Method of treating inflammatory conditions

Abstract

To provide a method for treating an inflammatory disease or a condition associated with or caused in part by elevated levels of IL-33 and IL-4, particularly an inflammatory lung disease.SOLUTION: The methods comprise administering to a subject in need thereof one or more therapeutically effective doses of an IL-33 antagonist, alone or in combination with one or more therapeutically effective doses of an IL - 4R antagonist. In certain embodiments, the methods of the invention encompass the use of the antagonist to treat any inflammatory disease or condition that is mediated in part by enhanced IL-33 - and IL-4-mediated signaling.SELECTED DRAWING: Figure 4A

Description

[Technical Field]

[0001] The present invention relates to a method for treating an inflammatory condition, comprising administering to a subject in need thereof a therapeutically effective amount of an interleukin-33 (IL-33) antagonist, alone or in combination with an interleukin-4 (IL-4) antagonist. More specifically, the present invention relates to treating an inflammatory or obstructive pulmonary disease or disorder by administering a therapeutically effective amount of an interleukin-33 (IL-33) antibody, alone or in combination with an interleukin-4R (IL-4R) antibody. [Background technology]

[0002] Inflammation is initiated as a defensive response by the host but can often lead to systemic pathology. In developed countries, inflammatory lung diseases such as asthma, allergies, and chronic obstructive pulmonary disease (COPD) are on the rise, significantly impacting healthcare costs. Several inflammatory cells and their mediators are involved in the development and progression of these diseases. In certain cases, these diseases reflect the outcome of type 2 immunity, characterized by tissue infiltration with eosinophils, basophils, mast cells, CD4+ T helper 2 (Th2) cells, group 2 innate lymphoid cells (ILC2s), interleukin-4 (IL-4) and / or IL-13-inducible macrophages, as well as elevated serum IgE and increased levels of the cytokines IL-4, IL-5, IL-9, and IL-13.

[0003] One cytokine thought to play a role in inflammatory lung diseases is interleukin-33 (IL-33), a proinflammatory cytokine released by damaged epithelial tissue in response to insults such as allergens, viruses, or smoke. IL-33 is a member of the interleukin-1 (IL-1) family that potently induces the production of T helper-2 (Th2)-associated cytokines (e.g., IL-4). IL-33 is expressed by a wide variety of cell types, including fibroblasts, mast cells, dendritic cells, macrophages, osteoblasts, endothelial cells, and epithelial cells. Interleukin-33 (IL-33) is a ligand for the Toll-like / interleukin-1 receptor superfamily member ST2 (sometimes referred to as "suppression of tumorigenicity 2"), which binds to the accessory protein IL-1RAcP ("interleukin-1 receptor accessory protein"; see, for reviews, e.g., J. Immunol. 2004, 103:111-114, 2004, 2005, 2006, 2007, 2008, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2021, 2022, 2023, 2024, 2025, 2026, 2027, 2028, 2030, 2031, 2032, 2033, 2034, 2035, 2036, 2037, 2038, 2040, 2041, 2042, 2043, 2044, 2045, 2046, 2047, 2048, 2049, 2050, 2051, 2052, 2053, 2054, 2055, 2056, 2057, 2058, 2060, 2061, 2062, 2063, 2064, 2065, 2066, 2067, 2068, 2069, 2070, 2071, 2072, Inhibitors of IL-33 signaling are described, for example, in U.S. Patent No. 6,273,629, ... and U.S. Patent No. 6,273,629.

[0004] Interleukin-4 (IL-4, also known as B cell stimulating factor or BSF-1) has also been implicated as a key cytokine driving allergic and T helper cell type 2 (Th2) polarized inflammatory processes. IL-4 has been shown to have a wide range of biological activities, including stimulating the proliferation of T cells, mast cells, granulocytes, megakaryocytes, and erythrocytes. IL-4 induces the expression of class II major histocompatibility complex molecules in resting B cells and the production of IgE and IgG1 isoforms by stimulated B cells. The biological activity of IL-4 is mediated by a specific cell surface receptor for IL-4. Human IL-4 receptor alpha (hIL-4R) (SEQ ID NO: 347) is described, for example, in U.S. Patent No. 5,623,299, U.S. Patent No. 5,623,299, and U.S. Patent No. 5,623,299. Antibodies against hIL-4R are described in U.S. Patent No. 5,623,299, U.S. Patent No. 5,623,299, and U.S. Patent No. 5,623,299. Methods of using antibodies against hIL-4R are described in U.S. Patent No. 5,623,299, ... and U.S. Patent No. 5,623,299.

[0005] Current therapies for treating pulmonary inflammatory diseases leave much room for improvement in safety and efficacy, especially in reducing exacerbations, for example, in patients with asthma and chronic obstructive pulmonary disease (COPD).Although many inhaled combinations of anti-inflammatory drugs and bronchodilators are available, many patients continue to experience exacerbations.Exacerbations may require the use of systemic corticosteroids, which are effective due to their broad immunoneutralizing capacity, but have undesirable side effects, including bone loss and infections.Several biological therapies, most of which target a single immune mediator, are in the late stages of development for asthma and COPD.However, the complexity of the inflammatory environment will likely limit the use of these drugs.

[0006] Thus, there is an unmet need in the art for novel combinations of therapies for treating and / or preventing inflammatory lung diseases, such as those described herein. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] US2010 / 0260770 [Patent Document 2] US2009 / 0041718 [Patent Document 3] US$9,453,072 [Patent Document 4] US8187596 [Patent Document 5] US2013 / 17373761 [Patent Document 6] US2014 / 0212412 [Patent Document 7] US2014 / 0271658 [Patent Document 8] US2014 / 0271642 [Patent Document 9] US2014 / 0004107 [Patent Document 10] WO2015 / 099175 [Patent Document 11] WO2015 / 106080 [Patent Document 12] WO2011 / 031600 [Patent Document 13] WO2014 / 164959 [Patent Document 14] WO2014 / 152195 [Patent Document 15] WO2013 / 165894 [Patent Document 16] WO2013 / 173761 [Patent Document 17] EP1725261 [Patent Document 18] EP10815921A1 [Patent Document 19] EP2850103A2 [Patent Document 20] U.S. Patent No. 5,599,905 [Patent Document 21] U.S. Patent No. 5,767,065 [Patent Document 22] U.S. Patent No. 5,840,869 [Patent Document 23] U.S. Patent No. 5,717,072 [Patent Document 24] U.S. Patent No. 7,186,809 [Patent Document 25] U.S. Patent No. 7,605,237 [Patent Document 26] U.S. Patent No. 5,714,146 [Patent Document 27] U.S. Patent No. 5,985,280 [Patent Document 28] U.S. Patent No. 6,716,587 [Patent Document 29] U.S. Patent No. 9,290,574 [Non-patent literature]

[0008] [Non-Patent Document 1] Kakkar and Lee, Nature Reviews-Drug Discovery7(10):827-840(2008) [Non-patent document 2] Schmitz et al., Immunity23:479-490(2005) [Non-patent document 3] Liew et al.,Nature Reviews-Immunology10:103-110(2010) Summary of the Invention

[0009] According to certain aspects of the present invention, there is provided a method for treating an inflammatory disease or disorder or at least one symptom associated with an inflammatory disease or disorder, comprising administering to a subject in need thereof one or more doses of a therapeutically effective amount of an interleukin-33 (IL-33) antagonist, alone or in combination with one or more doses of a therapeutically effective amount of an interleukin-4 (IL-4) antagonist, or administering to a patient in need thereof a pharmaceutical composition comprising an IL-33 antagonist and an IL-4α antagonist. In one embodiment, administration of the IL-33 antagonist in combination with the IL-4 antagonist results in enhanced therapeutic efficacy compared to the therapeutic efficacy observed with administration of the IL-33 antagonist alone or the IL-4 antagonist alone.

[0010] In certain embodiments, an IL-33 antagonist is any agent capable of blocking, attenuating, or disrupting IL-33 signaling and / or the interaction between IL-33 and a cellular receptor (e.g., ST2) or co-receptor (e.g., IL-1RAcP) or complexes thereof, any of which may block or inhibit at least one biological activity of IL-33.

[0011] In certain embodiments, the IL-33 antagonist is an antibody that binds to or interacts with IL-33, blocks the interaction of IL-33 with its receptor ST2, prevents or inhibits the interaction of ST2 with the co-receptor IL1-RAcP, or prevents the formation of a signaling complex. In one embodiment, the IL-33 antagonist is a monoclonal antibody that binds to or specifically interacts with human IL-33. In one embodiment, the IL-33 antagonist is a receptor-based trap that binds to or specifically interacts with human IL-33.

[0012] In one embodiment, the IL-4 antagonist is an interleukin-4 receptor (IL-4R) antagonist.

[0013] In one embodiment, the IL-4R antagonist is any agent that binds to or interacts with IL-4Rα or an IL-4R ligand and inhibits or attenuates the normal biological signaling function of type 1 and / or type 2 IL-4 receptors. In one embodiment, the IL-4R antagonist is a monoclonal antibody that specifically binds to human IL-4Rα. In one embodiment, the IL-4R antagonist is a monoclonal antibody that binds to IL-4Rα and blocks both IL-4- and IL-13-mediated signaling through either type I or type II receptors. In one embodiment, the monoclonal antibody that specifically binds to human IL-4Rα and blocks both IL-4- and IL-13-mediated signaling is dupilumab or a biological equivalent thereof. In one embodiment, the method for treating an inflammatory disorder or condition involves administering to a subject an antibody of SEQ ID NO: 274 / 2 This is achieved by using a combination of REGN3500, which has a heavy chain variable region / light chain variable region (HCVR / LCVR) amino acid sequence pair of 82, and dupilumab, which has the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 337 / 338.

[0014] In one embodiment, the inflammatory disease or disorder treatable by the methods of the invention is selected from the group consisting of asthma, chronic obstructive pulmonary disease (COPD), asthma and COPD overlap syndrome (ACOS), atopic dermatitis, nasal polyps, allergic reactions, chronic bronchitis, emphysema, chronic rhinosinusitis with or without nasal polyps, inflammatory bowel disease, Crohn's disease, ulcerative colitis, hypersensitivity pneumonitis, multiple sclerosis, arthritis (osteoarthritis, rheumatoid arthritis, and psoriatic arthritis), allergic rhinitis, fibrosis, eosinophilic esophagitis, vasculitis, urticaria, Churg-Strauss syndrome, inflammatory pain, and psoriasis.

[0015] In one embodiment, the asthma is eosinophilic asthma.

[0016] In one embodiment, the asthma is non-eosinophilic asthma.

[0017] In one embodiment, the asthma is allergic asthma.

[0018] In one embodiment, the asthma is non-allergic asthma.

[0019] In one embodiment, the asthma is severe refractory asthma.

[0020] In one embodiment, the asthma is steroid-resistant asthma.

[0021] In one embodiment, the asthma is steroid-sensitive asthma.

[0022] In one embodiment, the asthma is steroid-refractory asthma.

[0023] In one embodiment, the asthma is an asthma exacerbation.

[0024] In one embodiment, the inflammatory disease or disorder is alleviated or reduced in severity, duration, or frequency of occurrence, or at least one symptom associated with the inflammatory disease or disorder is alleviated or reduced in severity, duration, or frequency of occurrence.

[0025] In one embodiment, administering to a subject in need thereof one or more doses of a therapeutically effective amount of an IL-33 antagonist, alone or in combination with one or more doses of a therapeutically effective amount of an IL-4R antagonist, results in enhanced therapeutic efficacy as measured by any one or more of the following parameters: a) a decrease in the frequency of one or more of eosinophils, activated B cells, activated CD8+ T cells, or the CD4 / CD8+ T cell ratio in a tissue sample; b) a decrease in one or more of interleukin-1 beta (IL-1β), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-13 (IL-13), monocyte chemoattractant protein-1 (MCP-1), or tumor necrosis factor alpha (TNFα) levels in a tissue sample; or c) A decrease in the expression level of one or more of the genes Il4, Il5, Il6, Il9, Il13, Il1rl1, Il13ra2, tnf, Tgfb1, Ccl2, Ccl11, Ccl24, Col15a1, or Col24a1 in the tissue sample.

[0026] In one embodiment, the therapeutic effect is further measured by one or more of the following: Administering one or more doses of a therapeutically effective amount of an IL-33 antagonist alone or in combination with one or more doses of an IL-4R antagonist to a subject in need thereof results in enhanced therapeutic efficacy: d) decreased serum IgE levels; e) reduced goblet cell metaplasia in the lungs; f) Improvement in lung consolidation, or g) Decreased subepithelial fibrosis in the lungs.

[0027] In one embodiment, the tissue sample is obtained from the lung.

[0028] In one embodiment, the tissue sample is selected from the group consisting of liver, kidney, heart, or whole blood. In certain embodiments, blood cells, serum, or plasma may be used to measure one or more of the above parameters.

[0029] In one embodiment, the chronic obstructive pulmonary disease treatable by the methods of the invention is exacerbated by one or more of the following: asthma, a viral disease, a bacterial infection, exposure to an allergen, exposure to a chemical or chemical fumes, or exposure to an environmental irritant or air pollution.

[0030] In related embodiments, the asthma treatable by the methods of the invention is exacerbated by one or more of the following: a viral illness, a bacterial infection, exposure to an allergen, exposure to a chemical or chemical fumes, or exposure to an environmental irritant or air pollution.

[0031] In certain embodiments, the asthma treatable by the methods of the present invention is selected from the group consisting of eosinophilic asthma, noneosinophilic asthma, steroid-resistant asthma, and steroid-sensitive asthma.

[0032] In one embodiment, the chronic obstructive pulmonary disease treatable by the methods of the invention is caused by or exacerbated in part by tobacco smoke.

[0033] In one embodiment, patients suffering from chronic obstructive pulmonary disease treatable by the methods of the present invention may or may not exhibit elevated eosinophil counts.

[0034] In one embodiment, patients suffering from asthma and COPD overlap syndrome (ACOS) treatable by the methods of the present invention may or may not exhibit elevated eosinophil counts.

[0035] A second aspect of the present invention provides for treating an inflammatory disease or disorder, or at least one symptom associated with an inflammatory disease or disorder, by administering an effective amount of one or more additional therapeutic agents useful for alleviating the inflammatory disease or disorder, or at least one symptom associated with the inflammatory disease or disorder, in combination with a therapeutically effective amount of an interleukin-33 (IL-33) antagonist, e.g., an IL-33 antibody or IL-33 trap, and a therapeutically effective amount of an interleukin-4 (IL-4) antagonist, e.g., an IL-4R antibody such as dupilumab, or a therapeutic equivalent thereof.

[0036] In one embodiment, the one or more additional therapeutic agents are a nonsteroidal anti-inflammatory drug (NSAID), a corticosteroid (e.g., an inhaled corticosteroid or ICS), a long-acting beta-2 adrenergic agonist (LABA), a long-acting muscarinic antagonist (LAMA), a bronchodilator, an antihistamine, epinephrine, a decongestant, a thymic stromal lymphopoietin (TSLP) antagonist, an IL-1 antagonist, an IL-8 antagonist, an IL-13 antagonist, an IL-4 antagonist, an IL-4 / IL-13 antagonist, or an IL-14 antagonist. Dual antagonists, IL-33 / IL-13 dual antagonists, IL-5 antagonists, IL-6 antagonists, IL-12 / 23 antagonists, IL-22 antagonists, IL-25 antagonists, IL-17 antagonists, IL-31 antagonists, TNF inhibitors, IgE inhibitors, leukotriene inhibitors, oral PDE4 inhibitors, methylxanthines, nedocromil sodium, cromolyn sodium, long-acting beta 2 agonist, and another IL-33 antagonist (e.g., a different antibody to IL-33, a different IL-33 receptor-based trap, an ST2 antagonist (including an antibody to ST2), a soluble ST2 receptor, or an antagonist to another IL-33 receptor other than ST2, or an IL-1RAcP antagonist (including an antibody to IL-1RAcP), or an antibody that interacts with the IL-33 / ST2 complex).

[0037] In some embodiments, the invention provides methods for treating moderate to severe chronic obstructive pulmonary disease (COPD), comprising co-administering an IL-4R antagonist (e.g., dupilumab) and an IL-33 antagonist (e.g., REGN3500) in addition to background therapy including, for example, inhaled corticosteroids (ICS) and / or long-acting beta-2 adrenergic agonists (LABAs) and / or long-acting muscarinic antagonists (LAMAs).

[0038] In certain embodiments, the present invention provides a method for treating asthma by administering a "loss of asthma control" to a patient. The present invention provides a method for reducing the incidence of long-term asthma control (LOAC) events, comprising treating a patient suffering from asthma with an IL-4R antagonist (e.g., dupilumab) in combination with an IL-33 antagonist (e.g., REGN3500). In certain embodiments, the use of an IL-4R antagonist in combination with an IL-33 antagonist provides more effective results than administering either the IL-4R antagonist alone or the IL-33 antagonist alone.

[0039] In a related embodiment, administration of an IL-33 antagonist in combination with an IL-4R antagonist results in an increase in type 1 immune response and / or a decrease in type 2 immune response induced by the disease or by the causative agent of the disease or allergy.

[0040] A third aspect of the present invention provides a method for treating a fibrotic disease or disorder or at least one symptom associated with a fibrotic disease or disorder, comprising administering to a patient in need thereof a combination of an IL-33 antagonist (IL-33 antibody or IL-33 trap) that specifically binds to IL-33 and an antibody that specifically binds to IL-4Rα or an antigen-binding fragment thereof, or a pharmaceutical composition comprising an IL-33 antagonist and an IL-4α antagonist, wherein the fibrotic disease or disorder is alleviated or reduced in severity or duration, or at least one symptom associated with the fibrotic disease or disorder is alleviated or reduced in severity, duration, or frequency of occurrence. In one embodiment, treating a fibrotic disease with an IL-33 antagonist in combination with an IL-4R antagonist can result in the restoration of fibrotic tissue to its normal state.

[0041] In one embodiment, fibrotic diseases or disorders treatable by administering an anti-IL-33 and IL-4R antagonist of the invention, such as an IL-33 antibody or IL-33 trap in combination with an IL-4Rα antibody described herein, include pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis, bleomycin-induced pulmonary fibrosis, asbestos-induced pulmonary fibrosis, and bronchiolitis obliterans), chronic asthma, fibrosis associated with acute lung injury and acute respiratory distress (e.g., bacterial pneumonia-induced fibrosis, trauma-induced fibrosis, viral pneumonia-induced fibrosis, Included are ventilator-induced fibrosis, non-pulmonary sepsis-induced fibrosis, and aspiration-induced fibrosis, silicosis, radiation-induced fibrosis, chronic obstructive pulmonary disease (COPD, which may or may not be associated with, caused in part by, or attributable to exposure to first- or second-hand smoke), scleroderma, ocular fibrosis, dermal fibrosis (e.g., scleroderma), liver fibrosis (e.g., cirrhosis, alcohol-induced liver fibrosis, non-alcoholic steatohepatitis (NASH), bile duct injury, primary biliary cirrhosis, infection- or virus-induced liver fibrosis, autoimmune hepatitis, renal (kidney) fibrosis, cardiac fibrosis, atherosclerosis, stent restenosis, and bone marrow fibrosis.

[0042] A fourth aspect of the present invention provides a method for preventing or reducing the severity of an allergic reaction, comprising administering to a subject in need thereof one or more doses of a therapeutically effective amount of an IL-33 antagonist in combination with one or more doses of a therapeutically effective amount of an IL-4R antagonist, wherein administration of the combination results in enhanced therapeutic efficacy for preventing or reducing the severity of an allergic reaction compared to the therapeutic efficacy observed with administration of the IL-33 antagonist alone or the IL-4R antagonist alone. A subject treated with an IL-33 antagonist in combination with an IL-4R antagonist may exhibit reduced sensitivity or reduced allergic response to an allergen, or may not experience sensitivity or an allergic response or an anaphylactic response to an allergen, after administration of the combination of an IL-33 antagonist and an IL-4R antagonist or a composition comprising these antagonists.

[0043] In one embodiment, the IL-33 antagonist for use in the methods of the present invention is a monoclonal antibody or antigen-binding fragment thereof that binds to or specifically interacts with human IL-33. The IL-33 antibody or antigen-binding fragment thereof may block the interaction of IL-33 with ST2 or enable low-affinity binding of IL-33 to the ST2 receptor. In doing so, ST2 may be prevented from interacting with IL-1RAcP. Thus, the IL-33 antibodies of the present invention are particularly useful for inhibiting IL-33-mediated signal transduction and treating diseases and disorders caused by or associated with IL-33 activity and / or IL-33 signaling.

[0044] In one embodiment, the IL-33 antibody or antigen-binding fragment thereof reduces the frequency of one or more of eosinophils, CD4+ T cells, B cells, ST2+ / CD4+ cells in a T cell population, or reduces the CD4 / CD8 T cell ratio in the lung when administered to a mammal with allergen-induced pulmonary inflammation.

[0045] In one embodiment, the IL-33 antibody or antigen-binding fragment thereof reduces the expression levels of one or more of IL-4, IL-5, IL-6, IL-9, IL-13, Ccl2, Ccl11, Ccl24, or MCP-1 in the lung when administered to a mammal with allergen-induced pulmonary inflammation.

[0046] In one embodiment, the IL-33 antibody or antigen-binding fragment thereof reduces serum IgE levels, goblet cell metaplasia, or epithelial collagen thickness in the lung when administered to a mammal with allergen-induced pulmonary inflammation.

[0047] In certain embodiments, IL-33 antibodies or antigen-binding fragments thereof that specifically bind to human IL-33 that can be used in the methods of the present invention are those selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 220, 222, 224, 226, 228, 229, 230, 232, 234, 236, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, and three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, and 316.

[0048] In certain embodiments, an IL-33 antibody or antigen-binding fragment thereof that specifically binds human IL-33 that can be used in the methods of the present invention comprises a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, and 308, or a sequence substantially similar thereto with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0049] According to certain embodiments, the anti-IL-33 antibody or antigen-binding fragment thereof for use in the methods of the invention comprises a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, and 316, or a sequence substantially similar thereto with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0050] According to certain embodiments, an anti-IL-33 antibody or antigen-binding fragment thereof for use in the methods of the invention comprises a HCVR and LCVR (HCVR / LCVR) sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 106, 114 / 122, 130 / 138, 146 / 154, 162 / 170, 178 / 186, 194 / 202, 210 / 218, 226 / 234, 242 / 250, 258 / 266, 274 / 282, 290 / 298, and 308 / 316.

