culture supernatant was obtained after culturing for 5 days. Stable expression lines using CHO cells were established using the GS system (Lonza) with pConPlus vector and CHO K1SV cells. Stable expression lines of CHO cells were measured using WAVE Bioreactor SYSTEM 20 / 50 EHT (GE Healthcare) at 0.3 × 10⁶ 6 Culture was started at cells / mL, and the culture medium containing secreted IgG was collected. Using an AKTA explorer 100 (GE Healthcare), IgG was purified from the culture supernatant by affinity chromatography with Protein A resin (GE Healthcare, HiTrap MabSelect SuRe). IgG bound to Protein A resin was eluted with a pH 3.2 elution buffer, and the pH was quickly neutralized to near neutral before dialyzing with PBS (pH 7.2). To improve the purity of the purification, the IgG purified after Protein A column purification was purified with CHT (ceramic hydroxyapatite Type I resin) (BIORAD). IgG bound to CHT was eluted with a NaCl concentration gradient, the target fraction was collected, and dialyzed with PBS (pH 7.2). The antibody obtained by this purification method was designated as "neutral purified antibody". In addition to the elution step from the Protein A resin in the above purification method, a purification method was also performed that included a 6-minute washing step with 6 Column Volume 100 mM sodium carbonate buffer (pH 11.0). The antibody obtained by this purification method was designated as "alkaline purified antibody." The recovery rates of the alkaline purified antibody at each step are shown in Table 11. The purified alkaline antibody was concentrated by centrifugation using a VIVASPIN Turbo15 30000MWCO (Sartoeius).

[0159] [Table 11]

[0160] Example 9: Affinity for IL-33 protein The affinity of the test antibody (IgG) (meaning its molecular form is IgG, and the same notation applies hereafter) for human IL-33 protein was determined by measuring the dissociation constant (Kd) in PBS using a kinetic exclusion assay (KinExA) (Sapidyne, KinExA3200). Mixed samples were prepared by titrating a constant concentration of the test antibody (final concentration from tens of pM to hundreds of pM) with human IL-33 protein (ATGen, ILC0701) over a wide range (upper limit of final concentration from a few nM to tens of nM, resulting in a 2048-fold concentration range using a 12-step 2-fold dilution series) and incubated at room temperature until the antigen-antibody reaction reached equilibrium. After reaching equilibrium, the abundance of free anti-IL-33 antibody was measured using KinExA3200. Kd was calculated by fitting a plot of the abundance (vertical axis) and antigen concentration (horizontal axis) of anti-IL-33 antibody not bound to human IL-33 protein to a theoretical formula using the KinExA data analysis program (Sapidyne, KinExA Pro Software v3.5.3). Anti-IL-33 antibody capture beads were prepared by suspending 50 mg of Azlactone beads (Sapidyne) in 1 mL of coating solution (10 μg / mL human IL-33 protein (ATGen, ILC0701), 50 mM sodium carbonate pH 9.6) and incubating at room temperature for 1 hour. The antibody used for detection was anti-human F(ab)'2-DyLight649 (Jackson, 309-495-006). As shown in Table 12, when using neutral purified antibodies, the affinity of antibodies with improved complementarity-determining regions (A10-1C04, A23-1A05, A25-2C02, A25-3H04, A26-1F02) for human IL-33 protein ranged from Kd=231pM for the weakest antibody, A23-1A05, to Kd=720fM for the strongest antibody, A25-2C02.

[0161] Similarly, the affinity of alkali-purified antibodies to human IL-33 protein (residues 112-270) (ATGen, ILC0701) or full-length human IL-33 protein was measured by KinExA (Table 12). The affinity for human IL-33 protein (residues 112-270) was Kd=100.3pM for A10-1C04, Kd=195.3pM for A23-1A05, Kd=700pM for A25-2C02, Kd=7.7pM for A25-3H04, and Kd=5.3pM for A26-1F02. The affinity for full-length human IL-33 protein was Kd=179.8pM for A10-1C04 ​​and Kd=10.4pM for A26-1F02.

[0162] The recombinant proteins used as ligands were prepared as follows: Full-length human IL-33 protein was converted into an expression vector by inserting a NusA-tag-6His-tag-TEV Protease cleavage sequence, which was added to the N-terminus, into pET30a(+) to prepare the recombinant protein. After pre-culturing the BL21(DE3) strain containing the expression vector, it was inoculated into 50 mL of LB medium at a density of OD=0.5 and cultured with shaking at 37°C for 4 hours. After 4 hours, the culture temperature was changed to 13°C and cultured with shaking for 30 minutes. Then, IPTG was added to a final concentration of 0.1 mM, and the culture was continued with shaking at 13°C for 72 hours to obtain full-length IL-33 expressing E. coli. The full-length IL-33 expressing E. coli was lysed with BugBuster Master Mix (Novagen), and the supernatant fraction was obtained by centrifugation. The supernatant after collection 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 increase protein purity. The sample after anion exchange was concentrated by centrifugation by ultrafiltration using VIVASPIN6 (5,000MWCO). To 1750 μL of the concentrate, 100 μL of Turbo TEV protease (Nacalai Tesque) and 4.5 μL of 1M DTT were added and incubated at 4°C to cleave the NusTag and HisTag. To remove the NusTag and Turbo TEV protease (HisTag fusion) contained in the sample after tag cleavage, it was passed through a Ni Sepharose Excel column (GE Healthcare), and the pass-through fraction was collected. DTT was added to the pass-through fraction to a final concentration of 3.3 mM and used for KinExA measurement as full-length human IL-33 protein.