[0051] According to certain embodiments, the anti-IL-33 antibody or antigen-binding fragment thereof for use in the methods of the present invention comprises a heavy chain CDR3 (HCDR3) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216, 232, 248, 264, 280, 296, and 314, or at least 90%, at least 95%, at least 98%, or at least 99%, or at least 100%, or at least 101%, or at least 102%, or at least 103%, or at least 104%, or at least 105%, or at least 106%, or at least 108%, or at least 109%, or at least 110%, or at least 111%, or at least 112%, or at least 113%, or at least 114%, or at least 115%, or at least 116%, or at least 117%, or at least 118%, or at least 119%, or at least 119%, or at least 119%, or at least 120%, or at least 121%, or at least 122%, or at least 123%, or at least 124%, or at least 125%, or at least 126%, or at least 127%, or at least 128%, or at least 129%, or at least 130%, or at least 131%, or at least 132%, or at least 133%, or at least 134%, or at least 135%, or at least 135%, or at least 136%, or at least 137%, or at least 138%, or at least 1 and a light chain CDR3 (LCDR3) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 208, 224, 240, 256, 272, 288, 304, and 322, or a sequence substantially similar thereto with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0052] According to certain embodiments, an anti-IL-33 antibody or antigen-binding fragment thereof for use in the methods of the invention comprises an HCDR3 / LCDR3 amino acid sequence pair selected from the group consisting of SEQ ID NOs: 8 / 16, 24 / 32, 40 / 48, 56 / 64, 72 / 80, 88 / 96, 104 / 112, 120 / 128, 136 / 144, 152 / 160, 168 / 176, 184 / 192, 200 / 208, 216 / 224, 232 / 240, 248 / 256, 264 / 272, 280 / 288, 296 / 304, and 314 / 322.

[0053] According to certain embodiments, the anti-IL-33 antibody or antigen-binding fragment thereof for use in the methods of the present invention comprises a heavy chain CDR1 (HCDR1) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 228, 244, 260, 276, 292, and 310, or at least 90% substantially similar thereto. a heavy chain CDR2 (HCDR2) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214, 230, 246, 262, 278, 294, and 312, or a heavy chain CDR2 (HCDR2) domain having an amino acid sequence at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto; a light chain CDR1 (LCDR1) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220, 236, 252, 268, 284, 300, and 318, or a sequence substantially similar thereto with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and a light chain CDR2 (LCDR2) domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 206, 222, 238, 254, 270, 286, 302, and 320, or a sequence substantially similar thereto with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0054] Certain non-limiting exemplary anti-IL-33 antibodies and antigen-binding fragments that can be used in the methods of the present invention are those set forth in SEQ ID NOs: 4-6-8-12-14-16 (e.g., H1M9559N), 20-22-24-28-30-32 (e.g., H1M9566N), 36-38-40-44-46-48 (e.g., H1M9568N), 52-54-56-60-62-64 (e.g., H4H9629P), 68-70-72-76-78-80 (e.g., H4H9633P). , 84-86-88-92-94-96 (e.g., H4H9640P), 100-102-104-108-110-112 (e.g., H4H9659P), 116-118-120-124-126-128 (e.g., H4H9660P), 132-134-136-140-142-144 (e.g., H4H9662P), 148-150-152-156-158-160 (e.g., H4H9663P), 164-166-168-172-174-176 (e.g., For example, H4H9664P), 180-182-184-188-190-192 (for example, H4H9665P), 196-198-200-204-206-208 (for example, H4H9666P), 212-214-216-220-222-224 (for example, H4H9667P), 228-230-232-236-238-240 (for example, H4H9670P), 244-246-248-252-254-256 (for example, H4H9671P), 260-262- and HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains, each having an amino acid sequence selected from the group consisting of 264-268-270-272 (e.g., H4H9672P), 276-278-280-284-286-288 (e.g., H4H9675P), 292-294-296-300-302-304 (e.g., H4H9676P), and 310-312-314-318-320-322 (H1M9565N).

[0055] According to certain embodiments, the anti-IL-33 antibodies or antigen-binding fragments thereof for use in the methods of the invention, e.g., for treating an inflammatory condition, are selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 106, 114 / 122, 130 / 138, 146 / 154, 162 / 170, 178 / 186, 194 / 200, 210 / 212, 220 / 224, 230 / 238, 246 / 254, 256 / 260, 262 / 270, 278 / 286, 294 / 296, 300 / 310, 312 / 320, 322 / 330, 330 / 340, 340 / 350, 356 / 360, 370 / 380, 372 / 390, 384 / 396, 394 / 400, 410 / 410, 420 / 420, 430 / 440, 450 / 460, 470 / 480, 480 / 490, 490 / 500, 510 / 520, 530 / 540, 550 / 560, 570 / 580, 580 / 590, 600 / 610, 610 / 620, 620 / 630, 630 / 640, 640 / 650, 650 / 660, 660 / 700, 670 / 710, 670 / 720, 67 The heavy and light chain CDR domains contained within the heavy chain variable region and light chain variable region (HCVR / LCVR) sequences selected from the group consisting of 02, 210 / 218, 226 / 234, 242 / 250, 258 / 266, 274 / 282, 290 / 298, and 308 / 316. Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specific HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary rules that can be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. Generally, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997), and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within antibodies.

[0056] In one embodiment, an IL-33 antibody or antigen-binding fragment for use in the methods of the invention comprises the heavy and light chain CDRs of the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 274 / 282.

[0057] In a related embodiment, an IL-33 antibody or antigen-binding fragment for use in the methods of the invention comprises the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains of SEQ ID NOs: 276-278-280-284-286-288, respectively.

[0058] In one embodiment, an antibody or antigen-binding fragment thereof that specifically binds human interleukin-33 (IL-33) for use in the methods of the invention comprises (a) a heavy chain variable region (HCVR) having the amino acid sequence of SEQ ID NO: 274, and (b) a light chain variable region (LCVR) having the amino acid sequence of SEQ ID NO: 282.

[0059] In one embodiment, an IL-33 antibody or antigen-binding fragment for use in the methods of the invention comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 274 / 282.

[0060] In one embodiment, an IL-33 antibody or antigen-binding fragment thereof for use in the methods of the invention interacts with an amino acid sequence ranging from about position 1 to about position 12 of SEQ ID NO: 349 and / or an amino acid sequence ranging from about position 50 to about position 94 of SEQ ID NO: 349, as determined by hydrogen / deuterium exchange.

[0061] In one embodiment, an IL-33 antibody or antigen-binding fragment thereof for use in the methods of the invention interacts with an amino acid sequence ranging from about position 112 to about position 123 of SEQ ID NO: 348 and / or an amino acid sequence ranging from about position 161 to about position 205 of SEQ ID NO: 348, as determined by hydrogen / deuterium exchange.

[0062] In one embodiment, an IL-33 antibody or antigen-binding fragment thereof for use in the methods of the invention interacts with either the amino acid sequence of SEQ ID NO: 350 or the amino acid sequence of SEQ ID NO: 351, or both SEQ ID NOs: 350 and 351, as determined by hydrogen / deuterium exchange.

[0063] In one embodiment, an IL-33 antibody or antigen-binding fragment thereof for use in the methods of the invention competes for binding to IL-33 with a reference antibody comprising the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 274 / 282.

[0064] In one embodiment, an IL-33 antibody or antigen-binding fragment thereof for use in the methods of the invention binds to the same epitope on IL-33 as a reference antibody comprising the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 274 / 282.

[0065] In a fifth aspect, the present invention provides nucleic acid molecules encoding anti-IL-33 antibodies or antigen-binding fragments thereof for use in the methods of the present invention. Recombinant expression vectors harboring nucleic acids of the present invention and host cells into which such vectors have been introduced are also encompassed by the present invention, as are methods of producing the antibodies by culturing host cells under conditions that allow antibody production and recovering the produced antibodies.

[0066] In one embodiment, the invention provides a method of using an antibody or fragment thereof that specifically binds human IL-33, wherein the antibody or fragment thereof comprises an HCVR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 17, 33, 49, 65, 81, 97, 113, 129, 145, 161, 177, 193, 209, 225, 241, 257, 273, 289, and 307, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology thereto.

[0067] The present invention also provides methods of using antibodies or fragments thereof that specifically bind to human IL-33, wherein the antibodies or fragments thereof comprise an LCVR encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 9, 25, 41, 57, 73, 89, 105, 121, 137, 153, 169, 185, 201, 217, 233, 249, 265, 281, 297, and 315, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology thereto.

[0068] The present invention also relates to a HCDR3 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 7, 23, 39, 55, 71, 87, 103, 119, 135, 151, 167, 183, 199, 215, 231, 247, 263, 279, 295, and 313, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology thereto, and a HCDR3 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 15, 31, 47, 63, 79, 95, and an LCDR3 domain encoded by a nucleotide sequence selected from the group consisting of 111, 127, 143, 159, 175, 191, 207, 223, 239, 255, 271, 287, 303, and 321, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology thereto.

[0069] The present invention also relates to a method for detecting a HCDR1 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 19, 35, 51, 67, 83, 99, 115, 131, 147, 163, 179, 195, 211, 227, 243, 259, 275, 291, and 309, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology thereto, and a method for detecting a HCDR1 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 5, 21, HCDR2 domains encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 11, 27, 43, 59, 75, 91, 107, 37, 53, 69, 85, 101, 117, 133, 149, 165, 181, 197, 213, 229, 245, 261, 277, 293, and 311, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology thereto; and and a LCDR2 domain encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 13, 29, 45, 61, 77, 93, 109, 125, 141, 157, 173, 189, 205, 221, 237, 253, 269, 285, 301, and 319, or a substantially identical sequence having at least 90%, at least 95%, at least 98%, or at least 99% homology thereto.

[0070] According to certain embodiments, the methods of the present invention involve the use of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 and 9 (e.g., H1M9559N), SEQ ID NOs: 17 and 25 (e.g., H1M9566N), SEQ ID NOs: 33 and 41 (e.g., H1M9568N), SEQ ID NOs: 49 and 57 (e.g., H4H9629P), SEQ ID NOs: 65 and 73 (e.g., H4H9633P), SEQ ID NOs: 81 and 89 (e.g., H4H9640P), SEQ ID NOs: 97 and 105 (e.g., H4H9659P), SEQ ID NOs: 113 and 121 (e.g., H4H9660P), SEQ ID NOs: 129 and 137 (e.g., H4H9662P), SEQ ID NOs: 145 and 153 (e.g., H4H9663P), SEQ ID NOs: 161 and 169 (e.g., H4H9664P), SEQ ID NOs: The present invention provides the use of an antibody or fragment thereof that specifically binds to human IL-33, comprising heavy and light chain CDR sequences encoded by the nucleic acid sequence of SEQ ID NOs: 177 and 185 (e.g., H4H9665P), SEQ ID NOs: 193 and 201 (e.g., H4H9666P), SEQ ID NOs: 209 and 217 (e.g., H4H9667P), SEQ ID NOs: 225 and 233 (e.g., H4H9670P), SEQ ID NOs: 241 and 249 (e.g., H4H9671P), SEQ ID NOs: 257 and 265 (e.g., H4H9672P), SEQ ID NOs: 273 and 281 (e.g., H4H9675P), SEQ ID NOs: 289 and 297 (e.g., H4H9676P), or SEQ ID NO: 307 and 315 (H1M9565N).

[0071] In one embodiment, an IL-33 antagonist for use in the methods of the invention is an IL-33 receptor-based trap, such as those described herein (see FIG. 1).

[0072] In one embodiment, the IL-33 receptor-based trap comprises a first IL-33 binding domain (D1) linked to a multimerization domain (M), wherein D1 comprises the IL-33 binding portion of an ST2 protein.

[0073] In one embodiment, an IL-33 trap for use in the methods of the invention further comprises a second IL-33 binding domain (D2) attached to D1 and / or M, wherein D2 comprises the extracellular portion of an IL-1RAcP protein. In one embodiment, D1 is attached to the N-terminus of M. In one embodiment, D1 is attached to the C-terminus of M. In one embodiment, D2 is attached to the N-terminus of M. In one embodiment, D2 is attached to the C-terminus of M. In one embodiment, D1 is attached to the N-terminus of D2 and D2 is attached to the N-terminus of M.

[0074] In one embodiment, D1 comprises the amino acid sequence of SEQ ID NO: 328 or 329, or an amino acid sequence having at least 90% identity thereto. In one embodiment, D2 comprises the amino acid sequence of SEQ ID NO: 330 or 331, or an amino acid sequence having at least 90% identity thereto.

[0075] In one embodiment, the IL-33 antagonist for use in the methods of the present invention is and a second IL-33 binding domain (D2) linked to a second multimerization domain (M2), wherein the D1 and / or D2 domains comprise an IL-33 binding portion of a receptor selected from the group consisting of ST2 and IL-1RAcP.

[0076] In one embodiment, an IL-33 antagonist for use in the methods of the invention comprises a third IL-33 binding domain (D3) linked to either D1 or M1, wherein D3 comprises an IL-33 binding portion of a receptor selected from the group consisting of ST2 and IL-1RAcP.

[0077] In one embodiment, an IL-33 antagonist for use in the methods of the invention comprises a fourth IL-33 binding domain (D4) linked to either D2 or M2, wherein D4 comprises an IL-33 binding portion of a receptor selected from the group consisting of ST2 and IL-1RAcP.

[0078] In one embodiment, D1 is attached to the N-terminus of M1 and D2 is attached to the N-terminus of M2.

[0079] In one embodiment, D3 is attached to the N-terminus of D1.

[0080] In one embodiment, D3 is attached to the C-terminus of M1.

[0081] In one embodiment, D4 is attached to the N-terminus of D2.

[0082] In one embodiment, D4 is attached to the C-terminus of M2.

[0083] In one embodiment, D3 is attached to the N-terminus of D1, D1 is attached to the N-terminus of M1, D4 is attached to the N-terminus of D2, and D2 is attached to the N-terminus of M2.

[0084] In one embodiment, D3 is the same as or substantially the same as D4, and D1 is the same as or substantially the same as D2.

[0085] In one embodiment, D3 and D4 each comprise an IL-33 binding portion of an ST2 protein, and D1 and D2 each comprise an extracellular portion of an IL-1RAcP protein.

[0086] In one embodiment, an IL-33 trap for use in the methods of the invention comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 323, 324, 325, 326, and 327.

[0087] In one embodiment, the IL-4 antagonist for use in the methods of the invention is an interleukin-4 receptor (IL-4R) antagonist.

[0088] In one embodiment, the IL-4R antagonist for use in the methods of the invention is an antibody or antigen-binding fragment thereof that binds to IL-4Rα and prevents the interaction of IL-4 and / or IL-13 with type 1 or type 2 IL-4 receptors.

[0089] In a related embodiment, the IL-4R antibody or antigen-binding fragment thereof for use in the methods of the invention interferes with the interaction of IL-4 and / or IL-13 with both type 1 and type 2 IL-4 receptors.

[0090] In one embodiment, the IL-4R antagonist for use in the methods of the present invention is a monoclonal antibody that specifically binds to human IL-4Rα.

[0091] In one embodiment, the monoclonal antibody that specifically binds human IL-4Rα for use in the methods of the invention is dupilumab or a biological equivalent thereof.

[0092] In certain embodiments, an IL-4R antibody or antigen-binding fragment thereof for use in the methods of the present invention comprises a heavy chain complementarity-determining region (HCDR) of the heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 335 or SEQ ID NO: 337, and a light chain complementarity-determining region (LCDR) of the light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 336 or SEQ ID NO: 338.

[0093] In a related embodiment, an IL-4R antibody or antigen-binding fragment thereof for use in the methods of the invention comprises three HCDRs (HCDR1, HCDR2, and HCDR3) and three LCDRs (LCDR1, LCDR2, and LCDR3), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 339, HCDR2 comprises the amino acid sequence of SEQ ID NO: 340, HCDR3 comprises the amino acid sequence of SEQ ID NO: 341, LCDR1 comprises the amino acid sequence of SEQ ID NO: 342, LCDR2 comprises the amino acid sequence of SEQ ID NO: 343, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 344.

[0094] In one embodiment, an IL-4R antibody or antigen-binding fragment thereof for use in the methods of the invention comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 335 or SEQ ID NO: 337, and an LCVR comprising the amino acid sequence of SEQ ID NO: 336 or SEQ ID NO: 338.

[0095] In one embodiment, an IL-4R antibody or antigen-binding fragment thereof for use in the methods of the invention comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 335 / 336 or SEQ ID NOs: 337 / 338.

[0096] In a related embodiment, the IL-4R antagonist for use in the methods of the invention is dupilumab (SEQ ID NOs: 337 / 338) or a biological equivalent thereof.

[0097] In certain embodiments, the IL-33 antagonist and the IL-4R antagonist are administered in separate formulations.

[0098] In certain embodiments, the IL-33 antagonist and the IL-4R antagonist are co-formulated for administration to a patient in need thereof.

[0099] In certain embodiments, the IL-33 antagonist and the IL-4R antagonist are administered to the subject subcutaneously, intravenously, intramuscularly, or intranasally.

[0100] The IL-33 and IL-4R antibodies of the present invention can be full-length (e.g., IgG1 or IgG4 antibodies) or may comprise only the antigen-binding portion (e.g., a Fab fragment, a F(ab')2 fragment, or an scFv fragment) and may be modified to affect functionality, for example, to remove residual effector function (Reddy et al., 2000, J. Immunol. 164:1925-1933).

[0101] In one embodiment, a compound that specifically binds to human interleukin-33 or human IL-4R The antibody is isolated as a fully human monoclonal antibody.

[0102] In a sixth aspect, the present invention provides a pharmaceutical composition comprising a recombinant human antibody or fragment thereof or trap that specifically binds to IL-33, or an antibody that specifically binds to IL-4R, and a pharmaceutically acceptable carrier. In a related aspect, the present invention features a composition that is a combination of an anti-IL-33 antibody, an IL-33 trap, or an antibody that specifically binds to IL-4R, and one or more additional therapeutic agents. In one embodiment, the one or more additional therapeutic agents are any agent that is advantageously combined with either or both of an IL-33 antagonist and / or an IL-4R antagonist. Exemplary agents that may be advantageously combined with IL-33 antagonists and / or IL-4R antagonists include, but are not limited to, other agents that inhibit IL-33 activity and / or IL-4 activity (such as other antibodies or antigen-binding fragments thereof, peptide inhibitors, small molecule antagonists, etc.) and / or agents that do not directly bind to IL-33 or IL-4 or IL-4R but still interfere with, block, or attenuate IL-33- or IL-4-mediated signaling.In one embodiment, the one or more additional therapeutic agents are a nonsteroidal anti-inflammatory drug (NSAID), a corticosteroid (e.g., an inhaled corticosteroid), a bronchodilator, an antihistamine, epinephrine, a decongestant, a thymic stromal lymphopoietin (TSLP) antagonist, an IL-1 antagonist, an IL-8 antagonist, an IL-13 antagonist, a different IL-4 antagonist, an IL-4 / IL-13 dual antagonist, an IL-33 / IL-13 dual antagonist, an IL-5 antagonist, an IL-6 antagonist, an IL-12 / 23 antagonist, an IL-22 antagonist, or an IL-23 antagonist. The therapeutic agent may be selected from the group consisting of an IL-25 antagonist, an IL-17 antagonist, an IL-31 antagonist, a TNF inhibitor, an IgE inhibitor, a leukotriene inhibitor, an oral PDE4 inhibitor, a methylxanthine, nedocromil sodium, cromolyn sodium, a long-acting beta-2 agonist (LABA), a long-acting muscarinic antagonist (LAMA), an inhaled corticosteroid (ICS), and another IL-33 antagonist or an IL-4 antagonist or a different antibody to IL-33 or IL-4 or IL-4R, and another IL-33 antagonist.

[0103] In certain embodiments, the cytokine antagonist may be a small molecule inhibitor (synthetic or naturally occurring) or a protein (e.g., an antibody) that interacts with either the cytokine itself or its receptor, or a complex comprising both the cytokine and its receptor(s). Additional combination therapies and co-formulations involving the anti-IL-33 and / or IL-4R antibodies of the invention are disclosed elsewhere herein.

[0104] In yet another aspect, the present invention provides a therapeutic method for inhibiting IL-33 and / or IL-4 signaling activity using an anti-IL-33 antagonist (such as an IL-33 antibody or IL-33 trap) and an IL-4R antibody or antigen-binding fragments of one or more antibodies of the present invention, comprising administering a pharmaceutical composition containing a therapeutically effective amount of an IL-33 antibody or IL-33 trap, either alone or in combination with an IL-4R antibody or antigen-binding fragments of one or more antibodies of the present invention. The disorder being treated is any disease or condition that is ameliorated, inhibited, or prevented by IL-33 and / or IL-4 signaling. When used together, the anti-IL-33 and / or IL-4R antagonists of the present invention may function to block the interaction between IL-33 and an IL-33 binding partner and the interaction between IL-4 and an IL-4 binding partner, or otherwise inhibit the signaling activity of both IL-33 and IL-4. In one embodiment, the IL-4R antagonist is an antibody that binds to IL-4Rα and, in doing so, prevents both IL-4 and IL-13 signaling through either the type I or type II receptor. In one embodiment, the IL-4Rα antagonist is dupilumab or a biological equivalent thereof. Given the dual inhibitory activity of dupilumab against both IL-4 and IL-13, when used in combination with an IL-33 antagonist of the present invention, the combined treatment regimen is believed to result in enhanced inhibition of undesired inflammatory activity that is due in part to signaling through the IL-4, IL-13, and IL-33 signaling pathways that may occur during inflammation.

[0105] In one embodiment, the IL-33 antagonist is an antibody or antigen-binding fragment thereof that specifically binds to IL-33 and blocks the interaction of IL-33 with its receptor ST2 (also known as IL1RL1).

[0106] In one embodiment, an antibody or antigen-binding fragment thereof that specifically binds to IL-33 comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, and 308, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, and 316.

[0107] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to IL-33 comprises a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, and 308.

[0108] In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to IL-33 comprises a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, and 316.

[0109] In one embodiment, an antibody or antigen-binding fragment thereof that specifically binds to IL-33 is (a) an HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 228, 244, 260, 276, 292, and 310; (b) an HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214, 230, 246, 262, 278, 294, and 312; (c) an HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216, 232, 248, 264, 280, 296, and 314; (d) an LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220, 236, 252, 268, 284, and 318; (e) an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 206, 222, 238, 254, 270, 286, and 320; (f) SEQ ID NOs: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160 , 176, 192, 208, 224, 240, 256, 272, 288, and 322.

[0110] In one embodiment, an antibody or antigen-binding fragment thereof that specifically binds to IL-33 comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 106, 114 / 122, 130 / 138, 146 / 154, 162 / 170, 178 / 186, 194 / 202, 210 / 218, 226 / 234, 242 / 250, 258 / 266, 274 / 282, 290 / 298, and 308 / 316.