[0163] [Table 12]

[0164] Example 10: Evaluation of the neutralizing activity of human IL-33 in vitro using HUVEC The in vitro neutralizing activity of the test antibody (IgG) against human IL-33 was evaluated using HUVEC's IL-33-dependent IL-6 production as an indicator. A commercially available polyclonal anti-IL-33 antibody (R&D Systems, AF3625) was used as a positive control. HUVEC (LONZA, CLC2517A) was suspended in EGM-2 medium (LONZA, CLCC-3156, CLCC-4176) and seeded in 96-well microplates (IWAKI) (6x10⁻¹⁰⁻¹ 3 The cells were confirmed to be confluent (per well). A mixed solution of anti-IL-33 antibody (final concentration 1 μg / mL (approx. 6.7 nM)) and recombinant human IL-33 (ATGen, ILC0701) (final concentration 100 ng / mL (approx. 5 nM)) was added to the culture 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 cells at the time of medium collection was measured using a cell counting kit (Dojindo, 345-06463), confirming that the suppression of IL-6 production was not due to a decrease in the number of viable cells. The IL-33 neutralizing activity of the test antibody was calculated as the inhibition rate (%) of IL-6 production by IL-33 monotherapy. When using neutral purified antibodies, A10-1C04 ​​showed 67% inhibition, A23-1A05 74% inhibition, A25-2C02 96% inhibition, A25-3H04 97% inhibition, and A26-1F02 96% inhibition, demonstrating strong neutralizing activity. In contrast, the parent clones A00-0070 showed only 4% inhibition and A00-0036 showed only -2% inhibition, indicating very weak neutralizing activity. Increasing the concentration to 10 μg / mL resulted in moderate neutralizing activity, with A00-0070 showing 42% inhibition and A00-0036 showing 38% inhibition. On the other hand, a commercially available polyclonal antibody (R&D Systems, AF3625) showed moderate neutralizing activity, with 30% inhibition when added at a final concentration of 1 μg / mL. Similarly, a mixed solution of alkali-purified test antibody (final concentration 0.1-10 μg / mL (approx. 0.67-67 nM)) and recombinant human IL-33 (ATGen, ILC0701) (final concentration 100 ng / mL (approx. 5 nM)) was added to HUVEC, and the neutralizing activity of the antibody was measured to determine the inhibitory effect on IL-6 production by IL-33 treatment alone (IC). 50 The value was calculated. A10-1C04 ​​is IC 50 =0.35 μg / mL, A23-1A05 is IC 50 =0.27 μg / mL, A25-2C02 is IC 50 = 0.19 μg / mL, A25-3H04 is IC 50 =0.21 μg / mL, A26-1F02 is IC 50 The result was 0.23 μg / mL. Furthermore, a mixed solution of alkali-purified test antibody (final concentration 0.1-3 μg / mL) and recombinant cynomolgus monkey IL-33 (prepared by the method described in Example 7 and used without biotinylation) (final concentration 100 ng / mL) was added to HUVEC, and the neutralizing activity of the antibody was used to determine the inhibitory effect on IL-6 production by IL-33 treatment alone (IC). 50 The value was calculated using the IC A10-1C04. 50 The concentration was 0.43 μg / mL, confirming that A10-1C04 ​​neutralizes human IL-33 and cynomolgus monkey IL-33 with similar strength.

[0165] Example 11: Evaluation of the neutralizing activity of human IL-33 in vitro using KU-812 cells The in vitro neutralizing activity of the test antibody (IgG) against human IL-33 was evaluated using IL-33-dependent production of IL-5, IL-6, and IL-13 by KU-812 cells as an indicator. A commercially available polyclonal anti-IL-33 antibody (R&D Systems, AF3625) was used as a positive control. Human basophil cell line, KU-812 cells (ECACC, EC90071807), were seeded in 96-well microplates (Falcon) (1 x 10⁻¹⁴). 4( / well). Subsequently, a mixed solution of the test antibody (final concentration 3 μg / mL (approx. 20 nM)) and recombinant human IL-33 (ATGen, ILC0701) (final concentration 100 ng / mL (approx. 5 nM)) was added and incubated at 37°C for 24 hours. The concentrations of IL-5, IL-6, and IL-13 in RPMI-1640 medium containing 10% FBS were measured using the Human IL-5 Flex set, Human IL-6 Flex set, and Human IL-13 Flex set of the BD™ Cytometric Bead Array (BD Biosciences). In addition, the viability of cells at the time of medium collection was measured using a cell counting kit (Dojindo, 345-06463) to confirm that the suppression of IL-5, IL-6, and IL-13 production was not due to a decrease in the number of viable cells. When using a neutral purified antibody, A26-1F02 inhibited the production of IL-5, IL-6, and IL-13 by 70%, 82%, and 72%, respectively, in this evaluation system, demonstrating stronger neutralizing activity against each cytokine production than commercially available polyclonal antibodies (inhibiting 47%, 51%, and 41%, respectively).

[0166] Similarly, KU-812 cells were treated with alkali-purified test antibodies (final concentration 100-0.01 μg / mL (approx. 667-0.067 nM)), recombinant human IL-33 (ATGen, ILC0701) (final concentration 3 ng / mL (approx. 0.15 nM)), human IL-3 (PeproTech, 200-03, final concentration 10 ng / mL (approx. 0.67 nM)), and human complement C5a (Sigma-Aldrich, A mixed solution of C5788 (final concentration 1 nM) was added 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 viability of cells at the time of medium collection was measured using a cell counting kit to confirm that the inhibitory effect on IL-5 and IL-13 production was not due to a decrease in the number of viable cells. In this evaluation system, the alkali-purified test antibodies (A10-1C04, A23-1A05, A25-2C02, A25-3H04, A26-1F02) showed an inhibitory effect of 50% or more on IL-5 and IL-13 production at a final concentration of 1 μg / mL.

[0167] Example 12: Evaluation of the neutralizing activity of human IL-33 in vitro using human peripheral blood mononuclear cells The in vitro neutralizing activity of the test antibody (IgG) against human IL-33 was evaluated using IL-33-dependent IFN-γ production in human peripheral blood mononuclear cells (PBMCs) as an indicator. A commercially available polyclonal anti-IL-33 antibody (R&D Systems, AF3625) was used as a positive control. PBMCs were prepared and seeded in 96-well microplates (2 x 10⁻¹⁴). 5 Recombinant human IL-12 (Wako Pure Chemical Industries) (final concentration 10 ng / mL) was added to each well. A mixture of the test antibody and recombinant human IL-33 protein (10 ng / mL) was added and incubated at 37°C for 48 hours. Subsequently, the culture supernatant was collected, and the amount of IFN-γ produced in the medium was measured using the AlfaLISA™ human IFN-γ immunoassay kit (PerkinElmer) to evaluate the neutralizing activity of IL-33. In this evaluation system, the inhibition rates when the alkaline purified antibody was applied at a final concentration of 10 μg / mL were 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.

[0168] Example 13: Evaluation of the effect of intraperitoneal administration of human IL-33 on inflammation. Intraperitoneal administration of human IL-33 to mice induced various inflammatory changes. Specifically, increases in blood IgE, IgA, and IL-5 levels, increases in neutrophils, eosinophils, and basophils, an increase in splenocytes (increased spleen weight), and pathological changes in various mucosal organs occurred. These changes were used as indicators to evaluate the in vivo anti-inflammatory effect of the test antibody (IgG).

[0169] Male C57BL6 turtles (6-8 weeks old) (Charles River, Japan) were intraperitoneally administered 0.4 μg / individual human IL-33 protein (R&D Systems, 3625-IL-010) for 7 days (Day 0-Day 6). In addition, the test antibody (IgG) was administered intraperitoneally for 7 days (Day 0-Day 6). Seven days after the start of administration (Day 7), the average spleen weight of the group administered PBS (labeled "vehicle" in the figure) instead of human IL-33 protein was 76 ± 4 mg, while the average spleen weight of the group administered IL-33 protein was 90 ± 7 mg. Furthermore, the average spleen weight in the group that received intraperitoneal administration of human control IgG (MP Biomedicals, 55908) at 10 mg / kg (indicated as "mpk" in the figure) in addition to IL-33 protein administration was 93 ± 4 mg, while the average spleen weight in the group that received intraperitoneal administration of the neutral purified antibody A26-1F02 at 10 mg / kg in addition to IL-33 protein administration was 66 ± 3 mg.

[0170] Next, the alkaline purified antibody was evaluated by a single subcutaneous injection (sc, one shot) on the day before administration of human IL-33 protein (Day-1). Seven days after the start of administration (Day 7), the average spleen weight of the group administered PBS instead of human IL-33 protein was 70 mg, while the average spleen weight of the group administered IL-33 protein in addition to the aforementioned human control IgG (10 mg / kg) was 152 mg. In contrast, as shown in Figure 8, the spleen weights of the groups administered IL-33 protein in addition to A25-3H04 (1, 3, 5, and 10 mg / kg) were 143, 106, 109, and 78 mg, respectively, indicating that A25-3H04 suppressed the increase in spleen weight due to inflammation in a concentration-dependent manner. Similar to these anti-inflammatory effects on spleen weight, it was confirmed that serum IgA concentration, serum IgE concentration, blood neutrophil count, basophil count, eosinophil count, and serum IL-5 concentration, which increase with human IL-33 administration, were also suppressed by A25-3H04 (Figure 8). These results confirm that A25-3H04 exhibits an inhibitory effect on the in vivo inflammatory response induced by IL-33. Furthermore, when the blood concentration of A25-3H04 in mice was measured 7 days after the start of administration (Day 7), it was 0.6, 3.7, 6.5, and 20.3 μg / ml for 1, 3, 5, and 10 mg / kg administrations, respectively.