[0111] The present invention also includes the use of an IL-33 antagonist, alone or in combination with an IL-4R antagonist, in the manufacture of a medicament for the treatment of a disease or disorder associated with or caused by IL-33 and / or IL-4 activity or signaling in a patient. In one embodiment, the disease or disorder associated with or caused by IL-33 activity and / or IL-4 activity in the patient is an inflammatory disease or disorder selected from the group consisting of asthma (eosinophilic or non-eosinophilic), chronic obstructive pulmonary disease (COPD), asthma and COPD overlap syndrome (ACOS), atopic dermatitis, nasal polyps, allergic reactions, chronic bronchitis, emphysema, chronic rhinosinusitis with or without nasal polyps, inflammatory bowel disease, Crohn's disease, ulcerative colitis, hypersensitivity pneumonitis, multiple sclerosis, arthritis (osteoarthritis, rheumatoid arthritis, and psoriatic arthritis), allergic rhinitis, fibrosis, eosinophilic esophagitis, vasculitis, urticaria, Churg-Strauss syndrome, inflammatory pain, and psoriasis. The present invention also includes a therapeutically effective amount of an IL-33 antagonist in combination with a therapeutically effective amount of an IL-4R antagonist for use in treating an inflammatory disease or disorder or at least one symptom of an inflammatory disease or disorder, wherein administration of the IL-33 antagonist in combination with the IL-4R antagonist results in enhanced therapeutic efficacy compared to that observed with administration of the IL-33 antagonist alone or the IL-4R antagonist alone. Any of the methods discussed herein also encompass the use of IL-33 and / or IL-4R antagonists (e.g., antibodies) to treat or for the disease, disorder, and / or symptom discussed in connection with the method.

[0112] Other embodiments will become apparent from review of the following detailed description. [Brief explanation of the drawings]

[0113] [Figure 1]Four exemplary configurations of the individual components of an IL-33 antagonist relative to one another are shown. Panel A shows an arrangement in which a first IL-33-binding domain (D1) is attached to the N-terminus of a first multimerization domain (M1), and a second IL-33-binding domain (D2) is attached to the N-terminus of a second multimerization domain (M2). To indicate that D1 and D2 are derived from different IL-33-binding proteins, D1 is shown as an open box and D2 is shown as a closed box. Panel B shows an arrangement in which a first IL-33-binding domain (D1) is attached to the N-terminus of a first multimerization domain (M1), and a second IL-33-binding domain (D2) is attached to the C-terminus of a second multimerization domain (M2). To indicate that D1 and D2 are derived from different IL-33-binding proteins, D1 is shown as an open box and D2 is shown as a closed box. Panels C and D show configurations including four IL-33 binding domains, D1, D2, D3, and D4. In these configurations, D3-D1-M1 and D4-D2-M2 are linked in tandem, with D3 linked to the N-terminus of D1, D1 linked to the N-terminus of M1, D4 linked to the N-terminus of D2, and D2 linked to the N-terminus of M2. In panel C, D3 and D4 are identical or substantially identical to each other, and D1 and D2 are identical or substantially identical to each other. In panel D, D1 and D4 are identical or substantially identical to each other, and D3 and D2 are identical or substantially identical to each other. [Figure 2]These results demonstrate that HDM exposure induces similar increases in activated eosinophils in the lungs of IL-33, IL-4, and IL-4Rα triple-humanized and wild-type mice. Statistical significance was determined by two-way analysis of variance with Tukey's multiple comparison test. The following symbols were used to indicate statistical significance: an asterisk (*) represents a comparison between saline-exposed and HDM-exposed mice of the same genotype, and an ampersand (&) represents a comparison with the respective saline-exposed or HDM-exposed wild-type mice. Increasing values ​​indicate increasing significance: 1x = p ≤ 0.05; 2x = p ≤ 0.01; 3x = p ≤ 0.001; 4x = p ≤ 0.0001. Abbreviations: WT = wild-type. All mice were on a mixed C57BL / 6NTac / 129S6SvEvTac background. [Figure 3] This figure shows that administration of the combination of REGN3500 and dupilumab prevents HDM exposure-induced increases in relative lung weight. Relative lung weight is expressed as the ratio of wet lung weight (mg) to body weight (g). Statistical significance was determined by Kruskal-Wallis one-way analysis of variance with Dunn's multiple comparison post-hoc test. The following symbols were used to indicate statistical significance: an asterisk (*) indicates a comparison between all 19-week HDM-exposed groups, and a hashtag (#) indicates a comparison between untreated, saline-exposed animals and all other groups. Increasing values ​​indicate increasing significance: 1x = p ≤ 0.05; 2x = p ≤ 0.01; 3x = p ≤ 0.001. Abbreviations: wk = week; IgG4P = isotype control antibody REGN1945. [Figure 4A]The effects of REGN3500 and dupilumab, alone or in combination, on HDM exposure-induced pulmonary eosinophilic infiltration are shown. Statistical significance was determined by Kruskal-Wallis one-way analysis of variance with Dunn's multiple comparison post-hoc test. The following symbols were used to indicate statistical significance: "*" asterisk represents comparisons between all 19-week HDM-exposed groups, "#" hashtag represents comparisons between saline-exposed untreated animals and all other groups, and "+" plus sign represents comparisons between 11-week HDM-exposed untreated animals and all other groups. Increasing values ​​indicate increasing significance: 1x = p ≤ 0.05; 2x = p ≤ 0.01; 3x = p ≤ 0.001; 4x = p ≤ 0.0001. Abbreviations: wk = week; IgG4P = isotype control antibody REGN1945. [Figure 4B] The effects of REGN3500 and dupilumab, alone or in combination, on HDM exposure-induced pulmonary eosinophilic infiltration are shown. Statistical significance was determined by Kruskal-Wallis one-way analysis of variance with Dunn's multiple comparison post-hoc test. The following symbols were used to indicate statistical significance: "*" asterisk represents comparisons between all 19-week HDM-exposed groups, "#" hashtag represents comparisons between saline-exposed untreated animals and all other groups, and "+" plus sign represents comparisons between 11-week HDM-exposed untreated animals and all other groups. Increasing values ​​indicate increasing significance: 1x = p ≤ 0.05; 2x = p ≤ 0.01; 3x = p ≤ 0.001; 4x = p ≤ 0.0001. Abbreviations: wk = week; IgG4P = isotype control antibody REGN1945. [Figure 5]Figure 1 shows that REGN3500, alone or in combination with dupilumab, blocks HDM exposure-induced lung infiltration by ST2+ CD4+ T cells. Statistical significance was determined by one-way analysis of variance with Tukey's multiple comparison post-hoc test. The following symbols were used to indicate statistical significance: "*" asterisk represents a comparison between all 19-week HDM-exposed groups, "#" hashtag represents a comparison between saline-exposed, untreated animals and all other groups, and "+" plus sign represents a comparison between 11-week HDM-exposed, untreated animals and all other groups. Increasing values ​​indicate increasing significance: 1x = p ≤ 0.05; 2x = p ≤ 0.01; 3x = p ≤ 0.001; 4x = p ≤ 0.0001. Abbreviations: wk = week; IgG4P = isotype control antibody REGN1945. [Figure 6] This figure shows that administration of the combination of REGN3500 and dupilumab prevents HDM exposure-induced increases in MPO lung protein levels, an indicator of neutrophil infiltration. Statistical significance was determined by Kruskal-Wallis one-way analysis of variance with Dunn's multiple comparison post-hoc test. The following symbols were used to indicate statistical significance: an asterisk (*) indicates a comparison between all 19-week HDM-exposed groups, and a hashtag (#) indicates a comparison between saline-exposed, untreated animals and all other groups. Increasing numbers indicate increasing significance: 1x = p ≤ 0.05; 2x = p ≤ 0.01; 3x = p ≤ 0.001. Abbreviations: wk = week; IgG4P = isotype control antibody REGN1945. [Figure 7A]The effects of REGN3500 and dupilumab, alone or in combination, on HDM exposure-induced increases in lung IL-5 and IL-6 protein levels are shown. Lungs (anterior and middle lobes of the right lung) were harvested, and IL-5 (A) and IL-6 (B) protein levels were measured by multiplex immunoassay. IL-5 and IL-6 protein levels in lung tissue are expressed as pg of protein per lobe. Statistical significance was determined by Kruskal-Wallis one-way analysis of variance with Dunn's multiple comparison post-hoc test. The following symbols were used to indicate statistical significance: "*" asterisk indicates comparisons between all 19-week HDM-exposed groups, and "#" hashtag indicates comparisons between saline-exposed, untreated animals and all other groups. Increasing numerical values ​​indicate increasing significance: 1x = p ≤ 0.05; 2x = p ≤ 0.01; 3x = p ≤ 0.001. Abbreviations: wk = week, IgG4P = isotype control antibody REGN1945. [Figure 7B] The effects of REGN3500 and dupilumab, alone or in combination, on HDM exposure-induced increases in lung IL-5 and IL-6 protein levels are shown. Lungs (anterior and middle lobes of the right lung) were harvested, and IL-5 (A) and IL-6 (B) protein levels were measured by multiplex immunoassay. IL-5 and IL-6 protein levels in lung tissue are expressed as pg of protein per lobe. Statistical significance was determined by Kruskal-Wallis one-way analysis of variance with Dunn's multiple comparison post-hoc test. The following symbols were used to indicate statistical significance: "*" asterisk indicates comparisons between all 19-week HDM-exposed groups, and "#" hashtag indicates comparisons between saline-exposed, untreated animals and all other groups. Increasing numerical values ​​indicate increasing significance: 1x = p ≤ 0.05; 2x = p ≤ 0.01; 3x = p ≤ 0.001. Abbreviations: wk = week, IgG4P = isotype control antibody REGN1945. [Figure 8]These results demonstrate that REGN3500, alone or in combination with dupilumab, blocks HDM exposure-induced increases in circulating SAA protein levels. Four days after the final antibody injection, whole blood was collected by cardiac puncture, and serum was isolated. Circulating SAA protein levels were measured using a commercially available ELISA kit. Circulating SAA protein levels are expressed as μg of SAA protein per mL of serum. Statistical significance was determined by Kruskal-Wallis one-way analysis of variance with Dunn's multiple comparison post-hoc test. The following symbols were used to indicate statistical significance: an asterisk (*) indicates a comparison between all 19-week HDM-exposed groups, and a hashtag (#) indicates a comparison between saline-exposed, untreated animals and all other groups. Increasing values ​​indicate increasing significance: 1x = p ≤ 0.05; 2x = p ≤ 0.01; 3x = p ≤ 0.001. Abbreviations: wk = week, IgG4P = isotype control antibody REGN1945. [Figure 9] Circulating IgE protein levels increase in response to HDM exposure. Whole blood was collected by cardiac puncture, and serum was isolated. Circulating IgE protein levels were measured using a commercially available ELISA kit. Circulating IgE protein levels are expressed as μg of IgE protein per mL of serum. Statistical significance was determined by Kruskal-Wallis one-way analysis of variance with Dunn's multiple comparison post-hoc test. The following symbols were used to indicate statistical significance: "*" asterisk indicates comparisons between all 19-week HDM-exposed groups, and "#" hashtag indicates comparisons between saline-exposed, untreated animals and all other groups. Increasing numbers indicate increasing significance: 1x = p ≤ 0.05; 2x = p ≤ 0.01; 3x = p ≤ 0.001. Abbreviations: wk = week; IgG4P = isotype control antibody REGN1945. DETAILED DESCRIPTION OF THE INVENTION

[0114] Before describing the present invention, it is to be understood that the invention is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. It should also be understood that the terminology used herein is for the purpose of describing certain embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0115] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention belongs.As used herein, the term "about" when used in relation to a specific listed numerical value means that this value can vary by 1% or less from the listed value.For example, as used in the present invention, the expression "about 100" includes 99 and 101, and all values ​​therebetween (for example, 99.1, 99.2, 99.3, 99.4, etc.).

[0116] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All patents, applications, and non-patent publications mentioned herein are incorporated by reference in their entirety.

[0117] definition As used herein, "interleukin-33," "IL-33," or similar terms refer to human IL-33 protein and include the 270 amino acid, unprocessed, full-length IL-33 (e.g., SEQ ID NO: 348 or UniProtKB Accession No. 095760) and any form of IL-33 resulting from intracellular processing (see, e.g., SEQ ID NO: 349, which contains amino acid residues 112-270 of the full-length protein). Other processed forms of IL-33 are described in Lefrancais, et al. (Lefrancais, et al., (2012), Proc. Natl. Acad. Sci. 109(5):1693-1678). The term also encompasses naturally occurring variants of IL-33, such as splice variants (see, e.g., Hong, et al., (2011), J. Biol. Chem. 286(22):20078-20086, or any other isoforms of IL-33, such as those described in WO2016 / 156440. All references herein to proteins, polypeptides, and protein fragments are intended to refer to the human form of the respective protein, polypeptide, or protein fragment, unless expressly specified as being from a non-human species.

[0118] As used herein, the phrase "IL-33 antagonist" refers to any agent that can block, attenuate, or disrupt IL-33 signaling and / or the interaction between IL-33 and a cell surface receptor (e.g., ST2, also known as IL1RL1) or co-receptor (e.g., IL1-RAcP) or complexes thereof. For example, an "IL-33 antagonist," also referred to as an "IL-33 inhibitor" or "IL-33 blocker," includes any of the following: (1) an agent that binds to or interacts with IL-33, or (2) an agent that binds to or interacts with the IL-33 receptor (sometimes referred to as "suppressor of tumorigenesis" or "ST2," also known as "IL1RL1"), or (3) an agent that binds to or interacts with the IL-33 co-receptor (interleukin-1 receptor accessory protein or IL1-RAcP), or (4) an agent that binds to the IL-33 / ST2 complex, or (5) an agent that binds to or interacts with ST2 / IL-1RAcP. Any of the above includes, but is not limited to, an agent that binds to or interacts with IL-33 when bound to its receptor / co-receptor complex. The present invention may result in the inhibition or attenuation of at least one biological activity of IL-33, such as a biological signaling function occurring in the IL-33.

[0119] In one embodiment, an "IL-33 antagonist" is an antibody that specifically binds to or interacts with IL-33 and prevents IL-33 from binding to ST2, and in so doing, prevents the interaction of ST2 with its co-receptor, IL-1RAcP. In one embodiment, an "IL-33 antagonist" is an antibody that specifically binds to either ST2 or the ST2 / IL-1RAcP complex and prevents IL-33 from binding to ST2 or the ST2 / IL1-RAcP receptor complex. In one embodiment, an "IL-33 antagonist" is an antibody that binds to the IL-33 / ST2 complex and subsequently prevents the interaction of ST2 with the IL-1RAcP co-receptor. In one embodiment, an "IL-33 antagonist" is an antibody that can bind to IL-33 and enable low-affinity binding to ST2, while at the same time, such low-affinity binding can prevent the subsequent interaction of ST2 with its receptor, IL-1RAcP.

[0120] An "IL-33 antagonist" can also be an agent such as a soluble ST2 receptor or an IL-33 receptor-based trap, such as those described herein and disclosed in US 2014 / 0271642. Any agent that blocks IL-33-mediated signaling is considered an "IL-33 antagonist." An "IL-33 antagonist" can be a small organic molecule, a protein such as an antibody or fragment thereof, or a soluble IL-33 receptor-based trap (as described herein), or a nucleic acid such as an antisense molecule or siRNA. As used herein, "antibodies that bind IL-33" or "anti-IL-33 antibodies" include antibodies and antigen-binding fragments thereof that bind to human IL-33 protein or a biologically active fragment thereof (see SEQ ID NOs: 348, 349, 350, and 351).

[0121] As used herein, the expressions "interleukin-4 receptor" or "IL-4R" refer to the human IL-4Rα receptor having the amino acid sequence of SEQ ID NO:347.

[0122] As used herein, an "IL-4R antagonist" (also referred to herein as an "IL-4R inhibitor," "IL-4Rα antagonist," "IL-4R blocker," "IL-4Rα blocker," etc.) is any agent that binds to or interacts with IL-4Rα or an IL-4R ligand and inhibits or attenuates the normal biological signaling function of type 1 and / or type 2 IL-4 receptors. Type 1 IL-4 receptor is a dimeric receptor comprising an IL-4Rα chain and a γc chain. Type 2 IL-4 receptor is a dimeric receptor comprising an IL-4Rα chain and an IL-13Rα1 chain. Type 1 IL-4 receptor interacts with and is stimulated by IL-4, while type 2 IL-4 receptor interacts with and is stimulated by both IL-4 and IL-13. Thus, IL-4R antagonists that can be used in the methods of the present invention may function by blocking IL-4-mediated signaling, IL-13-mediated signaling, or both IL-4- and IL-13-mediated signaling. Thus, IL-4R antagonists of the present invention may interfere with the interaction of IL-4 and / or IL-13 with type 1 or type 2 receptors. Non-limiting examples of IL-4R antagonist classes include small molecule IL-4R inhibitors, anti-IL-4R aptamers, peptide-based IL-4R inhibitors (e.g., "peptibody" molecules), "receptor bodies" (e.g., engineered molecules containing the ligand-binding domain of an IL-4R component), and antibodies or antigen-binding fragments of antibodies that specifically bind to human IL-4Rα. As used herein, IL-4R antagonists also include antigen-binding proteins that specifically bind to IL-4 and / or IL-13.

[0123] As used herein, the term "antibody" refers to any antigen-binding molecule or molecular complex containing at least one complementarity-determining region (CDR) that specifically binds to or interacts with a particular antigen (e.g., IL-33 or IL-4R). The term "antibody" includes immunoglobulin molecules containing four polypeptide chains, two heavy (H) chains and two light (L) chains, inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain contains a heavy chain variable region (referred to herein as HCVR or V). H The heavy chain constant region comprises three domains: C H 1. C H 2, and C H Each light chain comprises a light chain variable region (referred to herein as LCVR or V L The light chain constant region comprises one domain (C L 1) V H Area and V L The regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). H and V L consists of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In different embodiments of the present invention, the FRs of an anti-IL-33 antibody (or antigen-binding portion thereof) or an anti-IL-4R antibody may be identical to human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a parallel analysis of two or more CDRs.

[0124] The term "antibody" as used herein also includes antigen-binding fragments of complete antibody molecules. "Antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and similar terms, as used herein, include naturally occurring, enzymatically obtained, synthetic, or genetically engineered polypeptides or glycoproteins that specifically bind antigens to form complexes. Antibody-binding fragments of antibodies can be derived from complete antibody molecules using any suitable standard techniques, such as, for example, proteolytic or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable domains and, optionally, constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or using molecular biology techniques, for example, to place one or more variable and / or constant domains in the appropriate configuration, or to introduce codons, generate cysteine ​​residues, modify, add, or delete amino acids, etc.

[0125] Non-limiting examples of antibody-binding fragments include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single-chain Fv (scFv) molecules, (vi) dAb fragments, and (vii) minimal recognition units consisting of amino acid residues mimicking a hypervariable region of an antibody (e.g., an isolated complementarity-determining region (CDR) such as a CDR3 peptide), or a constrained FR3-CDR3-FR4 peptide. Domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other engineered molecules such as shark variable IgNAR domains are also encompassed within the expression "antigen-binding fragment" as used herein.

[0126] Antigen-binding fragments of antibodies typically contain at least one variable domain, which may be of any size or amino acid composition and generally contains at least one CDR adjacent to or in-frame with one or more framework sequences. L V bound to the domain H In an antibody-binding fragment having a domain, V H Domains and V L The domains can be arranged relative to each other in any suitable configuration. For example, the variable region is a dimer and the V H -V H , V H -V L or V L -V L Alternatively, the antigen-binding fragment of an antibody may comprise a dimer of monomeric V H Domain or V L It may contain domains.

[0127] In certain embodiments, an antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody of the invention include: (i) V H -C H 1, (ii) V H -C H 2, (iii) V H -C H 3, (iv) V H -C H 1-C H 2. (v) V H -C H 1-C H 2-C H 3. (vi) V H -C H 2-C H 3, (vii)V H -C L , (viii) V L -C H 1, (ix)VL -C H 2. (x)V L -C H 3. (xi) V L -C H 1-C H 2, (xii)V L -C H 1-C H 2-C H 3, (xiii)V L -C H 2-C H 3, and (xiv) V L -C L In any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids that provide a flexible or semi-flexible connection between adjacent variable and / or constant domains in a single polypeptide molecule. Moreover, antigen-binding fragments of antibodies of the present invention may be linked to each other and / or to one or more monomeric V H Domain or V L The variable domain configurations may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above in non-covalent association with the domains (e.g., by disulfide bond(s)).

[0128] Like intact antibody molecules, antibody-binding fragments can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies will typically comprise at least two different variable domains, each capable of specifically binding to a separate antigen or to a different epitope on the same antigen. Any multispecific antibody format, including the exemplary bispecific antibody formats disclosed herein, can be adapted for use in conjunction with antigen-binding fragments of antibodies of the present invention using routine techniques available in the art. For example, the present invention includes methods involving the use of bispecific antibodies in which one immunoglobulin arm is an immunoglobulin specific for IL-4Rα or a fragment thereof or IL-33 or a fragment thereof, and the other immunoglobulin arm is specific for a second therapeutic target or is conjugated to a therapeutic moiety. Exemplary bispecific formats that can be used in the context of the present invention include, for example, scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadroma, knobs-into-holes, common light chain (such as common light chain with knobs-into-holes), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody, IgG1 / IgG2, dual acting Fab (DAF)-IgG, and Mab 2 These include, but are not limited to, bispecific formats (for a review of the aforementioned formats, see, e.g., Klein et al. 2012, mAbs 4:6, 1-11, and the references cited therein). Bispecific antibodies can also be constructed using peptide / nucleic acid linkages, for example, using unnatural amino acids with orthogonal chemical reactivity to generate site-specific antibody-oligonucleotide conjugates that then self-assemble into multimeric complexes with defined composition, valency, and geometry. (See, e.g., Kazane et al., J. Am. Chem. Soc. [Epub: December 4, 2012]).

[0129] In certain embodiments of the present invention, the anti-IL-33 antibody and the anti-IL-4R antibody of the present invention The antibody is a human antibody. As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present invention may include amino acid residues, for example in the CDRs, particularly CDR3, that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human framework sequences. The term includes antibodies recombinantly produced in non-human mammals or in the cells of non-human mammals. The term is not intended to include antibodies isolated from or generated in a human subject.

[0130] The antibodies of the present invention may, in some embodiments, be recombinant human antibodies. As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, generated, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described below), antibodies isolated from a recombinant combinatorial human antibody library (described below), antibodies isolated from an animal (e.g., a mouse) transgenic for human immunoglobulin genes (e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, generated, or isolated by any other means, including splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis), thus modifying the V and V regions of the recombinant antibody. H and V L The amino acid sequence of the region is human germline V H and V L These are sequences that are derived from and related to sequences, but may not naturally occur within the human antibody germline repertoire in vivo.

[0131] Human antibodies can exist in two forms related to hinge heterogeneity. In one form, the immunoglobulin molecule contains a stable four-chain construct of approximately 150-160 kDa in which dimers are held together by interchain heavy chain disulfide bonds. In the second form, the dimers are not linked via interchain disulfide bonds, forming approximately 75-80 kDa molecules consisting of covalently linked light and heavy chains (half antibodies). These forms have been extremely difficult to separate, even after affinity purification.

[0132] The frequency of occurrence of the second form in various intact IgG isotypes is due to, but not limited to, structural differences associated with the antibody hinge region isotype. A single amino acid substitution in the hinge region of a human IgG4 hinge can significantly reduce the occurrence of the second form to the level typically observed with a human IgG1 hinge (Angal et al. (1993) Molecular Immunology 30:105). The present invention provides a method for detecting the occurrence of the second form in various intact IgG isotypes. H 2 or C H Antibodies with one or more mutations in the three regions are included, which may be desirable, for example, in production to improve the yield of the desired antibody form.

[0133] The antibody of the present invention may be an isolated antibody. As used herein, "isolated antibody" refers to an identified antibody and an antibody that has been separated and / or recovered from at least one component of its natural environment. For example, an antibody that has been separated or removed from at least one component of an organism, or from a tissue or cell in which it naturally occurs or is naturally produced, is an "isolated antibody" for purposes of the present invention. Isolated antibodies also include antibodies in situ within recombinant cells. An isolated antibody is an antibody that has undergone at least one purification or isolation step. According to certain embodiments, an isolated antibody is an antibody that has been isolated from other cellular substances. The composition may be substantially free of materials and / or chemicals.

[0134] The present invention includes neutralizing and / or blocking anti-IL-33 and IL-4R antibodies. As used herein, a "neutralizing" or "blocking" antibody refers to an antibody whose binding to a target molecule, e.g., either IL-33 or IL-4R, (i) prevents the interaction between the target molecule and either its receptor (in the case of an IL-33 antibody) or its ligand (in the case of an IL-4R antibody), and / or (ii) results in the inhibition of at least one biological function of the target molecule, e.g., signal transduction. The inhibition caused by an IL-33 or IL-4R neutralizing or blocking antibody need not be complete, as long as the inhibition is detectable using an appropriate assay.

[0135] The antibodies disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences from which the antibody was derived. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein to germline sequences available, for example, from public antibody sequence databases. The present invention includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids in one or more framework and / or CDR regions are mutated to the corresponding residue(s) in the germline sequence from which the antibody was derived, or to the corresponding residue(s) in another human germline sequence, or to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are collectively referred to herein as "germline mutations"). Starting from the heavy and light chain variable region sequences disclosed herein, one skilled in the art can readily generate numerous antibodies and antibody-binding fragments containing one or more individual germline mutations or combinations thereof. In certain embodiments, V H and / or V LAll framework and / or CDR residues within a domain are mutated back to the residue found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, e.g., the mutated residue is found within the first 8 amino acids of FR1, or the mutated residue is found within the last 8 amino acids of FR4, or the mutated residue is found only in CDR1, CDR2, or CDR3. In other embodiments, one or more framework and / or CDR residue(s) are mutated to the corresponding residue(s) in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody was originally derived). Furthermore, antibodies of the present invention may contain any combination of two or more germline mutations within the framework and / or CDR regions, e.g., certain individual residues are mutated to the corresponding residue in a particular germline sequence, while certain other residues that differ from the original germline sequence are maintained or mutated to the corresponding residue in a different germline sequence. Once obtained, antibodies and antibody-binding fragments containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonist or agonist biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antibody-binding fragments obtained by this general method are encompassed within the scope of the present invention.

[0136] The present invention also includes antibodies comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present invention includes antibodies having HCVR, LCVR, and / or CDR amino acid sequences with, for example, 10 or fewer, 8 or fewer, 6 or fewer, or 4 or fewer conservative amino acid substitutions relative to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein.

[0137] The term "epitope" refers to a specific region in the variable region of an antibody molecule known as a paratope. An epitope refers to an antigenic determinant that interacts with a target antigen-binding site. A single antigen may have two or more epitopes. Thus, different antibodies may bind to different regions on an antigen and have different biological effects. Epitopes may be either conformational or linear. Conformational epitopes are generated by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. Linear epitopes are generated by adjacent amino acid residues in a polypeptide chain. In certain circumstances, epitopes may include carbohydrate, phosphoryl, or sulfonyl moieties on an antigen.

[0138] The terms "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicate that when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is at least about 95%, more preferably at least about 96%, 97%, 98%, or 99% nucleotide sequence identity of the nucleotide bases as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST, or Gap, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule can, in certain cases, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0139] When applied to polypeptides, the terms "substantial similarity" or "substantially similar" refer to two peptide sequences that share at least 95% sequence identity, and even more preferably at least 98% or 99% sequence identity, when optimally aligned, such as by the programs GAP or BESTFIT, using a predetermined gap weight. Preferably, non-identical residue positions differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24:307-331, incorporated herein by reference. Examples of groups of amino acids having side chains with similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine, (2) aliphatic-hydroxyl side chains: serine and threonine, (3) amide-containing side chains: asparagine and glutamine, (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan, (5) basic side chains: lysine, arginine, and histidine, (6) acidic side chains: aspartic acid and glutamic acid, and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445, which is incorporated herein by reference. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0140] Sequence similarity for polypeptides, also called sequence identity, is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity measures assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from organisms of different species, or between a wild-type protein and its mutant protein. These include programs such as Gap and Bestfit, which can perform the above-mentioned comparisons. See, for example, GCG version 6.1. Polypeptide sequences can also be compared using FASTA, a program in GCG version 6.1, with default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm for comparing the sequences of the present invention to a database containing multiple sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, with default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, each of which is incorporated herein by reference.

[0141] As used herein, a "disease or disorder" refers to any condition treatable with the IL-33 and IL-4 antagonists of the present invention. As used herein, an "inflammatory disease or disorder" refers to a disease, disorder, or pathological condition in which the pathology is due, in whole or in part, to, for example, changes in the number, migration rate, or activation of immune system cells. Immune system cells include, for example, T cells, B cells, monocytes or macrophages, innate lymphocytes, antigen-presenting cells (APCs), dendritic cells, microglia, NK cells, neutrophils, eosinophils, mast cells, or any other cells specifically associated with immunology, such as cytokine-producing endothelial or epithelial cells. As used herein, in one embodiment, an "inflammatory disease or disorder" is an immune disorder or condition selected from the group consisting of asthma (including steroid-resistant asthma, steroid-sensitive asthma, eosinophilic asthma, or noneosinophilic asthma), allergy, anaphylaxis, multiple sclerosis, inflammatory bowel disease (e.g., Crohn's disease or ulcerative colitis), chronic obstructive pulmonary disease (COPD, which may or may not be associated with, caused in part by, or resulting from exposure to first- or second-hand smoke), asthma and COPD overlap syndrome (ACOS), eosinophilic esophagitis, chronic bronchitis, emphysema, chronic rhinosinusitis with or without nasal polyps, lupus, atopic dermatitis, psoriasis, scleroderma and other fibrotic diseases, Sjogren's syndrome, vasculitis (Behcet's disease, giant cell arteritis, Henoch-Schönlein purpura, and Churg-Strauss syndrome), inflammatory pain, and arthritis. In one embodiment, the arthritis is selected from the group consisting of rheumatoid arthritis, osteoarthritis, and psoriatic arthritis.

[0142] In one embodiment, the "inflammatory disease or disorder" is an immune disorder or condition involving a type 1 and / or a type 2 response.

[0143] "Type 1 immune response" refers to T helper 1 (T H 1) Cells and T HType 1 immunity is defined by immune cells, cytotoxic T cells, group 1 and group 3 innate lymphoid cells (ILCs), and immunoglobulin M (IgM), IgA, and specific IgG antibody classes, as well as cytokines including TNF, IL-1β, and IL-6. This effector response mediates immunity to many microorganisms, including bacteria, viruses, fungi, and protozoa. Components of type 1 immunity also help maintain tumor immunosurveillance.

[0144] "Type 2 immune response" is defined as CD4+ T helper 2 (T H Type 2 immunity is characterized by the expression of immune cells, group 2 innate lymphoid cells (ILCs), eosinophils, basophils, mast cells, IL-4 and / or IL-13-activated macrophages, IgE antibody subclasses, and cytokines, including IL-4, IL-5, IL-9, IL-13, thymic stromal lymphopoietin, IL-25, and IL-33. Type 2 immunity provides protection against large extracellular parasites by enhancing barrier defense. Components of the type 2 immune response also help maintain metabolic homeostasis and promote tissue remodeling after injury. This type of response can also be initiated in response to allergens.

[0145] As used herein, the phrase "inhibiting or attenuating IL-33-mediated signaling" refers to the extent to which IL-33 stimulates signaling through its receptor, ST2, and co-receptor IL-1RAcP that is attenuated in the presence of an antagonist, such as an IL-33 antibody or IL-33 trap described herein, compared to the extent to which IL-33 stimulates signaling through ST2 and IL-1RAcP in the absence of the antagonist, such as an IL-33 antibody or IL-33 trap described herein. As used herein, the phrase "inhibiting or attenuating IL-4R-mediated signaling" refers to the extent to which IL-4 stimulates signaling through type 1 and / or type 2 IL-4 receptors that is attenuated in the presence of an antagonist, such as IL-4 or an IL-4R antibody described herein, compared to the extent to which IL-4 stimulates signaling through type 1 and / or type 2 IL-4 receptors in the absence of the antagonist, such as an IL-4 or IL-4R antibody described herein. To determine the degree of inhibition, a sample is treated with a potential inhibitor / antagonist and compared to a control sample not treated with the inhibitor / antagonist. The control sample, i.e., not treated with the antagonist, is assigned a relative activity value of 100%. Inhibition is achieved when the activity value compared to the control is less than about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or 20%. Inhibition endpoints can include a predetermined amount or percentage of inflammation or indicators of cell degranulation, secretion, or activation, such as cytokine release. Inhibition of IL-33 signaling via ST2 and IL-1RAcP can be determined by assaying for IL-33 signaling in in vitro assays, such as those known to those skilled in the art. Additionally, in vivo assays can be used to determine whether a molecule is an antagonist of IL-33. For example, an in vivo assay can be used to assess the effect of antibodies to IL-33 on pulmonary inflammation in allergen-sensitized animals homozygous for expression of human IL-33.After sensitizing animals with an allergen, some of the animals are treated with either an anti-IL-33 antibody of the present invention or a negative isotype control antibody. The animals are then sacrificed, and lungs are harvested for assessment of cellular infiltrates and cytokine measurements (IL-4 and IL-5). IL-33 antibodies that are effective as antagonists should demonstrate a trend toward a reduction in pulmonary inflammatory cells and cytokines such as IL-4 and IL-5. Similar assays can be performed to evaluate the ability of IL-4R antagonists to block signal transduction after IL-4 binds to type 1 and / or type 2 receptors in vitro or in vivo. Furthermore, any of the above assays can be modified to compare the effects of using either an IL-33 antagonist alone, an IL-4 or IL-4R antagonist alone, or the effects of using a combination of both an IL-33 antagonist and an IL-4 or IL-4R antagonist together.

[0146] In another aspect, the present invention provides a method for reducing the incidence or recurrence of asthma or COPD or an exacerbation of asthma or COPD in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising an interleukin-4 receptor (IL-4R) antagonist in combination with a pharmaceutical composition comprising an IL-33 antagonist. As used herein, the term "asthma or COPD exacerbation" refers to an increase in the severity and / or frequency and / or duration of one or more symptoms or signs of asthma or COPD. "Asthma or COPD exacerbation" also includes any deterioration in the subject's respiratory health that requires or is treatable by therapeutic intervention for asthma or COPD (e.g., steroid treatment, inhaled corticosteroid treatment, hospitalization, etc.).

[0147] "Reducing the incidence or recurrence" of asthma or COPD exacerbations means using a pharmaceutical composition of the present invention. "Reduced incidence or recurrence" of asthma or COPD exacerbations means that a subject has fewer asthma or COPD exacerbations after treatment than before treatment (i.e., a reduction in at least one exacerbation), or does not experience an asthma or COPD exacerbation for at least four weeks (e.g., four, six, eight, twelve, fourteen, or more weeks) after initiation of treatment with a pharmaceutical composition of the present invention. Alternatively, a "reduced incidence or recurrence" of asthma or COPD exacerbations means that a subject is at least 10% (e.g., 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more) less likely to experience an asthma or COPD exacerbation after administration of a pharmaceutical composition of the present invention, compared to a subject not receiving a pharmaceutical composition of the present invention.

[0148] As used herein, "fibrotic disease or disorder" refers to a condition involving excess fibrous connective tissue in a tissue or organ. "Fibrosis" refers to a pathological process involving scar formation and excessive production of extracellular matrix by connective tissue in response to tissue injury. As used herein, exemplary "fibrotic diseases or disorders" treatable by administering the anti-IL-33 and IL-4R antagonists of the present invention include pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis, bleomycin-induced pulmonary fibrosis, asbestos-induced pulmonary fibrosis, and bronchiolitis obliterans), chronic asthma, fibrosis associated with acute lung injury and acute respiratory distress (e.g., bacterial pneumonia-induced fibrosis, trauma-induced fibrosis, viral pneumonia-induced fibrosis, ventilator-induced fibrosis, non-pulmonary sepsis-induced fibrosis, and aspiration-induced fibrosis), silicosis, radiation-induced fibrosis, chronic obstructive pulmonary disease (COPD, which is caused by primary or secondary smoking). fibrosis, which may or may not be associated with, caused in part by, or attributable to exposure to secondhand smoke, scleroderma, ocular fibrosis, skin fibrosis (e.g., scleroderma), liver fibrosis (e.g., cirrhosis, alcohol-induced liver fibrosis, nonalcoholic steatohepatitis (NASH), bile duct injury, primary biliary cirrhosis, infectious or viral-induced liver fibrosis, autoimmune hepatitis, renal (kidney) fibrosis, cardiac fibrosis, atherosclerosis, stent restenosis, and bone marrow fibrosis. While asthma and COPD are generally considered to be inflammatory conditions, each is known to also exhibit fibrotic disease characteristics.

[0149] A patient "response" or patient "responsiveness" to a treatment or therapy, e.g., a treatment comprising an IL-33 antagonist (e.g., an IL-33 or ST2-binding antagonist) or an IL-4 antagonist, refers to the clinical or therapeutic benefit that accrues to a patient at risk for or having an IL-33-mediated disorder (e.g., asthma, COPD, ACOS, nasal polyps, or pulmonary fibrosis, e.g., idiopathic pulmonary fibrosis) from or as a result of the treatment. Such benefits include a cellular or biological response, a complete response, a partial response, stable disease (without progression or relapse), or a response with late relapse of the patient from or as a result of treatment with the antagonist. One of ordinary skill in the art will readily be in a position to determine whether a patient is responsive. For example, patients suffering from asthma who respond to treatment comprising an IL-33 antagonist and / or an IL-4 antagonist may exhibit an observable and / or measurable reduction or absence in one or more of the following exemplary symptoms: recurrent wheezing, cough, dyspnea, chest tightness, symptoms that develop or worsen at night, symptoms caused by cold air, exercise, or exposure to allergens.

[0150] Furthermore, "enhanced therapeutic efficacy" can be determined by assessing whether treatment with a combination of an IL-33 antagonist and an IL-4R antagonist results in a significant improvement in at least one symptom of a disease or disorder, or an improvement in at least one of the biological parameters as measured herein (e.g., pulmonary inflammation, cytokine release, etc.), when compared to the results achieved when using either the IL-33 antagonist or the IL-4R antagonist alone. Any of the biological measures of efficacy described in this application can be used to determine therapeutic efficacy or its enhancement.

[0151] IL-33 antagonist and IL-4R antagonist The methods of the invention involve administering to a patient suffering from an inflammatory disease or disorder a therapeutically effective amount of an IL-33 antagonist in combination with a therapeutically effective amount of an IL-4R antagonist.

[0152] IL-33 antagonist The term "human interleukin-33" or "human IL-33" or "hIL-33" or "IL-33" refers to the 270 amino acid full-length unprocessed IL-33 (see, e.g., SEQ ID NO: 348 or UniProtKB Accession No. O95760) or biologically active fragments thereof, and any form of IL-33 resulting from intracellular processing (see, e.g., SEQ ID NO: 349, which contains amino acid residues 112-270 of the full-length protein). The term also encompasses naturally occurring variants of IL-33, such as splice variants (see, e.g., Hong, et al., (2011), J. Biol. Chem. 286(22):20078-20086), allelic variants, or any other isoform of IL-33, such as the oxidized or reduced forms of IL-33 described in WO2016 / 156440. Activities of IL-33 that can be neutralized, inhibited, blocked, eliminated, attenuated, reduced, or prevented by the antibodies or antigen-binding fragments thereof of the present invention, or the IL-33 traps of the present invention include, but are not limited to, inhibition of signal transduction mediated by the IL-33 receptor or inhibition of inflammation mediated by IL-33.

[0153] As used herein, an "IL-33 antagonist" (also referred to herein as an "IL-33 inhibitor" or "IL-33 blocker") is any agent that can inhibit the interaction of IL-33 with one or more of its binding partners, and in so doing, inhibit IL-33-mediated signal transduction. For example, an "IL-33 antagonist" can bind to or interact with IL-33, or the IL-33 receptor termed "suppressor of tumorigenesis" ("ST2"), or the IL-33 receptor termed "interleukin-1 receptor accessory protein ("IL-1RAcP"), or either the IL-33 / ST2 or ST2 / IL-1RAcP complex, and in so doing, inhibit IL-33-mediated signal transduction.

[0154] Non-limiting examples of IL-33 antagonist classes include small molecule IL-33 inhibitors, or receptor antagonists, or nucleic acids that hybridize under stringent conditions to nucleic acid sequences encoding either IL-33 or an IL-33 receptor or co-receptor (e.g., small interfering RNA (siRNA) or clustered regularly interspaced short palindromic repeat RNA (CRISPR-RNA or crRNA), including single-stranded guide RNAs (sgRNAs) having crRNA and tracrRNA sequences as described in Mali et al. (Science. 339:823-26, 2013), which is incorporated herein by reference in its entirety). Other IL-33 antagonists include ligand-binding portions of IL-33 receptors (e.g., ST2), IL-33-binding scaffold molecules (e.g., DARPins, HEAT repeat proteins, ARM repeat proteins, tetratricopeptide repeat proteins, fibronectin-based scaffold constructs, and other naturally occurring repeat protein-based scaffolds, etc. [See, e.g., Boersma and Pluckthun, 2011, Curr. Opin. Biotechnol. 22:849-857, and references cited therein]), and anti-IL-33 aptamers or portions thereof.

[0155] IL-33 antibody According to certain embodiments, IL-33 antagonists or inhibitors that can be used in the context of the present invention are anti-IL-33 antibodies or antigen-binding fragments of antibodies that specifically bind to human IL-33. The amino acid sequence identifiers of exemplary anti-IL-33 antibodies for use in the methods described herein are set forth in Table 1, and the nucleic acid sequence identifiers encoding these IL-33 antibodies are set forth in Table 2.

[0156] In one embodiment, the anti-IL-33 antibodies described herein for use in the methods of the present invention are those disclosed in US Pat. No. 9,453,072, which is incorporated herein by reference in its entirety.

[0157] According to certain embodiments, the anti-IL-33 antibody used in the methods of the present invention specifically binds to IL-33. "Specifically bind" or similar terms means that the antibody or antigen-binding fragment thereof forms a complex with the antigen that is relatively stable under physiological conditions. Methods for determining whether an antibody specifically binds to an antigen are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc. For example, an antibody that "specifically binds" IL-33 as used in the context of the present invention has a K of less than about 1000 nM, less than about 500 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, or less than about 0.5 nM as measured in a surface plasmon resonance assay. D The present invention also includes antibodies that specifically bind to IL-33 or a biologically active portion thereof. However, an isolated antibody that specifically binds to human IL-33 may have cross-reactivity to other antigens, such as IL-33 molecules from other (non-human) species.

[0158] According to certain exemplary embodiments of the present invention, the IL-33 antagonist is an anti-IL-33 antibody or antigen-binding fragment thereof comprising a heavy chain variable region (HCVR), a light chain variable region (LCVR), and / or a complementarity determining region (CDR) comprising the amino acid sequence of any of the anti-IL-33 antibodies set forth in U.S. Patent No. 9,453,072 and Table 1 disclosed herein. In certain embodiments, the IL-33 antagonist is an anti-IL-33 antibody having the binding characteristics of the reference antibody described in U.S. Patent No. 9,453,072. In certain exemplary embodiments, an anti-IL-33 antibody or antigen-binding fragment thereof that can be used in connection with the methods of the present invention comprises a heavy chain complementarity determining region (HCDR) of the heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO:274, and a light chain complementarity determining region (LCDR) of the light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:282. According to certain embodiments, the anti-IL-33 antibody or antigen-binding fragment thereof comprises three HCDRs (HCDR1, HCDR2, and HCDR3) and three LCDRs (LCDR1, LCDR2, and LCDR3), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 276, HCDR2 comprises the amino acid sequence of SEQ ID NO: 278, HCDR3 comprises the amino acid sequence of SEQ ID NO: 280, LCDR1 comprises the amino acid sequence of SEQ ID NO: 284, LCDR2 comprises the amino acid sequence of SEQ ID NO: 286, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 288. In yet other embodiments, the anti-IL-33 antibody or antigen-binding fragment thereof comprises a HCVR comprising SEQ ID NO: 274 and a LCVR comprising SEQ ID NO: 282.

[0159] In one embodiment, the IL-33 antagonist is an IL-33 antibody designated REGN3500, which comprises an HCVR having the amino acid sequence of SEQ ID NO: 274, an LCVR having the amino acid sequence of SEQ ID NO: 282, heavy chain complementarity determining regions (HCDR1-HCDR2-HCDR3) having the amino acid sequences of SEQ ID NOs: 276-278-280, respectively, and light chain complementarity determining regions (LCDR1-LCDR2-LCDR3) having the amino acid sequences of SEQ ID NOs: 284-286-288, respectively.