[0171] The in vivo anti-inflammatory effects of other test antibodies (IgG) were also evaluated using a similar protocol via subcutaneous administration (10 mg / kg). As shown in Figure 9, the average spleen weight in the group subcutaneously administered human control IgG was 181 mg, while the spleen weights in the groups subcutaneously administered each of the alkali-purified antibodies (A10-1C04, A23-1A05, A25-2C02, A26-1F02) in addition to IL-33 protein were 82 mg, 92 mg, 100 mg, and 77 mg, respectively, indicating that the increase in spleen weight due to inflammation was suppressed. Similar to the anti-inflammatory effects on spleen weight, we confirmed that the alkali-purified antibodies (A10-1C04, A23-1A05, A25-2C02, A26-1F02) also suppressed serum IgA concentration, serum IgE concentration, blood neutrophil count, basophil count, and eosinophil count, which increase with human IL-33 protein administration (Figure 9). From these results, it was confirmed that, like A25-3H04, the other test antibodies (A10-1C04, A23-1A05, A25-2C02, A26-1F02) also exhibit anti-inflammatory effects against the in vivo inflammatory response induced by IL-33.

[0172] Example 14: Evaluation of the effect of human IL-33 intratracheal administration on lung injury. When human IL-33 protein is administered intratracheally to mice, and bronchoalveolar lavage fluid (BALF) is subsequently collected, the total cell count, eosinophil count, and neutrophil count in the BALF increase, and tracheal epithelial mucin proliferation is also observed. Furthermore, cytokines such as IL-4, 5, 6, and 13 are produced in the BALF. By administering the test antibody (IgG) intraperitoneally, subcutaneously, or intravenously to this system, the effect of the test antibody on lung damage can be evaluated.

[0173] Example 15: Evaluation of the effect of human IL-33 intranasal administration on airway hypersensitivity. Intranasal administration of IL-33 protein to mice subsequently induces airway hypersensitivity to inhaled methacholine. This evaluation system allows for the assessment of the effect of the test antibody (IgG) on airway hypersensitivity by intraperitoneal, subcutaneous, or intravenous administration.

[0174] Example 16: Evaluation of the effect on IL-33 using human IL-33 knock-in mice. When mite antigen or papain is administered nasally or intratracheally to human IL-33 knock-in mice, airway inflammation is induced, and when BALF is collected from these mice, the total number of cells in the BALF increases. It is known that airway inflammation induced by mite antigen or papain is caused by the release of IL-33 from airway epithelial cells due to the protease activity of the mite antigen or papain (Oboki et al., Proceedings of the National Academy of Sciences of the United States of America, 2010, vol. 107, p18581). By administering the test antibody (IgG) intraperitoneally, subcutaneously, or intravenously to this evaluation system, the effect of the test antibody on protease-induced airway inflammation and the effect of the test antibody on IL-33 induced in vivo can be evaluated.

[0175] Example 17: Evaluation of the effect on inflammation in a sepsis model administered intraperitoneally with LPS. While sepsis is induced in human IL-33 knock-in mice by intraperitoneal administration of LPS (Oboki et al., Proceedings of the National Academy of Sciences of the United States of America, 2010, vol. 107, p18581), the effect of the test antibody (IgG) on subsequent mortality can be evaluated by administering the test antibody intraperitoneally, subcutaneously, or intravenously before LPS administration. Furthermore, inflammatory cytokines such as IL-6 and TNF-α are detected at high concentrations in the blood within a few hours of LPS administration, and the anti-inflammatory effect of the test antibody can be evaluated by measuring these concentrations.

[0176] Example 18: Evaluation of the effect on cancer in vivo using tumor-bearing mice Mouse cancer cell lines or human cancer cell lines are transferred in appropriate cell numbers according to each cancer cell line, either orthotopically, subcutaneously, or intravenously, and human IL-33 is administered. A test antibody (IgG) is then administered to these mice intraperitoneally, subcutaneously, or intravenously. After the transfer of cancer cell lines, the number of cancer cells in the primary tumor site and metastatic organs is evaluated by volume and cell count, allowing for the assessment of the test antibody's effect on cancer.