[0160] Other anti-IL-33 antibodies and antigen-binding fragments thereof that can be used in the methods described herein are disclosed in EP1725261, US8187596, WO2011 / 031600, WO2015 / 099175, WO2015 / 106080(ANB020), US2016 / 0168242, WO2016 / 077381, WO2016 / 077366, or WO2016 / 156440, each of which is incorporated herein by reference in its entirety.

[0161] IL-33 Trap According to certain embodiments, IL-33 antagonists or inhibitors that can be used in the context of the present invention are receptor-based IL-33 traps, such as those described herein.

[0162] The IL-33 traps described herein contain at least one IL-33 binding domain, including the IL-33-binding portion of the IL-33 receptor protein designated ST2. In certain embodiments, the IL-33 traps further contain the extracellular portion of the IL-33 co-receptor designated IL-1 receptor accessory protein or IL-1RAcP. The IL-33 traps may also contain at least one multimerization component that functions to link the various components of the trap together. The various components of the IL-33 traps are described below and illustrated in FIG. 1.

[0163] In one embodiment, the IL-33 trap described herein for use in the methods of the present invention is disclosed in US2014 / 0271642 and WO2014 / 152195, which are incorporated by reference in their entireties.

[0164] Briefly, an IL-33 trap comprises a first IL-33 binding domain (D1) linked to a multimerization domain (M). In certain embodiments, an IL-33 antagonist of the present invention comprises a second IL-33 binding domain (D2) linked to D1 and / or M. According to certain embodiments, D1 comprises the IL-33-binding portion of the ST2 protein. According to certain embodiments, D2 comprises the extracellular portion of the IL-1RAcP protein.

[0165] The individual components of the IL-33 trap may be arranged relative to one another in a variety of ways to result in a functional antagonist molecule capable of binding to IL-33. For example, D1 and / or D2 may be attached to the N-terminus of M. In other embodiments, D1 and / or D2 are attached to the C-terminus of M. In yet other embodiments, D1 is attached to the N-terminus of D2 and D2 is attached to the N-terminus of M, resulting in a tandem fusion of the antagonist molecule from N to C-terminus represented by the formula D1-D2-M. Other orientations of the individual components are disclosed elsewhere herein in Figure 1.

[0166] Non-limiting examples of IL-33 traps for use in the methods of the invention are shown in Tables 3a and 3b, and include the IL-33 traps designated "hST2-hFc," "hST2-mFc," "hST2-hIL1RAcP-mFc," "hST2-hIL1RAcP-hFc," and "mST2-mIL1RAcP-mFc," which correspond to SEQ ID NOs: 323, 324, 325, 326, and 327, respectively. The invention includes IL-33 receptor-based traps having an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the exemplary IL-33 receptor-based traps shown herein (e.g., SEQ ID NOs: 323, 324, 325, 326, and 327).

[0167] Standard molecular biology techniques (eg, recombinant DNA techniques) can be used to construct any of the IL-33 traps of the present invention or variants thereof.

[0168] An IL-33 trap for use in the methods of the present invention comprises at least one IL-33-binding domain (sometimes referred to herein as "D" or "D1," "D2," etc.). In certain embodiments, the IL-33-binding domain comprises the IL-33-binding portion of the ST2 protein. The IL-33-binding portion of the ST2 protein can comprise or consist of all or a portion of the extracellular domain of the ST2 protein. In certain embodiments, the ST2 protein is a human ST2 protein. As used herein, "human ST2 protein" refers to the ST2 protein set forth in amino acids 1-556 of Accession No. NP_057316.3, also set forth as SEQ ID NO: 352. In certain embodiments, the ST2 protein is an ST2 protein from a non-human species (e.g., mouse ST2, monkey ST2, etc.). An exemplary IL-33-binding portion of an ST2 protein is set forth herein as the amino acid sequence of SEQ ID NO: 328 (corresponding to the extracellular domain of human ST2 [K19-S328 of NCBI Accession No. NP_057316.3]). Another example of an IL-33 binding portion of the ST2 protein is shown herein as the amino acid sequence of SEQ ID NO: 329 (corresponding to the extracellular domain of mouse ST2, [S27-R332 of NCBI Accession No. P14719]).

[0169] In certain embodiments, the IL-33-binding domain comprises the extracellular portion of an IL-1RAcP protein. In certain embodiments, the IL-1RAcP protein is a human IL-1RAcP protein. As used herein, "human IL-1RAcP protein" refers to an IL-1RAcP protein having the amino acid sequence of SEQ ID NO: 353. In certain embodiments, the IL-1RAcP protein is an IL-1RAcP protein derived from a non-human species (e.g., mouse IL-1RAcP, monkey IL-1RAcP, etc.). An exemplary extracellular portion of an IL-1RAcP protein is set forth herein as the amino acid sequence of SEQ ID NO: 330 (corresponding to the extracellular domain of human IL-1RAcP [S21-E359 of NCBI accession number Q9NPH3]). Another example of the extracellular portion of the IL-1RAcP protein is shown herein as the amino acid sequence of SEQ ID NO: 331 (corresponding to the extracellular domain of mouse IL-1RAcP [S21-E359 of NCBI accession number Q61730]).

[0170] The present invention includes IL-33 traps comprising D1 and / or D2 components having amino acid sequences at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the amino acid sequences of the exemplary IL-33 binding domain components set forth herein (e.g., SEQ ID NOs: 328, 329, 330, and 331).

[0171] The IL-33 antagonists of the present invention also comprise at least one multimerization domain (sometimes referred to herein by the abbreviations "M," "M1," "M2," etc.). Generally, the multimerization domain(s) of the present invention function to link various components of the IL-33 antagonist (e.g., IL-33 binding domain(s)) to one another. As used herein, a "multimerization domain" is any macromolecule that has the ability to associate (covalently or non-covalently) with a second multimerization domain of identical or similar structure or composition. For example, a multimerization domain may be a multimerization domain that binds to the IL-33 binding domain of immunoglobulin C. HA non-limiting example of a multimerization domain is the Fc portion of an immunoglobulin, such as the Fc domain of an IgG selected from the isotypes IgG1, IgG2, IgG3, and IgG4, as well as any allotype within each isotype group.

[0172] Non-limiting exemplary multimerization dopants that can be used in the IL-33 antagonists of the present invention include: Examples include human IgG1 Fc (SEQ ID NO: 332) or murine IgG2 Fc (SEQ ID NO: 333). The present invention includes IL-33 antagonists comprising an M component having an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the amino acid sequences of the exemplary M components set forth herein (e.g., SEQ ID NO: 332 or 333).

[0173] In certain embodiments, the IL-33 antagonist of the present invention comprises two multimerization domains, M1 and M2, where M1 and M2 are identical to each other. For example, M1 can be an Fc domain having a specific amino acid sequence, and M2 is an Fc domain having the same amino acid sequence as M1.

[0174] The individual components of the IL-33 antagonists of the present invention (e.g., D1, D2, M, etc.) can be arranged relative to one another in a variety of ways. Non-limiting examples of all of the above arrangements are illustrated in FIG.

[0175] Non-limiting examples of IL-33 traps for use in the methods of the invention that contain two multimerization domains (M1 and M2) and four IL-33 binding domains (D1, D2, D3, and D4) are also shown in FIG. 1 (configurations C and D).

[0176] The individual components of the IL-33 traps of the invention (e.g., D1, D2, M1, M2, etc.) can be directly coupled to one another (e.g., D1 and / or D2 can be directly coupled to M, etc.), or the individual components can be coupled to one another via a linker moiety (e.g., D1 and / or D2 can be coupled to M via a linker positioned between the individual components, D1 can be coupled to D2 via a linker, etc.). In any of the configurations disclosed herein in which one component is described as "coupled" to another component, the coupling can be via a linker (even if not specifically designated as such). As used herein, a "linker" is any molecule that joins two polypeptide components together.

[0177] The biological characteristics of IL-33 traps for use in the methods of the present invention are described in US2014 / 0271642 and WO2014 / 152195, which are incorporated by reference in their entireties.

[0178] Other IL-33 antagonists Polypeptides that bind to IL-33 and / or its receptors (ST2 and / or IL-1 RAcP) and block the ligand-receptor interaction are considered IL-33 antagonists and are described in WO2014 / 152195, which is incorporated by reference in its entirety. Other agents that act as IL-33 antagonists and may be used in the methods of the invention include immunoadhesins, peptibodies, and soluble ST2, or derivatives thereof, anti-IL-33 receptor antibodies (e.g., anti-ST2 antibodies, e.g., AMG-282 (Amgen) or STLM15 (Janssen)), or any of the anti-ST2 antibodies described in WO2012 / 113813, WO2013 / 173761, WO2013 / 165894, US8,444,987, or US7,452,980, each of which is incorporated by reference in its entirety. Other IL-33 antagonists for use in the methods of the present invention include ST2-Fc proteins such as those described in WO2013 / 173761 or WO2013 / 165894, each of which is incorporated by reference in its entirety.

[0179] IL-4R antagonist As used herein, an "IL-4R antagonist" (also referred to herein as an "IL-4R inhibitor," "IL-4Rα antagonist," "IL-4R blocker," "IL-4Rα blocker," etc.) is any agent that binds to or interacts with IL-4Rα or an IL-4R ligand and inhibits or attenuates the normal biological signaling function of type 1 and / or type 2 IL-4 receptors. As used herein, the term "human IL-4R" or "hIL-4R" refers to the IL-4R having the amino acid sequence of SEQ ID NO: 347 or a biologically active fragment thereof. Type 1 IL-4 receptor is a dimeric receptor comprising the IL-4Rα chain and the γc chain. Type 2 IL-4 receptor is a dimeric receptor comprising the IL-4Rα chain and the IL-13Rα1 chain. Type 1 IL-4 receptor interacts with and is stimulated by IL-4, while type 2 IL-4 receptor interacts with and is stimulated by both IL-4 and IL-13. Thus, IL-4R antagonists that can be used in the methods of the present invention can function by blocking IL-4-mediated signaling, IL-13-mediated signaling, or both IL-4- and IL-13-mediated signaling. Thus, the IL-4R antagonists of the present invention can interfere with the interaction of IL-4 and / or IL-13 with type 1 or type 2 receptors.

[0180] Non-limiting examples of classes of IL-4R antagonists include small molecule IL-4R antagonists, nucleic acid-based inhibitors of IL-4R expression or activity (e.g., siRNA or antisense), peptide-based molecules that specifically interact with IL-4R (e.g., peptibodies), "receptorbodies" (e.g., engineered molecules that contain the ligand-binding domain of an IL-4R component), IL-4R-binding scaffold molecules (e.g., DARPins, HEAT repeat proteins, ARM repeat proteins, tetratricopeptide repeat proteins, fibronectin-based scaffold constructs, and other scaffolds based on naturally occurring repeat proteins [e.g., Boersma and See Pluckthun, 2011, Curr. Opin. Biotechnol. 22:849-857, and references cited therein], and anti-IL-4R aptamers or portions thereof. According to certain embodiments, the IL-4R antagonist that can be used in the context of the present invention is an anti-IL-4R antibody or an antigen-binding fragment of an antibody that specifically binds to human IL-4R.

[0181] In one embodiment, the anti-IL-4R antibody disclosed herein for use in the methods of the invention is dupilumab (see also U.S. Patent Nos. 7,605,237, 7,608,693, and 9,290,574).

[0182] Anti-IL-4R antibody According to certain exemplary embodiments of the present invention, the IL-4R antagonist is an anti-IL-4Rα antibody or antigen-binding fragment thereof that specifically binds to IL-4Rα. "Specifically binds" or similar terms means that the antibody or antigen-binding fragment thereof forms a complex with the antigen that is relatively stable under physiological conditions. Methods for determining whether an antibody specifically binds to an antigen are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc. For example, an antibody that "specifically binds" IL-4Rα as used in the context of the present invention has a K of less than about 1000 nM, less than about 500 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, or less than about 0.5 nM as measured by a surface plasmon resonance assay. D The present invention includes antibodies that specifically bind to IL-4Rα or a biologically active portion thereof. However, an isolated antibody that specifically binds to human IL-4Rα may have cross-reactivity to other antigens, such as IL-4Rα molecules from other (non-human) species.

[0183] According to certain exemplary embodiments of the present invention, the IL-4R antagonist is an anti-IL-4Rα antibody or antigen-binding fragment thereof comprising a heavy chain variable region (HCVR), a light chain variable region (LCVR), and / or a complementarity-determining region (CDR) comprising any of the amino acid sequences of the anti-IL-4R antibodies set forth in U.S. Patent Nos. 7,605,237 and 7,608,693. In certain embodiments, the IL-4R antagonist is an anti-IL-4R antibody having the binding characteristics of the reference antibody referred to herein as dupilumab (see U.S. Patent Nos. 7,605,237 and 7,608,693). In certain exemplary embodiments, an anti-IL-4Rα antibody or antigen-binding fragment thereof that can be used in connection with the methods of the present invention comprises a heavy chain complementarity-determining region (HCDR) of the heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 337, and a light chain complementarity-determining region (LCDR) of the light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 338. According to certain embodiments, the anti-IL-4Rα antibody or antigen-binding fragment thereof comprises three HCDRs (HCDR1, HCDR2, and HCDR3) and three LCDRs (LCDR1, LCDR2, and LCDR3), wherein HCDR1 comprises the amino acid sequence of SEQ ID NO: 339, HCDR2 comprises the amino acid sequence of SEQ ID NO: 340, HCDR3 comprises the amino acid sequence of SEQ ID NO: 341, LCDR1 comprises the amino acid sequence of SEQ ID NO: 342, LCDR2 comprises the amino acid sequence of SEQ ID NO: 343, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 344. In yet other embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof comprises a HCVR comprising SEQ ID NO: 337 and a LCVR comprising SEQ ID NO: 338. In yet other embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof comprises a HCVR comprising SEQ ID NO: 335 and a LCVR comprising SEQ ID NO: 336. In yet other embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof comprises a heavy chain (HC) amino acid sequence set forth in SEQ ID NO: 345 and a light chain (LC) amino acid sequence set forth in SEQ ID NO: 346. According to certain exemplary embodiments, the methods of the present invention involve the use of an anti-IL-4Rα antibody known in the art and designated dupilumab, or a biological equivalent thereof.Dupilumab comprises an HCVR having the amino acid sequence of SEQ ID NO: 337, an LCVR having the amino acid sequence of SEQ ID NO: 338, heavy chain complementarity determining regions (HCDR1-HCDR2-HCDR3) having the amino acid sequences of SEQ ID NOs: 339-340-341, respectively, and light chain complementarity determining regions (LCDR1-LCDR2-LCDR3) having the amino acid sequences of SEQ ID NOs: 342-343-344, respectively.

[0184] Other anti-IL-4Rα antibodies that can be used in connection with the methods of the present invention include, for example, the antibody known in the art designated AMG317 (Corren et al., 2010, Am J Respir Crit Care Med., 181(8):788-796)), or MEDI9314, or any of the anti-IL-4Rα antibodies set forth in U.S. Pat. Nos. 7,186,809, 7,605,237, 7,638,606, 8,092,804, 8,679,487, or 8,877,189.

[0185] pH-dependent properties of anti-IL-4 and / or anti-IL-33 antibodies The anti-IL-4Rα and IL-33 antibodies used in the present methods may have pH-dependent binding properties. For example, an anti-IL-4Rα antibody or anti-IL-33 antibody for use in the present methods may exhibit reduced binding to IL-4Rα or IL-33, respectively, at acidic pH compared to neutral pH. Alternatively, an anti-IL-4Rα antibody or anti-IL-33 antibody of the present invention may exhibit enhanced binding to its antigen at acidic pH compared to neutral pH. The term "acidic pH" includes pH values ​​below about 6.2, such as about 6.0, 5.95, 5.9, 5.85, 5.8, 5.75, 5.7, 5.65, 5.6, 5.55, 5.5, 5.45, 5.4, 5.35, 5.3, 5.25, 5.2, 5.15, 5.1, 5.05, 5.0, or less. As used herein, the term "neutral pH" refers to a pH of about 7.0 to about 7.4. Includes pH values ​​of 0.5, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, and 7.4.

[0186] In certain instances, "reduced binding to IL-4Rα at acidic pH compared to neutral pH" or "reduced binding to IL-33 at acidic pH compared to neutral pH" refers to the K of an antibody that binds to IL-4Rα or IL-33, respectively, at neutral pH. D K values ​​for antibodies binding to IL-4Rα or IL-33, respectively, at acidic pH D For example, an antibody or antigen-binding fragment thereof is, for purposes of the present invention, expressed in terms of a ratio of values ​​(or vice versa) of the antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof has an acidic / neutral K of about 3.0 or greater. D When a ratio is presented, it can be considered to indicate "reduced binding to IL-4Rα at acidic pH compared to neutral pH" or "reduced binding to IL-33 at acidic pH compared to neutral pH." In certain exemplary embodiments, the acidic / neutral K of an antibody or antigen binding of the invention D The ratio can be about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 20.0, 25.0, 30.0, 40.0, 50.0, 60.0, 70.0, 100.0, or more.

[0187] Antibodies with pH-dependent binding properties can be obtained, for example, by screening a population of antibodies for decreased (or increased) binding to a specific antigen at acidic pH compared to neutral pH. Furthermore, modification of the antigen-binding domain at the amino acid level can produce antibodies with pH-dependent characteristics. For example, by substituting one or more amino acids in the antigen-binding domain (e.g., within the CDR) with histidine residues, an antibody can be obtained that has decreased antigen binding at acidic pH compared to neutral pH. As used herein, the term "acidic pH" refers to a pH of 6.0 or below.

[0188] Biological effects of IL-33 and IL-4R antagonists used in combination therapy The present invention includes the use of an IL-33 antagonist in combination with an IL-4R antagonist to treat inflammatory conditions. In one embodiment, the use of an anti-IL-33 antibody in combination with an anti-IL4R antibody in an animal model of fibrosis and pulmonary inflammation shows enhanced efficacy compared to the results obtained when each antibody is used alone as monotherapy.

[0189] For example, in the animal model described herein (referred to as the House Dust Mite (HDM) model of pulmonary inflammation and fibrosis), the levels of certain cytokines in the lungs are significantly elevated. These include elevated IL-4, IL-5, IL-6, IL-1β, and MCP-1. A trend toward increased IL-13 and TNFα levels in the lungs of mice was also observed after administration of house dust mite allergen. However, when tested in this model, the use of a combination of IL-33 and IL-4R antibodies resulted in reduced levels of the cytokines IL-4, IL-5, IL-6, IL-13, IL-1β, MCP-1, and TNFα in the lungs of treated mice. As shown in Example 4, the effect on lung cytokine levels observed with the combination of anti-IL-33 and anti-IL-4R antibodies was greater than that observed with treatment with either individual antibody when used alone.

[0190] Furthermore, mice exposed to house dust mite allergen showed increased levels of cytokine, chemokine, and collagen genes, including Il4, Il13, Il6, Ccl2, Tgfb1, Il13ra2, and Col24a1. This model also showed a trend toward increased levels of Il5, Il9, Ccl11, Ccl24, Tnf, Il1rl1, and Col15a1. Treatment with a combination of anti-IL-33 and anti-IL-4R antibodies significantly reduced the expression of Il6, Ccl2, Ccl11, and Ccl24 compared to the levels observed with either antibody alone. Mice exposed to anti-IL- Treatment with 33 antibody and anti-IL-4R antibody also showed a trend toward decreased expression of Il4, Il5, Il13, Il9, Tnf, Tgfb1, Il1rl1, Il13ra2, Col15a1, and Col24a1 compared with treatment with either antibody alone.

[0191] When lung cellular infiltrates in the house dust mite model were analyzed, additional biological effects associated with the combined use of anti-IL-33 and anti-IL-4R antibodies were observed. As shown in Example 4, the frequencies of eosinophils, activated B cells, activated CD8 cells, ST2+ CD4+ T cells, and the CD4 / CD8 T cell ratio were significantly higher in mice receiving house dust mite allergen. A trend toward increased frequencies of activated CD4+ T cells in the lungs of mice given house dust mite allergen was also observed. Mice treated with both anti-IL-33 and anti-IL-4R antibodies showed a trend toward decreased frequencies of eosinophils, activated B cells, activated CD8 cells, ST2+ CD4+ T cells, and the CD4 / CD8 T cell ratio compared to those observed when either antibody was used alone.

[0192] Furthermore, mice exposed to house dust mite allergen also exhibited increased goblet cell metaplasia in their lungs. Similarly, this mouse model also exhibited increased lung consolidation (accumulation of solid or liquid material in the alveolar spaces) and subepithelial fibrosis (excessive interstitial collagen deposition beneath the lung epithelium). Treatment of these mice with an anti-IL-33 antibody in combination with an anti-IL-4R antibody resulted in a significant reduction in goblet cell metaplasia and subepithelial collagen thickness and lung consolidation compared to the results observed when either of the two antibodies was used alone.

[0193] Mice receiving house dust mite allergen also showed increased circulating levels of IgE and a trend toward increased levels of house dust mite (HDM)-specific IgG1. Administration of both anti-IL-33 and IL-4R antibodies resulted in a significant decrease in serum IgE levels and a trend toward decreased levels of HDM-specific IgG1 compared to the levels of IgE and HDM-specific IgG1 observed when either of these antibodies was used alone.

[0194] IL-33 antagonists and IL-4R antagonists, such as the antibodies described herein, for use as a combination therapy to treat inflammatory lung disorders or conditions can inhibit or attenuate IL-33-mediated signaling and IL-4R-mediated signaling and can exhibit one or more of the biological properties observed in the models described herein, e.g., (1) a reduction in levels of cytokines, e.g., IL-4 or IL-5, that are elevated in mammals as a result of exposure to an allergen; (2) an inhibition of pulmonary inflammation resulting from acute or chronic exposure to an allergen (e.g., house dust mite (HDM)); (3) a reduction in cellular pulmonary infiltrates resulting from acute or chronic exposure to an allergen (e.g., house dust mite (HDM)); and (4) an improvement in composite macroscopic lung pathology.

[0195] Inhibition of IL-33-mediated signaling or IL-4R-mediated signaling can be measured in a cell-based bioassay, and means that the anti-IL-33 antibody or antigen-binding fragment thereof, or the anti-IL-4R antibody or antigen-binding fragment thereof, inhibits or reduces the signal generated in cells expressing the IL-33 receptor or IL-4 receptor and a reporter element that produces a detectable signal in response to IL-33 binding or IL-4 binding. For example, the present invention provides compounds with an IC of less than about 2 nM, less than about 1 nM, less than about 900 pM, less than about 800 pM, less than about 700 pM, less than about 600 pM, less than about 500 pM, less than about 400 pM, less than about 350 pM, less than about 300 pM, less than about 250 pM, less than about 200 pM, less than about 150 pM, less than about 100 pM, less than about 90 pM, less than about 80 pM, less than about 70 pM, less than about 60 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM, or less than about 10 pM in cells expressing human ST2 or cells expressing the IL-4 receptor, respectively, as measured in a cell-based inhibition bioassay. 50 Antibodies and methods for blocking IL-33-mediated or IL-4-mediated signaling in mice and an antigen-binding fragment of

[0196] The antibodies of the present invention may exhibit one or more of the above-mentioned biological effects, or any combination thereof. Other biological effects of the antibodies of the present invention will be apparent to those skilled in the art upon review of this disclosure, including the Examples herein. The use of other IL-33 antagonists in combination with IL-4 antagonists may exhibit similar effects.