[0177] Example 19: Evaluation of the colloidal stability of antibodies The colloidal stability of the test antibody (IgG) was evaluated by the presence or absence of aggregates determined by dynamic light scattering. Each alkali-purified antibody was concentrated to approximately 50 mg / mL using VIVASPIN or VIVASPIN TURBO (sartorius, 10,000 to 50,000 MWCO). Centrifuge was performed at 4°C, with rotation speed and time adjusted as appropriate. Dynamic light scattering (Nikkiso, Nanotrac UPA UT-151) was measured using 200 to 250 μL samples while sequentially diluting the test antibody solution, and data were obtained in the concentration range from approximately 1 mg / mL to approximately 50 mg / mL. The particle size distribution of the antibody protein was calculated from the cumulative data over 200 seconds, and the presence or absence of aggregates was evaluated. The test antibodies (A10-1C04, A23-1A05, A25-2C02, A25-3H04, A26-1F02) showed only a very slight shift in the particle size distribution around 10 nm towards higher particle sizes with increasing antibody concentration. Furthermore, there were no peaks of several tens of nanometers or larger, which are thought to originate from irreversible aggregates independent of antibody concentration. These results confirm the excellent colloidal stability of the test antibodies.

[0178] To quantitatively evaluate colloidal stability, the interaction parameter (k DThe following calculation was performed. The interaction parameter, which represents the concentration dependence of the diffusion coefficient (inversely proportional to particle size), is an important indicator used in the formulation design of high-concentration protein preparations such as antibodies. It has been reported that if the value of the interaction parameter is higher than -12.4 mL / g, it indicates a repulsive interaction, excellent colloidal stability, and low self-association (Saito et al., Pharm.Res., 2013.Vol.30 p1263). The test antibody solution dissolved in PBS (pH 7.2) was concentrated to several tens of mg / mL using an ultrafiltration membrane, and the particle size of the samples, which were sequentially diluted 2-fold in the same solvent, was measured using a dynamic light scattering analyzer (Nanotrac UPA UT151 Nikkiso). The diffusion coefficient was calculated from the obtained particle sizes using the following Stokes-Einstein equation.

[0179]

number

[0180] In the above equation, D is the diffusion coefficient (cm²). 2 / sec), K B Here, is the Boltzmann constant (J / K), T is the thermodynamic temperature (K), π is pi, η is the viscosity of the diluent P (poise), and d is the particle size (nm). The interaction parameters were determined by plotting the concentration dependence of the diffusion coefficient and fitting it using the following formula.

[0181]

number

[0182] D is the diffusion coefficient obtained from the Stokes-Einstein equation, D0 is the diffusion coefficient at infinite dilution, and c is the concentration of the test antibody at the time of measurement (g / mL). From this equation, the interaction parameter (k), which is the slope of the fitting line, is obtained. D The result was calculated that A10-1C04 ​​is k D = -8.1 mL / g (analysis range is 0.41-63.7 mg / mL), A23-1A05 is k D= -5.6 mL / g (analysis range is 0.40-61.8 mg / mL), A25-2C02 is k D = -6.2 mL / g (analysis range is 0.43-66.3 mg / mL), A25-3H04 is k D = -7.5 mL / g (analysis range is 0.34-56.5 mg / mL), A26-1F02 is k D The interaction parameters were -6.7 mL / g (analysis range 0.35-62.7 mg / mL), and all antibody interaction parameters were higher than -12.4 mL / g, indicating excellent colloidal stability.

[0183] Example 20: Evaluation of the thermodynamic stability of antibodies The thermodynamic stability of the test antibody (IgG) was evaluated by the temperature (Tm) at which the immunoglobulin domain folding breaks down. Protein Thermal Shift Dye (Life Technologies) was added to a test antibody solution of several tens of μg / mL according to the package insert, and fluorescence intensity was measured while increasing the temperature at approximately 1°C / min using real-time PCR 7500 Fast (Life Technologies). The obtained data was analyzed using Protein Thermal Shift (Life Technologies) to determine Tm. If multiple Tm values ​​were observed, they were designated as Tm1 and Tm2, ​​starting with the lowest temperature. As a result, when using neutral purified antibodies, the Tm=73.9°C was for A10-1C04, Tm1=69.3°C and Tm2=77.6°C for A23-1A05, Tm1=69.3°C and Tm2=80.3°C for A25-2C02, Tm1=70.0°C and Tm2=76.4°C for A25-3H04, and Tm=74.5°C for A26-1F02. Furthermore, when alkali-purified antibodies were used, the following temperatures were observed: A10-1C04 ​​had a Tm of 73.7°C, A23-1A05 had Tm1=69.5°C and Tm2=77.5°C, A25-2C02 had Tm1=69.5°C and Tm2=80.4°C, A25-3H04 had Tm1=70.1°C and Tm2=76.4°C, and A26-1F02 had Tm=74.4°C. All antibodies had a Tm of 65°C or higher, indicating good thermodynamic stability.

[0184] Example 21: Evaluation of the storage stability of antibodies To evaluate the storage stability of the test antibody (IgG), each alkali-purified antibody was dissolved in citrate buffer (50 mM citrate, 150 mM NaCl (pH 6.3)) at a concentration of approximately 10 mg / mL and stored at 40°C for 4 weeks. To evaluate the antibody monomer purity after storage, monomer purity was measured by gel filtration analysis (SEC) and microchip capillary SDS electrophoresis (mCE-SDS), and antigen binding activity was measured using surface plasmon resonance.

[0185] A column consisting of two linked TSKgel G3000SWXL (Tosoh) columns was mounted on an HPLC system (Beckman System Gold, 126 solvent manager, 166 detector, 508 auto sampler), and gel filtration analysis was performed. Separation was performed at a flow rate of 0.5 mL / min using 0.1 M phosphate buffer (pH 6.7) containing 0.1 M sodium sulfate as the mobile phase solvent, and detection was performed under UV 215 nm. A 10 mg / mL antibody storage solution was diluted 100-fold to prepare the analytical sample, and 50 μL of this sample was injected. The monomer purity obtained by gel filtration analysis is shown in Table 13. All of the test 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, demonstrating good storage stability.

[0186] Capillary SDS electrophoresis was performed using the Lab Chip GX II (PerkinElmer). Reduction was carried out under denaturation conditions using the HT Protein Express Reagent (PerkinElmer) reagent kit, according to the manufacturer's standard protocol. Approximately 2 μL of antibody storage solution at 10 mg / mL was added as the analytical sample. The reagents used for electrophoresis were added to the dedicated chip, HT Protein Express Lab Chip, version 2 (PerkinElmer), from the aforementioned kit, and measurements were performed using the built-in antibody analysis protocol, HT Antibody 200. As shown in Table 13, under denaturation and reduction conditions, all test antibodies (A10-1C04, A23-1A05, A25-2C02, A25-3H04, A26-1F02) maintained monomer purity of over 90% after 4 weeks of storage at 40°C, demonstrating good storage stability.

[0187] To investigate the presence or absence of irreversible aggregate formation independent of antibody concentration after storage, particle size measurements were performed. Particle size measurements were performed on analytical samples prepared by diluting the antibody storage solution 10-fold with citrate buffer (50 mM citrate, 150 mM NaCl (pH 6.3)) (final concentration: approximately 1 mg / mL) using dynamic light scattering (Nikkiso, Nanotrac UPA UT-151). The integration time was 200 seconds. No aggregates were detected after 4 weeks of storage at 40°C for any of the test antibodies (A10-1C04, A23-1A05, A25-2C02, A25-3H04, A26-1F02, A00-0070, A00-0036), demonstrating the acquisition of antibodies with excellent storage stability.

[0188] To investigate the presence or absence of antigen-binding ability after storage, antigen-binding activity was measured using a surface plasmon resonance spectrometer, Biacore T200 (GE Healthcare). Human IL-33 protein (ATGen, ILC0701) was immobilized on Sensor Chip CM5 (GE Healthcare) using an amine coupling kit (GE Healthcare) (immobilization amount: approximately 3000 to 6000 RU). Next, the antibody storage solution was diluted 10-fold with citrate buffer (50 mM citrate, 150 mM NaCl (pH 6.3)), and the total protein concentration in the solution was measured using a micro-spectrophotometer, Astragene II (Astranet) (protein concentration: approximately 1 mg / mL). The antibody solution with 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)) to prepare the analyte solution. Measurements were performed at 25°C. Various analyte solutions were added for 36 seconds to obtain conjugated phase sensorgrams. Flow rates were 5 μL / min and 100 μL / min. The concentrations of antibodies with antigen-binding activity were determined from the sensorgrams obtained at the two flow rates by 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 various test antibodies stored at 4°C for 4 weeks was similarly measured, and the percentage of antigen-binding activity of the test antibodies stored at 40°C for 4 weeks was calculated. As shown in Table 13, all test antibodies (A10-1C04, A23-1A05, A25-2C02, A25-3H04, A26-1F02) retained more than 90% of their antigen-binding activity after storage at 40°C for 4 weeks, demonstrating good storage stability.

[0189] [Table 13]

[0190] Example 22: Evaluation of antibody stability by forced oxidation The effect of oxidation of the test antibody (IgG) on its antigen-binding activity was investigated. Various alkaline purified antibodies at a final concentration of approximately 1 mg / mL were mixed with hydrogen peroxide (1% final concentration) and oxidized at 37°C for 24 hours. Subsequently, 80 mM methionine solution was added to terminate the oxidation. Next, the test antibody solution was replaced with PBS using a desalting column (Zebaspin, Thermo Scientific). The antigen-binding activity of the oxidized test antibody was examined using a surface plasmon resonance spectrometer (Biacore T200, GE Healthcare) in the same manner as in Example 21. When the ratio of antigen-binding activity after oxidation treatment to the antigen-binding activity of the unoxidized test antibodies was calculated, A10-1C04 ​​retained 83%, A23-1A05 95%, A25-2C02 100.5%, A25-3H04 98.7%, and A26-1F02 89.5% of their binding activity. These results indicate that all of the test antibodies (A10-1C04, A23-1A05, A25-2C02, A25-3H04, A26-1F02) showed stability in retaining more than 80% antigen-binding activity after forced oxidation by 1% hydrogen peroxide treatment.