[0197] Pharmaceutical Compositions and Administration The present invention provides pharmaceutical compositions comprising the IL-33 antagonist and / or IL-4R antagonist of the present invention. The IL-33 antagonist and IL-4R antagonist may be formulated in separate compositions or co-formulated in a single composition. The pharmaceutical compositions of the present invention are formulated with suitable carriers, excipients, and other agents that provide improved migration, delivery, tolerance, etc. Many suitable formulations can be found in formularies known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipid (cationic or anionic) containing vesicles (e.g., LIPOFECTIN™, Life Technologies, Carlsbad, CA), DNA complexes, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, emulsion carbowax (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. See Powell et al., "Compendium of excipients for parenteral formulations," PDA (1998) J Pharm Sci Technol 52:238-311.

[0198] The dose of an antibody administered to a patient may vary depending on the patient's age and size, the target disease, condition, route of administration, etc. Preferred doses are typically calculated according to body weight or body surface area. When the antibodies of the present invention are used to treat conditions or diseases associated with IL-33 activity or IL-4 in adult patients, it may be advantageous to administer the antibody at a single dose of typically about 0.01 to about 20 mg / kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg / kg body weight. The frequency and duration of treatment can be adjusted depending on the severity of the condition. Effective doses and schedules for administering anti-IL-33 antibodies can be determined empirically; for example, the patient's progress can be monitored by periodic evaluation and the dose adjusted accordingly. Furthermore, interspecies scaling of dosages can be performed using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0199] Various delivery systems are known and can be used to administer the pharmaceutical compositions of the present invention, such as liposome encapsulation, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis (see, e.g., Wu et al., J. Immunol. 1999, 103:111-114). (See, e.g.,

[0010] et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered by any convenient route, for example, by infusion or bolus injection, absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other biologically active agents. Administration can be systemic or local.

[0200] The pharmaceutical compositions of the present invention can be delivered subcutaneously or intravenously using a standard needle and syringe. In addition, for subcutaneous delivery, pen delivery devices readily find application in delivering the pharmaceutical compositions of the present invention. Such pen delivery devices may be reusable or disposable. Reusable pen delivery devices generally use a replaceable cartridge containing the pharmaceutical composition. Once the pharmaceutical composition in the cartridge has been administered and the cartridge is emptied, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In disposable pen delivery devices, there is no replaceable cartridge. Rather, the disposable pen delivery device is pre-filled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0201] Numerous reusable pen delivery devices and autoinjector delivery devices have application in the subcutaneous delivery of the pharmaceutical compositions of the present invention. Examples include the AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), the DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), the HUMALOG MIX 75 / 25™ pen, the HUMALOG™ pen, and the HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), the NOVOPEN™ I, II, and III (Novo Nordisk, Copenhagen, Denmark), the NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), the BD™ pen (Becton Dickinson, Franklin Lakes, NJ), the OPTIPEN™, and the OPTIPEN™. Disposable pen delivery devices include, but are not limited to, PRO (TM), OPTIPEN STARLET (TM), and OPTICLIK (TM) (sanofi-aventis, Frankfurt, Germany), to name just a few. Examples of disposable pen delivery devices that are suitable for delivering the pharmaceutical compositions of the present invention include, but are not limited to, SOLOSTAR (TM) pen (sanofi-aventis), FLEXPEN (TM) (Novo Nordisk), and KWIKPEN (TM) (Eli Lilly), SURECLICK (TM) auto-injector (Amgen, Thousand Oaks, CA), PENLET (TM) (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA (TM) pen (Abbott Labs, Abbott Park, IL), to name just a few.

[0202] In certain circumstances, pharmaceutical compositions can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201). In another embodiment, a polymeric material can be used. Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Florida. In yet another embodiment, a sustained-release system can be placed in proximity of the target of the composition, thereby requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications (See, "Controlled Release," supra, vol. 2, pp. 115-138.) Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0203] The injectable preparations may include dosage forms for intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, drip infusion, etc. These injectable preparations may be prepared by publicly known methods. For example, the injectable preparations may be prepared by, for example, conventionally used disinfectants for injection. The antibody or its salt can be prepared by dissolving, suspending, or emulsifying it in an aqueous or oily medium. Aqueous media for injection include, for example, saline, glucose-containing isotonic solutions, and other adjuvants. These may be used in combination with suitable solubilizers, such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants (e.g., polysorbate 80, HCO-50 (a polyoxyethylene (50 mole) adduct of hydrogenated castor oil)). Oily media include, for example, sesame oil and soybean oil, which may be used in combination with solubilizers, such as benzyl benzoate and benzyl alcohol. The injections prepared in this manner are preferably filled into appropriate ampoules.

[0204] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared into unit dosage forms suitable for the dosage of the active ingredient. Examples of such unit dosage forms include tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the antagonist contained is generally about 5 to about 500 mg per unit dosage form. In particular, in the form of injection, the antagonist is preferably contained in an amount of about 5 to about 100 mg, and in other dosage forms, the antagonist is preferably contained in an amount of about 10 to about 250 mg.

[0205] Dosage The amounts of IL-33 and IL-4R antagonists administered to a subject according to the methods of the present invention are generally therapeutically effective amounts. As used herein, the phrase "therapeutically effective amount" refers to the amount of IL-33 antagonist and IL-4R antagonist that, when used in combination, results in a significant change in one or more of the following as described herein: (a) prevention of inflammation; (b) treatment or reduction in severity of inflammation; (c) reduction in the frequency of one or more of eosinophils, activated B cells, activated CD8 T cells, or the CD4 / CD8 T cell ratio in the lung; (d) reduction in the expression of interleukin-1 beta (IL-1β), interleukin-4 (IL- 4), a decrease in one or more of interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-13 (IL-13), monocyte chemoattractant protein-1 (MCP-1), or tumor necrosis factor alpha (TNFα); (e) a decrease in the gene expression levels of one or more of Il4, Il5, Il6, Il9, Il13, Il1rl1, Il13ra2, tnf, Tgfb1, Ccl2, Ccl11, Ccl24, Col15a1, or Col24a1 in the lung; (f) a decrease in serum IgE levels; (g) a decrease in goblet cell metaplasia in the lung; or (h) a decrease in lung consolidation. While administration of either an IL-33 antagonist alone or an IL-4R antagonist can result in a positive therapeutic effect as measured using one or more of the above parameters, the use of an IL-33 antagonist and an IL-4R antagonist in combination shows a significant improvement (e.g., an additive or synergistic effect) in any one or more of those parameters compared to that observed using monotherapy with either an IL-33 antagonist alone or an IL-4R antagonist alone.

[0206] In the case of an IL-33 antagonist or an IL-4R antagonist, the therapeutically effective amount is about 0.05 mg to about 600 mg, for example, about 0.05 mg, about 0.1 mg, about 1.0 mg, about 1.5 mg, about 2.0 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, about 550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg, about 600 mg, about 610 mg, about 620 mg, about 630 mg, about 640 mg mg, about 150mg, about 160mg, about 170mg, about 180mg, about 190mg, about 200mg, about 210mg, about 220mg, about 230mg, about 240mg, about 250mg, about 260mg, about 27 0mg, about 280mg, about 290mg, about 300mg, about 310mg, about 320mg, about 330mg, about 340mg, about 350mg, about 360mg, about 370mg, about 380mg, about 390mg, about 4 The dosage may be about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, about 500 mg, about 510 mg, about 520 mg, about 530 mg, about 540 mg, about 550 mg, about 560 mg, about 570 mg, about 580 mg, about 590 mg, or about 600 mg. In certain embodiments, 75 mg, 150 mg, 200 mg, or 300 mg of an IL-4R antagonist is administered to a subject in combination with an IL-33 antagonist. In certain embodiments, 75 mg, 150 mg, 200 mg, or 300 mg of an IL-33 antagonist is administered to a subject in combination with an IL-4R antagonist.

[0207] The amount of IL-33 antagonist or IL-4R antagonist contained in an individual dose can be expressed in milligrams of antibody per kilogram of patient body weight (i.e., mg / kg). For example, the IL-33 antagonist or IL-4R antagonist can be administered to a patient at a dose of about 0.0001 mg / kg to about 25 mg / kg of patient body weight. In certain embodiments, the IL-4R and IL-33 antagonists can be administered at doses of about 0.1 mg / kg, 0.3 mg / kg, 1.0 mg / kg, 3.0 mg / kg, or 10 mg / kg, respectively.

[0208] The combination of the IL-33 antagonist and the IL-4R antagonist can be administered to a subject subcutaneously, intravenously, intramuscularly, or intranasally. They can be administered simultaneously or sequentially.

[0209] Therapeutic Uses of Antibodies Our experiments using mouse model systems have helped identify various diseases and conditions that can be treated, prevented, and / or ameliorated by a combination of IL-33 and IL-4R antagonism. For example, in a house dust mite model of pulmonary inflammation and fibrosis, treatment with a combination of an IL-33 antibody and an IL-4R antibody resulted in reduced cytokine levels in the lung, reduced lung cellular infiltrates (eosinophils, activated B cells, activated CD8+ cells, ST2+CD4+ T cells, and the CD4 / CD8 T cell ratio) in the lung, and improved lung consolidation and subepithelial fibrosis compared to the effects achieved when each antibody was used alone as a monotherapy.

[0210] The antibodies of the present invention are particularly useful for the treatment, prevention, and / or amelioration of any disease or disorder associated with or mediated by IL-33 expression and IL-4 expression, signaling, or activity, or any disease or disorder treatable by blocking the interaction between IL-33 and an IL-33 receptor (e.g., ST2), or by blocking the interaction between IL-4 and an IL-4 receptor, or by inhibiting the activity and / or signaling of IL-33 and IL-4. In certain embodiments, the IL-4R antagonist is an antibody that binds to or interacts with IL-4Rα, thereby blocking both the IL-4 and IL-13 signaling pathways via the IL-4R type 1 and type 2 receptors. Thus, the use of this dual IL-4 and IL-13 antagonist in combination with an IL-33 antagonist may provide additional clinical benefit when administered to patients with inflammatory conditions mediated in part by all three signaling pathways. For example, the present invention provides a method for treating asthma (allergic asthma, non-allergic asthma, severe refractory asthma, asthma exacerbation, steroid-resistant or steroid-refractory asthma, steroid-sensitive asthma, eosinophilic asthma or non-eosinophilic asthma, etc.), chronic obstructive pulmonary disease (COPD) and COPD exacerbation, asthma and COPD overlap syndrome (ACOS), chronic bronchitis, emphysema, hypersensitivity pneumonitis, atopic dermatitis, urticaria, psoriasis, allergy, allergic rhinitis, chronic rhinosinusitis with or without nasal polyps, eosinophilic esophagitis, anaphylaxis, cardiovascular disease, central nervous system disease, pain (including inflammatory pain), arthritis (e.g., rheumatoid arthritis, osteoarthritis, psoriatic arthritis, etc.), giant cell arteritis, Methods are provided for treating vasculitis (Behcet's disease and Churg-Strauss syndrome), Henoch-Schönlein purpura, multiple sclerosis, inflammatory bowel disorders (e.g., Crohn's disease or ulcerative colitis), lupus, Sjogren's syndrome, and other inflammatory diseases or disorders mediated in part by IL-33 and / or IL-4 signaling.

[0211] The antibodies of the present invention are also useful for the treatment, prevention, and / or amelioration of one or more fibrotic diseases or disorders. Exemplary fibrotic diseases or disorders treatable by administering the anti-IL-33 and IL-4R antagonists of the present invention include pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis, bleomycin-induced pulmonary fibrosis, asbestos-induced pulmonary fibrosis, and bronchiolitis obliterans syndrome), fibrosis associated with acute lung injury and acute respiratory distress (e.g., bacterial pneumonia-induced fibrosis, trauma-induced fibrosis, viral pneumonia-induced fibrosis, ventilator-induced fibrosis, non-pulmonary fibrosis, and pulmonary fibrosis). fibrosis, including fibrosis caused by liver cirrhosis, alcohol-induced liver fibrosis, non-alcoholic steatohepatitis (NASH), bile duct injury, primary biliary cirrhosis, infection- or virus-induced liver fibrosis, autoimmune hepatitis, renal (kidney) fibrosis, cardiac fibrosis, atherosclerosis, stent restenosis, and bone marrow fibrosis.

[0212] In the context of the treatment methods described herein, the anti-IL-33 antibody and the IL-4R antibody may be administered together (i.e., as the sole treatment regimen) or in combination with one or more additional therapeutic agents (examples of which are described elsewhere herein).

[0213] Combination therapy The present invention includes compositions and therapeutic formulations comprising any of the anti-IL-33 antagonists and IL-4R antagonists described herein in combination with one or more additional therapeutically active ingredients, as well as methods of treatment comprising administering such combinations to a subject in need of treatment. As used herein, the term "in combination" means that the additional therapeutic agent is administered before, after, or simultaneously with a pharmaceutical composition comprising an IL-33 antagonist and an IL-4R antagonist. The term "in combination" also includes sequential or simultaneous administration of an IL-4R antagonist and an IL-33 antagonist and one or more additional therapeutic agents. The present invention includes pharmaceutical compositions in which the IL-33 antagonists and IL-4R antagonists of the present invention are co-formulated with one or more additional therapeutically active ingredients.

[0214] For example, when administered "before" a pharmaceutical composition comprising an IL-33 antagonist and an IL-4R antagonist, an additional therapeutic agent can be administered about 72 hours, about 60 hours, about 48 hours, about 36 hours, about 24 hours, about 12 hours, about 10 hours, about 8 hours, about 6 hours, about 4 hours, about 2 hours, about 1 hour, about 30 minutes, about 15 minutes, or about 10 minutes before administration of the pharmaceutical composition comprising an IL-33 antagonist and an IL-4R antagonist. When administered "after" the pharmaceutical composition comprising an IL-33 antagonist and an IL-4R antagonist, the additional therapeutic agent may be administered about 10 minutes, about 15 minutes, about 30 minutes, about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, or about 72 hours after administration of the pharmaceutical composition comprising an IL-33 antagonist and an IL-4R antagonist. Administration "concurrently with" or together with a pharmaceutical composition comprising an IL-33 antagonist and an IL-4R antagonist means that the additional therapeutic agent is administered to the subject in a separate dosage form within less than 5 minutes of (before, after, or simultaneously with) administration of the pharmaceutical composition comprising an IL-33 antagonist and an IL-4R antagonist, or is administered to the subject as a single combined dosage formulation comprising both the additional therapeutic agent and the IL-33 antagonist and IL-4R antagonist.

[0215] The additional therapeutic agent may be, for example, another IL-33 antagonist, another IL-4R antagonist, an IL-1 antagonist (including, for example, the IL-1 antagonists set forth in US 6,927,044), an IL-6 antagonist, an IL-6R antagonist (including, for example, the anti-IL-6R antibodies set forth in US 7,582,298), an IL-13 antagonist, a TNF antagonist, an IL-8 antagonist, an IL-9 antagonist, an IL-17 antagonist, an IL-5 antagonist (e.g., mepolizumab or NUCALA®), an IgE antagonist (e.g., omalizumab or XOLAIR®), a CD48 antagonist, or an IL-13 antagonist. The therapeutic agent may be an antihistamine, an IL-31 antagonist (including, for example, those set forth in US 7,531,637), a thymic stromal lymphopoietin (TSLP) antagonist (including, for example, those set forth in US 2011 / 027468), interferon-gamma (IFNγ), an antibiotic, a corticosteroid (including an inhaled corticosteroid or ICS), a long-acting beta-2 adrenergic agonist (LABA), a long-acting muscarinic antagonist (LAMA), tacrolimus, pimecrolimus, cyclosporine, azathioprine, methotrexate, cromolyn sodium, a proteinase inhibitor, an antihistamine, or a combination thereof.

[0216] Dosage regimen According to certain embodiments of the present invention, multiple doses of an IL-33 antagonist and an IL-4R antagonist (or a pharmaceutical composition comprising a combination of an IL-33 antagonist, an IL-4R antagonist, and any of the additional therapeutically active agents mentioned herein) may be administered to a subject over a defined time course. The method according to this aspect of the present invention comprises sequentially administering multiple doses of an IL-33 antagonist and an IL-4R antagonist of the present invention to a subject. As used herein, "sequentially administering" means that each dose of an IL-33 antagonist and an IL-4R antagonist is administered to a subject at different time points, e.g., on different days, separated by a predetermined interval (e.g., hours, days, weeks, or months). The present invention includes methods comprising sequentially administering to a patient one primary dose of an IL-33 antagonist and an IL-4R antagonist, followed by one or more secondary doses of an IL-33 antagonist and an IL-4R antagonist, optionally followed by one or more tertiary doses of an IL-33 antagonist and an IL-4R antagonist.

[0217] The terms "primary dose," "secondary dose," and "tertiary dose" refer to the time sequence of administration of the IL-33 antagonist and IL-4R antagonist of the present invention. Thus, a "primary dose" is a dose administered at the beginning of a treatment regimen (also referred to as a "baseline dose"), a "secondary dose" is a dose administered after the primary dose, and a "tertiary dose" is a dose administered after the secondary dose. The primary, secondary, and tertiary doses may all contain the same amount of IL-33 antagonist and IL-4R antagonist, but generally may differ from one another in terms of frequency of administration. However, in certain embodiments, the amounts of IL-33 antagonist and IL-4R antagonist contained in the primary, secondary, and / or tertiary doses vary from one another (e.g., adjusted up or down as appropriate) during the course of treatment. In certain embodiments, two or more (e.g., two, three, four, or five) doses are administered as a "loading dose" at the beginning of a treatment regimen, followed by subsequent doses (e.g., "maintenance doses") administered on a less frequent basis.

[0218] In certain exemplary embodiments of the invention, each secondary dose and / or tertiary dose is administered 1 to 26 weeks (e.g., 1, 1½, 2, 2½, 3, 3½, 4, 4½, 5, 5½, 6, 6½, 7, 7½, 8, 8½, 9, 9½, 10, 10½, 11, 11½, 12, 12½) after the immediately preceding administration. , 13, 13½, 14, 14½, 15, 15½, 16, 16½, 17, 17½, 18, 18½, 19, 19½, 20, 20½, 21, 21½, 22, 22½, 23, 23½, 24, 24½, 25, 25½, 26, 26½ weeks or more later). As used herein, the phrase "immediately preceding" refers to a dose of an IL-33 antagonist and an IL-4R antagonist administered to a patient in a multiple dose series prior to administration of the next dose in the series without any intervening doses.

[0219] The method according to this aspect of the invention can include administering any number of secondary and / or tertiary doses of the IL-33 antagonist and the IL-4R antagonist to the patient. For example, in certain embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., two, three, four, five, six, seven, eight, or more) secondary doses are administered to the patient. Similarly, in certain embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g., two, three, four, five, six, seven, eight, or more) tertiary doses are administered to the patient.

[0220] In embodiments comprising multiple secondary doses, each secondary dose may be administered at the same frequency as the other secondary doses. For example, each secondary dose may be administered to the patient 1-2 weeks or 1-2 months after the immediately preceding dose. Similarly, in embodiments comprising multiple tertiary doses, each tertiary dose may be administered at the same frequency as the other tertiary doses. For example, each tertiary dose may be administered to the patient 2-12 weeks after the immediately preceding dose. In certain embodiments of the invention, the frequency with which the secondary and / or tertiary doses are administered to the patient may vary over the course of the treatment regimen. The administration frequency may also be adjusted by the physician during the course of treatment depending on the individual patient's needs after clinical testing.

[0221] The present invention includes dosing regimens in which two to six loading doses are administered to a patient at a first frequency (e.g., once per week, once every two weeks, once per three weeks, once per month, once every two months, etc.), followed by two or more maintenance doses administered to the patient less frequently. For example, according to this aspect of the invention, if the loading dose is administered monthly, maintenance doses may be administered to the patient every six weeks, every two months, every three months, etc. [Example]

[0222] The following examples are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors regard as the invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise specified, parts are parts by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric.

[0223] Example 1. Generation of human antibodies against human IL-33 Human anti-IL-33 antibodies were produced as described in U.S. Patent No. 9,453,072. Table 1 shows the heavy and light chain variable region amino acid sequence pairs and CDR sequences of selected anti-IL-33 antibodies and their corresponding antibody identifiers. Table 2 shows the heavy and light chain variable region amino acid sequence pairs and nucleic acid sequences encoding the CDR sequences of selected anti-IL-33 antibodies and their corresponding antibody identifiers. [Table 1] [Table 2]

[0224] Antibodies are typically referred to herein according to the following nomenclature: an Fc prefix (e.g., "H1M" or "H4H") followed by a numerical identifier (e.g., "9559," "9566," or "9629" as shown in Table 1), followed by a "P" or "N" suffix. Thus, according to this nomenclature, antibodies may be referred to herein as, for example, "H1M9559N," "H1M9566N," "H4H9629P," etc. The H1M and H4H prefixes in antibody names used herein indicate the particular Fc region isotype of the antibody. For example, an "H1M" antibody has a murine IgG1 Fc, whereas an "H4H" antibody has a human IgG4 Fc. As will be appreciated by those of skill in the art, an antibody having a particular Fc isotype can be converted to an antibody having a different Fc isotype (e.g., an antibody having a murine IgG1 Fc can be converted to an antibody having a human IgG4 Fc, etc.), but in all events, the variable domains (including the CDRs) indicated by the numerical identifiers shown in Table 1 will remain the same, and the binding characteristics are expected to be the same or substantially similar regardless of the nature of the Fc domain.

[0225] Example 2: Construction of IL-33 antagonist (IL-33 trap) Human anti-IL-33 traps were generated as described in U.S. Patent Publication No. 2014 / 0271642. Table 3a provides a summary of the amino acid sequence identifiers for the various components of the IL-33 trap, and Table 3b provides the full-length amino acid sequences of the traps.