[0191] Example 23: Evaluation of aggregate formation due to physical stress (agitation) The test antibody (IgG) was diluted to 0.2 mg / mL in PBS and subjected to physical stress by agitation in a batch cell mounted on an Aggregates Sizer (Shimadzu Corporation). After 30 minutes of up-and-down movement of the agitator (190 times / min) at room temperature, the aggregate concentration from 40 nm to 20 μm was measured using the Aggregates Sizer. When alkali-purified antibodies were used, the aggregate concentrations formed by agitation were 17.2 μg / mL for A10-1C04, 16.4 μg / mL for A23-1A05, 13.3 μg / mL for A25-2C02, 23.4 μg / mL for A25-3H04, and 17.0 μg / mL for A26-1F02. For all antibodies, the aggregation induced by physical stress was less than 15%, indicating that all test antibodies were stable against physical stress.

[0192] Example 24: Evaluation of antibody blood concentration profiles in mice Male C57BL6 mice (8-10 weeks old) (Charles River, Japan) were intravenously administered (3 mg / kg) with fluorescently labeled test antibodies (IgG). The concentration of the test antibodies was then measured by detecting fluorescence in the plasma. As shown in Figure 10, when using alkaline-purified antibodies, all test antibodies (A10-1C04, A23-1A05, A25-2C02, A25-3H04, A26-1F02) had an elimination half-life of 100 hours or more, indicating good blood stability.

[0193] Example 25: Evaluation of blood concentration profiles in monkeys Male cynomolgus monkeys (2-3 years old) (Hamley) were intravenously administered the test antibody (IgG) (1 mg / kg), and the concentration of the test antibody in the serum was measured using the Human Therapeutic IgG1 EIA Kit (Cayman Chemical, 500910). Alkali-purified antibody A10-1C04 ​​was administered to two cynomolgus monkeys (No. 201 and 202), and alkali-purified antibody A23-1A05 was administered to one cynomolgus monkey (No. 301). As shown in Figure 11, the elimination half-lives of A10-1C04 ​​were 16.56 days (No. 201) and 11.40 days (No. 202), and the clearance values ​​were 3.598 mL / day / kg (No. 201) and 5.451 mL / day / kg (No. 202). Furthermore, the elimination half-life of A23-1A05 was 10.87 days, and the clearance value was 10.07 mL / day / kg. All tested antibodies showed good blood stability in cynomolgus monkeys.

[0194] Example 26: Evaluation of antibody immunogenicity An in vitro T cell assay (Lonza) was performed to evaluate the immunogenicity of the test antibody (IgG). To represent the target population, 50 donors were selected. Dendritic cells derived from human peripheral blood collected from these donors were treated with 50 μg / mL of various alkaline purified antibodies, which were then incorporated into the dendritic cells. Meanwhile, CD4-positive T cells derived from human peripheral blood collected from the same donors were isolated. Subsequently, both groups, i.e., dendritic cells incorporating the test antibody, were co-cultured with CD4-positive T cells, and the response (proliferation) of the CD4-positive T cells was measured. As a negative control, the response of CD4-positive T cells obtained by performing the same procedure with a buffer (PBS) without the test antibody was compared to assess the immunogenicity risk when the antibody is administered to humans. As a result, no difference in T cell response was observed between any of the test antibodies (A10-1C04, A25-2C02, A25-3H04, A26-1F02) and the negative control.

[0195] Example 27: Evaluation of human tissue cross-reactivity The cross-reactivity of the test antibody (IgG) with human tissues (frozen sections of 35 tissues from 1 donor that meet the FDA and EMA guidelines) was evaluated by immunohistochemical staining (Covance Laboratories Ltd.). The 35 tissues include 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, 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 the alkali-purified antibody, strong staining was confirmed in vascular endothelial cells (positive control) where IL-33 expression is widely known for all test antibodies (A10-1C04, A23-1A05, A26-1F02, A25-2C02). Also, cross-reactivity to the cytoplasm or nucleus was confirmed in various tissues such as epithelial, stromal, nerve, muscle, and blood cells, but no cross-reactivity to the cell membrane was observed in any of the tissues. According to the ICH S6(R1) guideline and other papers (Toxicologic Pathology 2010, 38(7):1138-1166), cross-reactivity to the cytoplasm and nucleus where the antibody is less likely to reach in vivo is considered to have low toxicological significance. Therefore, no toxicity concerns were found for any of the test antibodies (A10-1C04, A23-1A05, A26-1F02, A25-2C02).