[0226] Five different exemplary IL-33 antagonists of the present invention were constructed using standard molecular biology techniques. The first IL-33 antagonist (hST2-hFc, SEQ ID NO: 3) The first IL-33 antagonist (hST2-mFc, SEQ ID NO: 323) consists of the soluble extracellular region of human ST2 (SEQ ID NO: 328) fused at its C-terminus to the N-terminus of a human IgG1 Fc region (SEQ ID NO: 332). The second IL-33 antagonist (hST2-mFc, SEQ ID NO: 324) consists of the soluble extracellular region of human ST2 (SEQ ID NO: 328) fused at its C-terminus to the N-terminus of a mouse IgG2a Fc region (SEQ ID NO: 333). The third IL-33 antagonist (hST2-hIL1RAcP-mFc, SEQ ID NO: 325) consists of a tandem fusion of human ST2 (SEQ ID NO: 328) at its N-terminus, followed by the extracellular region of human IL-1RAcP (SEQ ID NO: 330), followed by mouse IgG2a Fc (SEQ ID NO: 333) at its C-terminus. The fourth IL-33 antagonist (mST2-mIL1RAcP-mFc, SEQ ID NO: 326) consists of a tandem fusion of mouse ST2 (SEQ ID NO: 329) at its N-terminus, followed by the extracellular region of mouse IL-1RAcP (SEQ ID NO: 331), followed by mouse IgG2a Fc (SEQ ID NO: 333) at its C-terminus. The fifth IL-33 antagonist (hST2-hIL1RAcP-hFc, SEQ ID NO: 327) consists of a tandem fusion of human ST2 of SEQ ID NO: 328 at its N-terminus, followed by the extracellular region of human IL-1RAcP (SEQ ID NO: 330), followed by human IgG1 Fc (SEQ ID NO: 332) at its C-terminus. Table 3a provides an overview of the different IL-33 antagonists and their component parts. Table 3b provides the amino acid sequences of the IL-33 antagonists and their component parts. [Table 3] [Table 4] [Table 5] [Table 6] [Table 7]

[0227] Example 3: IL-4R antagonist antibodies Human anti-IL-4R antibodies were generated as described in U.S. Patent No. 7,608,693. The exemplary IL-4R antibody used in the following examples is a murine antibody specific for murine IL-4R and has the following amino acid sequences: a heavy chain variable region (HCVR) comprising SEQ ID NO: 335 and a light chain variable domain (LCVR) comprising SEQ ID NO: 336. The human anti-IL-4R antibody designated dupilumab specifically binds to human IL-4Rα and has a heavy chain variable region (HCVR) comprising SEQ ID NO: 337, a light chain variable region (LCVR) comprising SEQ ID NO: 338, a heavy chain complementarity-determining region 1 (HCDR1) comprising SEQ ID NO: 339, and a HCDR2 comprising SEQ ID NO: 340. and a light chain complementarity determining region 1 (LCDR1) comprising SEQ ID NO: 342, an LCDR2 comprising SEQ ID NO: 343, and an LCDR3 comprising SEQ ID NO: 344. The full-length heavy chain of dupilumab is set forth as SEQ ID NO: 345, and the full-length light chain is set forth as SEQ ID NO: 346.

[0228] Example 4: Chronic House Dust Mite (HDM)-Induced Fibrosis and Severe Lung Inflammation Model to Study the Role of IL-33 in Lung Inflammation - Comparison of the Effects of Anti-IL-33 Antibody, IL-4R Antibody, or a Combination of Both Chronic inflammatory airway diseases are the result of recurrent episodes of airway inflammation, primarily due to repeated exposure to allergens or other pathogens. In humans, such chronic injury induces a variety of pathologies, including pulmonary infiltration by immune cells, increased cytokine production, mucus production, and collagen deposition (Hirota, (2013) Chest. Sep;144(3):1026-32; Postma, (2015) N Engl J Med., Sep 24;373(13):1241-9). This increase in inflammatory cytokines and immune cell infiltration is accompanied by severe airway remodeling, leading to airway narrowing, hypersensitivity to inhaled provocative agents such as allergens or pathogens, airway obstruction, and loss of lung function.

[0229] To determine the effects of anti-IL-33 inhibition in a relevant in vivo model, chronic house dust mite extract (HDM)-induced fibrosis and severe lung inflammation and remodeling studies were conducted in mice homozygous for expression of human IL-33 instead of mouse IL-33 (IL-33HumIn mice; see U.S. Patent Publication Nos. 2015 / 0320021 and 2015 / 0320022). Chronic HDM extract exposure induces severe lung inflammation, resulting in significant cellular infiltration, cytokine expression, and remodeling. The effects of anti-IL-33 antibodies, anti-mouse IL-4Rα antibodies, or a combination of both were compared in this model. The anti-mouse IL-4Rα antibody used in this study was designated M1M1875N and contains the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 335 / 336. The anti-IL-33 antibody used in this study was designated H4H9675P and comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 274 / 282.

[0230] IL-33HumIn mice were intranasally administered either 50 μg of house dust mite extract (HDM; Greer, #XPB70D3A2.5) diluted in 20 μL of 1× phosphate-buffered saline (PBS) or 20 μL of 1× PBS, 3 days per week for 15 weeks. A second control group of IL-33HumIn mice received 50 μg of HDM extract diluted in 20 μL of 1× PBS, 3 days per week for 11 weeks, and disease severity was assessed at the start of antibody treatment. Four groups of HDM-challenged mice were subcutaneously injected with 25 mg / kg of either the anti-IL-33 antibody H4H9675P, the anti-mouse IL-4Rα antibody M1M1875N, a combination of both antibodies, or an isotype control antibody, twice per week, starting 11 weeks after HDM challenge and continuing until the end of HDM challenge (4 weeks of antibody treatment). All mice were sacrificed and their lungs harvested on day 108 of the study. The experimental dosing and treatment protocols for the mouse groups are shown in Table 4. [Table 8]

[0231] Lung collection for cytokine analysis: Elevated pulmonary levels of key mediators, such as the prototypic type 2 cytokines IL-4, IL-5, and IL-13, as well as cytokines more characteristic of type 1 immune responses, such as IL-1β or TNFα, have been implicated in the development of human lung disease (Gandhi, (2016) Nat Rev Drug Discov Jan;15(1):35-50.; Barnes, (2008) Nat Rev Immunol Mar;8(3):183-92). Pulmonary levels of these inflammatory cytokines were measured in this study.

[0232] After exsanguination, the anterior and median lobes of the right lung were removed from each mouse and placed in a tube containing a solution of tissue protein extraction reagent (1x T-PER Reagent, Pierce, #78510) supplemented with 1x Halt protease inhibitor cocktail (Thermo Scientific, #87786). All further steps were performed on ice. The volume of T-PER Reagent (containing the protease inhibitor cocktail) was adjusted for each sample to match a tissue-to-T-PER ratio of 1:7 (w / v). Lung samples were mechanically disrupted using a TissueLyser II (Qiagen, #85300). The resulting lysate was centrifuged to pellet debris. The supernatant containing the soluble protein extract was transferred to a new tube and stored at 4°C until further analysis.

[0233] The Bradford assay was used to measure the total protein content in lung protein extracts. For this assay, 10 μL of diluted extract samples were plated in duplicate in a 96-well plate and mixed with 200 μL of 1× Dye Reagent (Biorad, #500-0006). Serial dilutions of bovine serum albumin (BSA, Sigma, #A7979) starting at 700 μg / mL in 1× T-Per Reagent were used to measure the protein content of the extracts. The BSA standard was used to determine the protein concentration. After 5 minutes of incubation at room temperature, absorbance at 595 nm was measured using a Molecular Devices SpectraMax M5 plate reader. Data analysis to determine total lung extract protein content based on BSA standards was performed using GraphPad Prism™ software.

[0234] Cytokine concentrations in lung protein extracts were measured using the Proinflammatory Panel 1 (Mouse) Multiplex Immunoassay Kit (MesoScale Discovery, #K15048G-2) and the Custom Mouse 6plex Multi-Spot® Immunoassay Kit (MesoScale Discovery, #K152A41-4) according to the manufacturer's instructions. Briefly, 50 μL / well of calibrators and samples (diluted in Diluent 41) were added to plates pre-coated with capture antibodies and incubated at room temperature with shaking at 700 rpm for 2 hours. The plates were then washed three times with 1× PBS containing 0.05% (w / v) Tween-20, followed by the addition of 25 μL of detection antibody solution diluted in Diluent 45. After 2 hours of incubation at room temperature with shaking, the plates were washed three times, and 150 μL of 2× Read Buffer was added to each well. Electrochemiluminescence was immediately read on an MSD Spector® instrument. Data analysis was performed using GraphPad Prism software.

[0235] The concentration of each cytokine in the total lung protein extracts from all mice in each group was normalized to the total protein content of the extracts as measured by Bradford assay and expressed for each group as the mean pg of cytokine per mg of total lung protein (pg / mg lung protein, ±SD), as shown in Table 5.

[0236] Pulmonary cytokine analysis: As shown in Table 5, the levels of cytokines and chemokines IL-4, IL-5, IL-6, IL-1β, and MCP-1 released in the lungs of IL-33HumIn mice receiving HDM for 15 weeks and treated with or without an isotype control antibody were significantly higher than those of IL-33HumIn mice challenged with 1x PBS alone. Similarly, a trend toward increased release of cytokines IL-13 and TNFα was observed in the lungs of IL-33HumIn mice receiving HDM for 15 weeks. In contrast, the levels of IL-6, IL-13, and MCP-1 in the lungs of IL-33HumIn mice treated with a combination of anti-IL-33 and anti-mouse IL-4Rα antibodies during the final 4 weeks of chronic HDDM challenge were significantly decreased compared to IL-33HumIn mice administered HDM with an isotype control antibody during this period. Lung levels of IL-4, IL-5, IL-1β, and TNFα in HumIn mice tended to be reduced compared with IL-33HumIn mice administered HDM with an isotype control antibody during this period. The effect on lung cytokines observed with the combination of anti-IL-33 and anti-mouse IL-4Rα antibodies was greater than treatment with either individual antibody alone. [Table 9]

[0237] Sampling of lungs for gene expression analysis After exsanguination, the accessory lobe of the right lung was removed from each mouse, placed in a tube containing 400 μL of RNA Later (Ambion, #AM7020), and stored at −20°C until processing. The samples were homogenized in TRIzol, and chloroform was used for phase separation. The aqueous phase, containing total RNA, was purified using the MagMAX™-96 for Microarrays Total RNA Isolation Kit (Ambion by Life Technologies, #AM1839) according to the manufacturer's specifications. Genomic DNA was removed from the MagMAX™ kit using MagMAX™ Turbo™ DNase Buffer and TURBO DNase. mRNA (up to 2.5 μg) was reverse transcribed into cDNA using SuperScript® VILO™ Master Mix (Invitrogen by Life Technologies, #11755500). cDNA was diluted to 2 ng / μL, and 10 ng of cDNA was incubated with TaqMan® Gene Expression Master Mix (Applied Biosystems by Life Technologies, #4369542) and relevant probes (Life Technologies; mouse B2m: Mm00437762_m1; mouse Il4: Mm00445259_m1; mouse Il5: Mm00439646_m1; mouse Il13: Mm00434204_m1; mouse Il9: Mm00434305_m1; mouse Il6: Mm00446190_m1; mouse Ccl2: Mm00441242_m1; mouse Ccl11: Mm00441238_m1 The following cDNA fragments were amplified using an ABI 7900HT sequence detection system (Applied Biosystems): mouse Ccl24: Mm00444701_m1; mouse Tnf: Mm00443258_m1; mouse Tgfb1: Mm01178820_m1; mouse Il1rl1: Mm00516117_m1; mouse Il13ra2: Mm00515166_m1; mouse Col15a1: Mm00456584_m1; mouse Col24a1: Mm01323744_m1). B2m was used as an internal control gene to normalize for differences in cDNA input. The reference group used for normalization of all samples was the average of the group 1 samples ("1x PBS challenge").As shown in Table 6, the expression of each gene was normalized to B2m expression within the same sample and expressed relative to its normalized expression in the reference group (mean ± SD).

[0238] Lung gene expression analysis As shown in Table 6, the expression levels of the cytokine, chemokine, and collagen genes Il4, Il13, Il6, Ccl2, Tgfb1, Il13ra2, and Col24a1 in the lungs of IL-33HumIn mice that received HDM for 15 weeks and were treated with or without an isotype control antibody were significantly increased compared to IL-33HumIn mice challenged with 1x PBS alone. Similarly, there was a trend toward increased expression of the genes Il5, Il9, Ccl11, Ccl24, Tnf, Il1rl1, and Col15a1 in the lungs of IL-33HumIn mice that received HDM for 15 weeks.

[0239] In contrast, the levels of Il6, Ccl2, Ccl11, and Ccl24 were significantly reduced in IL-33HumIn mice treated with a combination of anti-IL-33 and anti-mouse IL-4Rα antibodies during the final 4 weeks of chronic HDDM challenge compared with IL-33HumIn mice administered HDM with an isotype control antibody during this period. In contrast, the expression levels of Il4, Il5, Il13, Il9, Tnf, Tgfb1, Il1rl1, Il13ra2, Col15a1, and Col24a1 tended to decrease in mice treated with a combination of anti-IL-33 and anti-mouse IL-4Rα antibodies during the final 4 weeks of chronic HDDM challenge compared with IL-33HumIn mice administered HDM with an isotype control antibody during this period. The effect on gene expression observed with the combination of anti-IL-33 and anti-mouse IL-4Rα antibodies was greater than that observed with either individual antibody alone. [Table 10] [Table 11]

[0240] Sampling of lungs for lung cellular infiltrate analysis Pulmonary infiltration by immune cells is observed in multiple airway inflammatory diseases, including asthma and COPD. Neutrophilic lung inflammation is associated with decreased lung function and severe tissue remodeling in asthmatic patients. It is associated with increased lung damage in COPD patients (Wenzel et al., (2012), Nat Med 18(5):716-725) and with increased lung damage in COPD patients (Meijer, et.al., (2013), Expert Rev. Clin. Immunol. 9(11):1055-1068). Eosinophilic lung inflammation is a hallmark of type 2 inflammation, commonly seen in atopic diseases (Jacobsen, et.al., (2014), Clin. Exp. Allergy, 44(9):1119-1136). In humans, high CD4 / CD8 ratios are observed in patients with granulomatous lung disease and other chronic inflammatory conditions (Costabel, et.al., (1997), Eur. Respir. J. 10(12):2699-2700; Guo, et.al., (2011), Ann. Clin. Biochem, 48(Pt4):344-351). In this study, we used flow cytometry to determine the level of cellular infiltration in the lungs of HDM-exposed mice.

[0241] After exsanguination, the caudal lobe of the right lung was removed from each mouse and minced into cubes approximately 2-3 mm in size. The pieces were then placed in a tube containing a solution of 20 μg / mL DNAse (Roche, #10104159001) and 0.7 U / mL Liberase TH (Roche, #05401151001) diluted in Hank's Balanced Salt Solution (HBSS) (Gibco, #14025). The tubes were incubated in a 37°C water bath for 20 min, vortexing every 5 min. The reaction was stopped by adding ethylenediaminetetraacetic acid (EDTA, Gibco, #15575) to a final concentration of 10 mM. Each lung was homogenized using a gentleMACS® homogenizer (Miltenyi Biotec, #130-095-937), then filtered through a 70 μm filter and centrifuged. The resulting lung pellet was resuspended in 1 mL of 1x erythrocyte lysis buffer (Sigma, #R7757) to remove red blood cells. After 3 minutes of incubation at room temperature, 3 mL of 1x DMEM was added to inactivate the erythrocyte lysis buffer. The cell suspension was then centrifuged, and the resulting cell pellet was resuspended in 5 mL of MACS buffer (autoMACS Running Buffer, Miltenyi Biotec, #130-091-221). The resuspended sample was filtered through a 70 μm filter, and 1 x 10 cells were collected per well. 6 Cells were plated in 96-well V-bottom plates. The cells were then centrifuged and the pellet washed in 1x PBS. After a second centrifugation, the cell pellet was washed with 100 μL of LIVE / DEAD® Fixable Blue Dead diluted 1:500 in 1x PBS. Cell viability was determined by resuspension in Cell Stain (Life Technologies, #L23105) and incubation for 20 minutes at room temperature, protected from light. After washing once in 1x PBS, cells were incubated in MACS buffer containing 10 μg / mL purified rat anti-mouse CD16 / CD32 Fc Block (clone: ​​2.4G2, BD Biosciences, #553142) for 10 minutes at 4°C. Protected from light, cells were then incubated in the appropriate 2x antibody mix (listed in Table 7) diluted in MACS buffer for 30 minutes at 4°C. After antibody incubation, cells were washed twice in MACS buffer, resuspended in BD CytoFix (BD Biosciences, #554655), and then incubated for 15 minutes at 4°C, protected from light. Cells were subsequently washed, resuspended in MACS buffer, and transferred to a BD FACS tube (BD Biosciences, #352235) for analysis of cellular infiltrates by flow cytometry.

[0242] CD4 and CD8 T cells were identified as live cells, CD45 + , S.S.C. Lo , FSC Lo , CD3 + , CD19 - , CD4 + , CD8 - , and live cell CD45 + , S.S.C. Lo , FSC Lo , CD3 + , CD19 - , CD4 - , CD8 + Activated CD4 T cells were defined as living cells with CD45 + , S.S.C. Lo , FSC Lo , CD3 + , CD19 - , CD4 + , CD8 - , and CD69 + Activated CD8 T cells were defined as live cells with CD45 + , S.S.C. Lo , FSCLo , CD3 + , CD19 - , CD4 - , CD8 + , and CD69 + Activated B cells were defined as living cells C D45 + , S.S.C. Lo , FSC Lo , CD3 - , CD19 + , and CD69 + ST2+ CD4+ T cells were defined as viable cells with CD45 + , S.S.C. Lo , FSC Lo , CD3+, CD19-, ST2+ and CD4 + Eosinophils were defined as living CD45 + , GR1 - , CD11c lo , SiglecF hi Alveolar macrophages were defined as living CD45 + , GR1 - , CD11c Hi , SiglecF hi The data for activated cells were calculated based on the percentage of activated cells (CD69) in the parent population (CD4, ±SD). + The data for ST2+CD4+ T cells are expressed as the frequency of T cells (viable cells CD45 + , S.S.C. Lo , FSC Lo Data are expressed as the frequency of CD4 T cells (defined as CD3+ and CD19-). Data for eosinophils and alveolar macrophages are expressed as the frequency of live cells. The CD4 / CD8 T cell ratio is calculated as the ratio of the frequency of CD4 T cells to the frequency of CD8 T cells in the live population. All data are shown in Table 8. [Table 12]

[0243] Lung cell infiltration analysis: As shown in Table 8, the frequencies of eosinophils, activated B cells, activated CD8 cells, ST2+Cd4+ T cells, and CD4 / CD8 T cell ratios in the lungs of IL-33HumIn mice receiving HDM for 15 weeks and treated with or without an isotype control antibody were significantly higher than those in IL-33HumIn mice challenged with 1x PBS alone. Similarly, a trend toward increased frequencies of activated CD4 T cells was observed in the lungs of IL-33HumIn mice receiving HDDM for 15 weeks. A trend toward decreased frequencies of alveolar macrophages, as detected by flow cytometry, was observed in the lungs of IL-33HumIn mice receiving HDDM for 15 weeks in the absence or presence of isotype control antibody treatment. The frequency of alveolar macrophages was significantly increased in the lungs of IL-33HumIn mice treated with a combination of anti-IL-33 and anti-mouse IL-4Rα antibodies during the final 4 weeks of chronic HDDM challenge compared to IL-33HumIn mice receiving HDM with an isotype control antibody during this period. Similarly, in the lungs of mice treated with a combination of anti-IL-33 and anti-mouse IL-4Rα antibodies during the final 4 weeks of chronic HDM challenge, there was a trend toward decreased frequencies of eosinophils, activated CD4 and CD8 T cells, activated B cells, ST2+ CD4+ T cells, and the CD4 / CD8 T cell ratio. The effects on the frequency of activated CD8 T cells, ST2+CD4+ T cells, and CD4 / CD8 ratio showed a trend toward greater effects than treatment with either individual antibody alone. [Table 13]

[0244] Lung sampling for quantification of histopathology: The inflammatory pattern observed in this model is accompanied by widespread and severe structural changes in HDM-exposed lungs, with signs of goblet cell metaplasia, increased subepithelial collagen deposition, and marked lung consolidation. These lesions are known hallmarks of human inflammatory respiratory diseases, leading to reduced lung function and airway hyperresponsiveness (James, (2007) Eur Respir J., Jul;30(1):134-55; Jeong, (2007) Radiographics May-Jun;27(3):617-37).

[0245] After exsanguination, the left lung was removed and placed in a plate containing 3 mL of 4% (w / v) paraformaldehyde (Boston Bioproducts, #BM-155) in 1x phosphate-buffered saline and stored at room temperature for 3 days. Lung samples were then blotted dry and transferred to tubes containing 70% ethanol for histological analysis. Samples were sent to Histoserv, Inc. (Germantown, MD) for paraffin embedding, sectioning, and periodic acid-Schiff (PAS) or hematoxylin and eosin (H&E) staining.

[0246] Quantification of goblet cell metaplasia: Goblet cell metaplasia and mucus hypersecretion are hallmarks of many lung diseases, including asthma, chronic obstructive pulmonary disease, and cystic fibrosis (Boucherat, (2013) Exp Lung Res. 2013 May-Jun;39(4-5):207-16). Excessive mucus production causes airway obstruction and affects several important outcomes, such as lung function, health-related quality of life, exacerbations, hospitalizations, and human mortality (Ramos, FL, et. al., (2014), Int J Chron Obstruct Pulmon Dis, Jan. 24;9:139-150). PAS-positive goblet cells and total epithelial cells were counted per millimeter of the main bronchus. As shown in Table 9, goblet cell metaplasia is expressed as the frequency of PAS-positive cells per millimeter of bronchial epithelium (%, ±SD).

[0247] Quantification of lung consolidation: "Lung consolidation" is defined as the accumulation of solid or liquid material in the alveolar space. Lung consolidation is a composite endpoint that can reflect a combination of cellular infiltration, hyperplasia, and mucus production, and is used here as a measure of gross lesions. ImageJ software (NIH, Bethesda, MD) was used to quantify the percentage of lung area occupied by crystal bodies on Movat pentachrome-stained paraffin-embedded lung sections. The particle analysis function was used to measure the total lung area of ​​the section and the consolidation area of ​​the section. As shown in Table 9, the percentage of lung consolidation area is given by the ratio of both measurements.