[0196] Example 28:Narrowing down the epitope regions of A10-1C04 and A25-3H04 The anti-IL-33 monoclonal antibodies A10-1C04 and A25-3H04 bound to the PEP14 epitope described in Example 1 above. Regarding shorter continuous amino acid sequences contained in PEP14 consisting of 20 amino acid residues, through experiments using a phage display library presenting such amino acid sequences, two types of epitopes (LEDESYEIYV (SEQ ID NO: 426 in the Sequence Listing) and EDESYEIYV (SEQ ID NO: 427 in the Sequence Listing)) were found. 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 human IL-33 shown in SEQ ID NO: 226 in the Sequence Listing. These peptides were synthesized, and the affinity with the alkali-purified antibody was calculated as K d as shown in (Table 14).

[0197] [Table 14] [Industrial Applicability]

[0198] The antibody having a neutralizing effect in the present invention can be used as a pharmaceutical composition for the diagnosis, treatment, prevention or alleviation of IL-33-related diseases.

Claims

1. It binds to an epitope consisting of an amino acid sequence containing at least six amino acid residues selected from the amino acid residues consisting of the aspartic acid residue at position 131, the glutamine residue at position 132, the serine residue at position 133, the threonine residue at position 135, the alanine residue at position 137, the leucine residue at position 138, the glutamic acid residue at position 139, the serine residue at position 142, the tyrosine residue at position 143, the glutamic acid residue at position 144, the isoleucine residue at position 145, the tyrosine residue at position 146, the glutamic acid residue at position 148, the aspartic acid residue at position 149, and the leucine residue at position 150 of sequence number 226 in the sequence listing. An IL-33 neutralizing monoclonal antibody that binds to a peptide consisting of the amino acid sequence from positions 131 to 150 of sequence number 226 in the sequence listing.

2. The IL-33 neutralizing monoclonal antibody according to claim 1, wherein the epitope is an epitope comprising an amino acid sequence containing at least six amino acid residues selected from amino acid residues consisting of a leucine residue at position 138, a glutamic acid residue at position 139, a serine residue at position 142, a tyrosine residue at position 143, a glutamic acid residue at position 144, an isoleucine residue at position 145, and a tyrosine residue at position 146.

3. The IL-33 neutralizing monoclonal antibody is one of the CDR sets listed in Table 1 below: Table 1 An IL-33 neutralizing monoclonal antibody according to claim 1 or 2, comprising the features of the IL-33 neutralizing monoclonal antibody according to claim 1 or 2.

4. The IL-33 neutralizing monoclonal antibody is A10-1C04 ​​or A25-3H04. A10-1C04 ​​includes light chain complementarity determination region 1 (LCDR1), light chain complementarity determination region 2 (LCDR2), light chain complementarity determination region 3 (LCDR3), heavy chain complementarity determination region 1 (HCDR1), heavy chain complementarity determination region 2 (HCDR2), and heavy chain complementarity determination region 3 (HCDR3), LCDR1 is the amino acid sequence of SEQ ID NO: 1, LCDR2 is the amino acid sequence of SEQ ID NO:

11. LCDR3 is the amino acid sequence of SEQ ID NO:

22. HCDR1 is the amino acid sequence of SEQ ID NO: 43, HCDR2 is the amino acid sequence of SEQ ID NO: 51, HCDR3 is the amino acid sequence of SEQ ID NO:

65. A25-3H04 includes light chain complementarity determination region 1 (LCDR1), light chain complementarity determination region 2 (LCDR2), light chain complementarity determination region 3 (LCDR3), heavy chain complementarity determination region 1 (HCDR1), heavy chain complementarity determination region 2 (HCDR2), and heavy chain complementarity determination region 3 (HCDR3), LCDR1 is the amino acid sequence of SEQ ID NO: 4, LCDR2 is the amino acid sequence of SEQ ID NO:

17. LCDR3 is the amino acid sequence of SEQ ID NO:

34. HCDR1 is the amino acid sequence of SEQ ID NO: 49, HCDR2 is the amino acid sequence of SEQ ID NO:

58. HCDR3 is the amino acid sequence of SEQ ID NO:

69. An IL-33 neutralizing monoclonal antibody according to any one of claims 1 to 3.

5. The antibody according to any one of claims 1 to 4, wherein the antibody is a chimeric antibody.

6. A pharmaceutical composition for the treatment, prevention, or alleviation of IL-33-related disease, comprising the antibody described in any one of claims 1 to 5.

7. The pharmaceutical composition according to claim 6, wherein the IL-33-related 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, bullous pemphigoid, scleroderma, ankylosing spondylitis, hepatic fibrosis (including primary biliary cirrhosis), pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), acute kidney injury, vasculitis, and cancer.

8. A cytokine expression inhibitor comprising the antibody described in any one of claims 1 to 5.

9. A cytokine expression inhibitor according to claim 8, which suppresses the expression of TNF-α, IFN-γ, IL-1β, IL-4, IL-5, IL-6, or IL-13.

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