[0248] Quantification of subepithelial fibrosis "Subepithelial fibrosis" is defined as excessive interstitial collagen deposition beneath the lung epithelium (Redington, et al., (1997), Thorax, April; 52(4):310-312). Increased subepithelial fibrosis has been reported to be specifically associated with human asthma (Boulet, et al., (1997), Chest, July; 112(1):45-52; James, AL and Wenzel, S., (2007), Eur Respir J, July, 30(1):134-155). In this model, subepithelial fibrosis was measured on Masson's trichrome-stained paraffin-embedded lung sections using HaLo software (Indica Labs, NM). Using the "thickness" tool, the thickness of the collagen layer beneath the bronchial epithelium was recorded multiple times at approximately 30 μm intervals across 1 mm of the main bronchus. Subepithelial fibrosis is expressed as the mean thickness of the subepithelial collagen layer (μm, ±SD), as shown in Table 9.

[0249] Analysis of lung histopathology As shown in Table 9, the mice received HDM for 15 weeks and were treated with an isotype control antibody. There was a trend toward increased goblet cell metaplasia in the lungs of untreated IL-33HumIn mice compared with IL-33HumIn mice challenged with 1x PBS alone. Similarly, IL-33HumIn mice receiving HDM for 15 weeks had significantly increased lung consolidation and subepithelial collagen thickness.

[0250] In contrast, lungs from IL-33HumIn mice treated with a combination of anti-IL-33 and anti-mouse IL-4Rα antibodies during the final 4 weeks of chronic HDDM challenge showed trends toward decreased goblet cell metaplasia and subepithelial collagen thickness, as well as significantly decreased lung consolidation, compared with IL-33HumIn mice administered HDM with an isotype control antibody during this period. The effects of the combination of anti-IL-33 and anti-mouse IL-4Rα antibodies on goblet cell metaplasia, lung consolidation, and subepithelial collagen thickness tended to be more effective than treatment with either individual antibody alone. [Table 14]

[0251] Serum collection for IgE and HDM-specific IgG1 levels: To determine the total IgE concentration in serum samples from each mouse, a sandwich ELISA OPTEIA kit (BD Biosciences, #555248) was used according to the manufacturer's instructions. Serum samples were diluted and incubated with an anti-IgE capture antibody coated on a 96-well plate. Total IgE was detected with a biotinylated anti-mouse IgE secondary antibody. Purified horseradish peroxidase (HRP)-labeled mouse IgE was used as a standard. HRP activity was detected using the chromogen 3,3',5,5'-tetramethylbenzidine (TMB) (BD OPTEIA Substrate Reagent Set, BD, #555214). A stop solution of 1 M sulfuric acid was then added, and the absorbance at 450 nm was measured using a Molecular Devices SpectraMax M5 plate reader. Data analysis was performed using Prism™ software. The results are shown in Table 10. As shown, the mean amount of circulating IgE levels in serum for each experimental group is expressed as ng / mL (±SD).

[0252] ELISA was used to determine the HDM-specific IgG1 levels in serum samples from each mouse. HDM (Greer, #XPB70D3A2.5)-coated plates were incubated with serially diluted mouse serum samples, followed by incubation with an antibody conjugated to rat anti-mouse IgG1-HRP (BD Biosciences, #559626). All samples were developed with TMB solution and analyzed as described above. The relative levels of circulating IgG1 in serum are expressed as titer units (titer units were calculated by multiplying the measured OD by the dilution factor required to achieve an OD450 greater than two times background). As shown in Table 10, the mean circulating HDM-specific IgG1 levels in serum from each experimental group were calculated as titer × 10 6 Expressed as (±SD).

[0253] Analysis of circulating levels of IgE and HDM-specific IgG1 As shown in Table 10, circulating IgE levels were significantly increased in the serum of IL-33HumIn mice that underwent HDM for 15 weeks and were treated with or without an isotype control antibody compared to IL-33HumIn mice challenged with 1x PBS alone. Similarly, a trend toward increased levels of circulating HDM-specific IgG1 was observed in the serum of IL-33HumIn mice that underwent HDM for 15 weeks. In contrast, a significant decrease in circulating IgE levels and a trend toward decreased levels of circulating HDM-specific IgG1 were observed in the serum of IL-33HumIn mice that were treated with a combination of anti-IL-33 and anti-mouse IL-4Rα antibodies during the final 4 weeks of chronic HDDM challenge compared to IL-33HumIn mice that received HDM with an isotype control antibody. [Table 15]

[0254] The combination of H4H9675P and anti-mIL-4Rα treatment, initiated in the context of severe mixed inflammation, improved all measured inflammatory parameters, reducing most to baseline levels. Furthermore, additive effects were observed on several of the most aggressive endpoints, including composite lung macroscopic lesions, goblet cell metaplasia, pneumocyte infiltration, and cytokine levels. Thus, simultaneous blockade of both pathways has the potential to affect multiple inflammatory mediators and normalize multiple parameters to baseline in the context of severe mixed inflammation and tissue lesions.

[0255] Example 5: Mapping the H4H9675P binding epitope to IL33 by hydrogen-deuterium exchange To determine the epitope of human IL33 recognized by the anti-IL33 antibody H4H9675P, hydrogen-deuterium (H / D) exchange studies were performed on the antibody co-complexed with human IL33. For the experiments, recombinant human IL33 (SEQ ID NO: 356) expressed with a C-terminal hexahistidine tag was used. A general description of the H / D exchange method is provided in Ehring et al. (1999) Analytical Biochemistry 267(2):252-259 and Engen and Smith (2001) Anal. Chem. 73:256A-265A. H / D exchange experiments were performed on an integrated Waters HDX / MS platform, consisting of a Leaptec HDX PAL system for deuterium labeling, a Waters Acquity M-Class (Auxiliary solvent manager) for sample digestion and loading, a Waters Acquity M-Class (μBinary solvent manager) for analytical column gradients, and a Synapt G2-Si mass spectrometer for digested peptide mass measurement.

[0256] The labeling solution was prepared in 10 mM PBS buffer in DO at pH 7.0 (corresponding to pH 6.6). For deuterium labeling, 3.8 μL of hIL33-MMH (96 pmol / μL) or hIL33-MMH premixed with antibody at a 1:1 molar ratio was incubated with 56.2 μL of DO labeling solution for various times (2 min, 10 min, undeuterated control = 0 s). Deuteration was quenched by transferring 50 μL of sample to 50 μL of pre-chilled 0.2 M TCEP, 6 M guanidine chloride in 100 mM phosphate buffer (pH 2.5) (quench buffer), and the mixed sample was incubated at 1.0 °C for 2 h. The quenched sample was then injected into the Waters HDX Manager for online pepsin / protease XIII digestion. Digested peptides were trapped on an ACQUITY UPLC BEH C18 1.7-μm, 2.1x5mm VanGuard precolumn at 0°C and eluted onto an ACQUITY UPLC BEH C18 1.7-μm, 1.0x50mm column using a 9-minute gradient separation of 5% to 40% B (mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile). The mass spectrometer was set to a cone voltage of 37 V, a scan time of 0.5 s, and a mass / charge range of 50 to 1700 Th.

[0257] To identify peptides derived from hIL33-MMH, LC-MS analysis was performed on non-deuterated samples. E The data were processed and searched against a database containing human IL33, pepsin, and their randomized sequences using Waters ProteinLynx Global Server (PLGS) software. The identified peptides were imported into DynamX software and filtered by two criteria: 1) a minimum product per amino acid of 0.3, and 2) a replication file threshold of 3. DynamX software then automatically determined the deuterium incorporation of each peptide based on retention time and high mass accuracy (<10 ppm) across multiple time points, with three replicates per time point.

[0258] MS E Using the online Pepsin / Protease XIII column coupled with data acquisition, a total of 68 peptides from hIL33-MMH were identified in the absence or presence of h4H9675P, representing 95% sequence coverage. Eleven peptides significantly reduced deuterium incorporation upon binding to H4H9675P (centroid delta values ​​>0.4 Daltons, p-values ​​<0.05) and are listed in Table 11. Peptide masses reported are the centroids of the MH peptides obtained from three replicates. + These peptides, corresponding to amino acids 1-12 and 50-94 of SEQ ID NO: 349, had slower deuteration rates upon binding of H4H9675P. These identified residues also correspond to residues 112-123 and 161-205 of human IL-33, as defined by Uniprot entry O95760 (IL33_human; see also SEQ ID NO: 348). These data provide support for amino acid residues 112-123 and 161-205 of SEQ ID NO: 348, or amino acid residues 1-12 and 50-94 of SEQ ID NO: 349, at least partially defining the binding region in IL-33 for antibody H4H9675P. [Table 16]

[0259] Example 6: Effects of an IL-33 antibody (REGN3500) and an IL-4R antibody (dupilumab), alone or in combination, in a 19-week model of allergen-induced lung inflammation using IL-33-, IL-4-, and IL-4Rα-humanized mice The effects of an IL-33 antibody alone, an IL-4R antibody alone, or a combination of both antibodies were first tested in Example 4 above using mice homozygous for expression of human IL-33 instead of mouse IL-33 (IL-33 Humln mice; see U.S. Patent Publication Nos. 2015 / 0320021 and 2015 / 0320022). A fully human anti-IL-33 antibody (REGN3500), and an anti-mouse IL-4Rα antibody, or a combination of both, were compared in this model, and the results are described in Example 4.

[0260] Because neither the human anti-IL-33 antibody (REGN3500) nor the human anti-IL-4R antibody (dupilumab) bind to their respective mouse target proteins, we compared the ectodomains of mouse IL-33, IL-4, and IL-4Rα with the corresponding human sequences (Il4ra hu / hu Il4 hu / hu Il33 hu / hu ) replaced by a genetically modified Il4ra hu / hu Il4 hu / hu Il33 hu / hu The mouse strain was validated as a tool to study the effects of REGN3500 and dupilumab treatment using a 4-week HDM exposure-induced lung inflammation model. In this model, HDM-exposed Il4ra mice were significantly increased in pulmonary inflammation as assessed by eosinophilic lung infiltration. hu / hu Il4 hu / hu Il33 hu / hu The mice displayed an immune response similar to that of wild-type mice.

[0261] To determine whether simultaneous blockade of the IL-33 and IL-4 / IL-13 pathways could have a greater effect on lung inflammation than blockade of either pathway alone, the following study was performed. hu / hu Il4 hu / hu Il33 hu / hu Mice were exposed intranasally (IN) to HDM or saline for 19 weeks. hu / hu Il4 hu / hu Il33 hu / huControl groups of mice were sacrificed after 11 weeks of HDM exposure to assess disease severity at the start of antibody treatment. Mice exposed to 19 weeks of HDM either received no antibody treatment or received subcutaneous (SC) antibody injections twice weekly for a total of 8 weeks and 16 doses during weeks 12–19 of HDM exposure. The following antibodies were administered at a final protein dose of 11 mg / kg: (a) 11 mg / kg isotype control antibody, (b) 1 mg / kg REGN3500 + 10 mg / kg isotype control antibody, (c) 10 mg / kg dupilumab + 1 mg / kg isotype control antibody, or (d) 1 mg / kg REGN3500 + 10 mg / kg dupilumab. The effects of REGN3500 and dupilumab treatment, alone or in combination, on HDM-exposed mice were assessed for the following pathological markers of airway inflammation: - Gross lesions (relative lung weight) -Type 1 inflammatory cells (neutrophils, quantified by lung protein levels of the neutrophil marker myeloperoxidase [MPO]) and type 2 inflammatory cells (all activated [CD11c Hi ] eosinophils and ST2 + CD4 + Lung tissue infiltration by T cells (quantified by flow cytometry) -inflammatory cytokine lung protein levels (human IL-4 and murine IL-5, IL-6, IL-1β, TNFα, IFNγ, GROα, and MCP-1, quantified by immunoassay) Circulating levels of serum amyloid A [SAA] protein, a systemic marker of inflammation (quantified by immunoassay)

[0262] material and method Test System IL-33, IL-4, and IL-4Rα ectodomain humanized mice Neither REGN3500 nor dupilumab binds to mouse IL-33 or mouse IL-4Rα, respectively. Therefore, to test REGN3500 and dupilumab synergistically and in combination, the ectodomains of mouse IL-33, IL-4, and IL-4Rα were cloned into the corresponding human sequences (Il4rahu / hu Il4 hu / hu Il33 hu / hu ) to generate a genetically modified mouse line. This triple-humanized mouse line was developed using Regeneron Pharmaceuticals' VelociGene® technology (Valenzuela, DM, et al. Nat Biotechnol. (2003), Jun;21(6):652-9, Poueymirou, WT, et al. Nat Biotechnol. (2007) Jan;25(1):91-9) was used to generate the IL-4 / IL-4Rα ectodomain-humanized mouse strain Il4ra hu / hu Il4 hu / hu and the IL-33-humanized line Il33 characterized above. hu / hu It was generated by crossing

[0263] Pulmonary inflammation mouse model A murine lung inflammation model was developed using HDM extract (Johnson, et al.; American Journal of Respira- tion) as a source of house dust mite allergens. Respiratory and Critical Care Medicine;(2004)Feb1;169(3):378-85), a significant cause of indoor allergy in humans (Calderon, et al.,(2015),Respiratory allergy caused by house dust mites: What do we really know? J Allergy Clin Immunol.2015Jul;136(1):38-48)). Chronic exposure to HDM has been reported to induce severe lung inflammation, leading to severe lung cell infiltration, cytokine expression, and remodeling. Specifically, mice chronically exposed to HDM exhibit a mixed type 1 / type 2 phenotype of lung inflammation, including tissue infiltration by type 1 and type 2 inflammatory cells (neutrophils and eosinophils, respectively), increased serum IgE, increased serum HDM-specific IgG1, and induction of type 2 inflammatory cytokines such as IL-5 and IL-13 (Johnson, et al. (2004), American Journal of Respiratory and Critical Care Medicine; Feb 1; 169(3):378-85; Johnson, et al. (2011), PloS ONE. Jan 20; 6(1):e16175; Llop-Guevara, et al., (2008), PloS ONE, Jun 11; 3(6):e2426).

[0264] Experimental design 4-week HDM exposure-induced lung inflammation model Female mice of the genotypes shown in Table 12 were randomly divided into two groups per genotype. Saline (20 μL) or 50 μg of HDM diluted in 20 μL of saline solution was administered intravenously three times per week for 4 weeks. All mouse strains were on a mixed C57BL / 6NTac / 129S6SvEvTac background. Four days after the last challenge, mice were sacrificed, lungs were harvested, and eosinophilic pulmonary infiltration was determined. [Table 17]

[0265] 19-week HDM exposure-induced lung inflammation model Il4ra used in this study hu / hu Il4 hu / hu Il33 hu / hu Mice were of mixed background C57BL / 6NTac (72%) / 129S6SvEvTac (28%), and female mice were randomly divided into seven separate groups. The HDM exposure and treatment or control administration protocols for mice in each group are shown in Table 13. Saline (20 μL) or 50 μg HDM diluted in 20 μL saline solution was administered three times per week for 19 weeks. IN administration. Il4ra hu / hu Il4 hu / hu Il33 hu / hu A control group of mice was sacrificed after 11 weeks of HDM exposure to assess disease severity at the start of antibody treatment. As shown in Table 13, 19-week HDM-exposed mice either received no antibody treatment or received subcutaneous (SC) injections of HDM twice weekly for a total of 16 antibody doses between weeks 12 and 19 of exposure. Briefly, the following antibodies were administered at a final protein dose of 11 mg / kg: 11 mg / kg isotype control antibody (Group D), 1 mg / kg REGN3500 + 10 mg / kg isotype control antibody (Group E), 10 mg / kg dupilumab + 1 mg / kg isotype control antibody (Group F), or 1 mg / kg REGN3500 + 10 mg / kg dupilumab (Group G). For the purposes of this document, dual antibody-treated groups (D-G) are identified solely by the therapeutic antibody (REGN3500 and / or dupilumab). On day 134 of the study, four days after the final IN exposure and antibody injection, all mice were sacrificed, blood was collected by cardiac puncture, and lungs were harvested for analysis. [Table 18]

[0266] Mouse Management Animals remained housed in the Regeneron animal facility under standard conditions for the entire duration of each experiment and were allowed to acclimate for at least 7 days before being subjected to study. All animal experiments were conducted in accordance with the guidelines of the Regeneron Institutional Animal Care and Use Committee.

[0267] Identification Procedures Relative lung weight measurement Terminal body weight measurements were recorded before sacrifice. After exsanguination, the left lung of each mouse was removed and placed in a tube containing 4% paraformaldehyde solution. The wet weight of the left lung of each mouse was recorded on a Mettler Toledo New Classic MS scale. To determine relative lung weight, the ratio of wet lung weight (mg) to body weight (g) was calculated by dividing the wet lung weight by body weight.

[0268] Analysis of cellular lung infiltrates After exsanguination, the caudal lobe of the right lung was removed from each mouse, placed in a tube containing a solution of 20 μg / mL DNase I and 0.7 U / mL Liberase TH diluted in Hank's Balanced Salt Solution (HBSS), and chopped into small pieces approximately 2-3 mm in size. The tube containing the small diced lung lobes was then incubated in a 37°C water bath for 20 minutes. The reaction was stopped by adding EDTA to a final concentration of 10 mM. The samples were then transferred to gentleMACS C tubes. 2 mL of autoMACS buffer was then added, followed by disruption of the samples using a gentleMACS™ disrupter (Miltenyi Biotec) to form a single-cell suspension. The tubes were then centrifuged, and the resulting pellet was resuspended in 4 mL of 1x red blood cell lysis buffer to lyse the red blood cells. After a 3-minute incubation at room temperature, 2.5 volumes of 1x DPBS were added to inactivate the red blood cell lysis buffer. The cell suspension was then centrifuged, and the resulting cell pellet was resuspended in 1 mL of DPBS. Each resuspended sample was filtered through a 50 μm cup-type filcon and transferred to a 2 mL deep-well plate. The plate was centrifuged at 400 × g for 4 minutes, and each sample was resuspended in 1 mL of DPBS. Approximately 1.5 × 10 cells per well were collected.6 Cells were plated in 96-well U-bottom plates. Cells were then centrifuged, and the cell pellet was resuspended in 100 μL of LIVE / DEAD Fixable Dead Cell Stain diluted 1:500 in 1x DPBS to determine cell viability. Cells were incubated with a viability dye for 15 minutes at room temperature, protected from light. After one wash in 1x DPBS, cells were incubated with purified rat anti-mouse CD16 / CD32 Fc Block diluted 1:50 in 50 μL of autoMACS buffer for 15 minutes at 4°C. Cells were then incubated in the appropriate 2x antibody mix diluted in Brilliant Stain buffer (listed in Table 14) for 30 minutes at 4°C, protected from light. After antibody incubation, cells were washed twice in autoMACS buffer, resuspended in BD CytoFix diluted 1:4 in 1x DPBS, and then incubated for 15 minutes at 4°C, protected from light. Cells were then washed and resuspended in autoMACS buffer. The cell suspension was then filtered through an AcroPrep Advance 96 filter plate, 30-40 μm, into a new U-bottom plate. Sample data were acquired on an LSR Fortessa X-20 cell analyzer using an HTS attachment (BD Biosciences). Data analysis was performed using FlowJo X software (Tree Star, OR), and statistical analysis was performed using GraphPad Prism™ (GraphPad Software, CA).

[0269] Eosinophil Gating Strategy (Total Activation) Eosinophils as intact, single, live cells (low LIVE / DEAD viability dye signal) CD45 + , F4 / 80 + , Ly6G - , SiglecF + Data for eosinophils were expressed as the frequency of live cells. Within the eosinophil population, activated eosinophils were defined as intact, single, live CD45 + , F4 / 80 + , Ly6G- , SiglecF + , CD11c Hi and expressed as the frequency of total eosinophils.

[0270] ST2 + CD4 + Gating strategy for T cells ST2 + CD4 + Intact, single, live CD45 T cells + , CD3 + , CD19 - , CD4 + , CD8 - , ST2 + It was defined as ST2 + CD4 + Data on T cells and CD4 + T cells (intact, single, live CD45 + , CD3 + , CD19 - , CD4 + , CD8 - ) frequency. [Table 19]

[0271] Measurement of lung protein concentration After exsanguination, the anterior and medial lobes of the right lung from each mouse were removed, weighed, and placed in a tube containing a solution of tissue protein extraction reagent (T-PER) supplemented with a protease inhibitor cocktail. To achieve a final 1:8 (w / v) lung tissue weight to T-PER volume ratio, 8 μL of T-PER solution (containing a protease inhibitor cocktail) was added per mg of tissue. Lung samples were mechanically homogenized using a TissueLyser II. The resulting lysate was centrifuged to pellet debris. The supernatant, containing the soluble protein extract, was transferred to a new tube and stored at 4°C until further analysis. Cytokine and MPO concentrations were expressed as the total amount of protein per examined lobe (ng / lobe and μg / lobe, respectively).

[0272] Cytokine Multiplex Immunoassay Mouse cytokine (IL-5, IL-13, IL-6, IL-1β, IL-12p70, TNFα, IFNγ, GROα, and MCP-1) concentrations in lung protein extracts were measured using a multiplex immunoassay kit (Custom Mouse 10-Plex, MSD) according to the manufacturer's instructions. Briefly, lung homogenate samples were diluted and incubated on plates precoated with capture antibodies. Protein calibrators provided by the manufacturer were used as standards. Cytokines in the homogenates were detected with tagged detection antibodies incubated with Read Buffer. Electrochemiluminescence was immediately read on an MSD Spector® instrument. Data analysis was performed using GraphPad Prism software. The lowest concentration of standard within the linear range of each assay was defined as the lower limit of quantification (LLOQ) of the assay for each cytokine. The LLOQ values ​​for the individual cytokines tested were as follows: IIFNγ = 0.2 pg / mL, IL-1β = 1.6 pg / mL, IL-5 = 0.2 pg / mL, IL-6 = 1.4 pg / mL, IL-12p70 = 125.8 pg / mL, IL-13 = 24.4 pg / mL, GROα: 0.5 pg / mL, MCP- 1=9.8pg / mL, TNFα=2.4pg / mL.

[0273] Human IL-4 ELISA Human IL-4 concentrations in lung protein extracts were measured using a sandwich ELISA kit (Human IL-4 Quantikine ELISA, R&D Systems) according to the manufacturer's instructions. Briefly, lung homogenates were diluted and incubated on 96-well plates precoated with anti-human IL-4 capture antibody. Purified human IL-4 was used as a standard. Captured human IL-4 was detected using an HRP-conjugated anti-human IL-4 detection antibody. HRP activity was detected using the chromogen 3,3',5,5'-tetramethylbenzidine (TMB). Stop solution was then added, and the optical density at 450 nm (OD ) was measured. 450) was measured on a Molecular Devices SpectraMax M5 plate reader. Data analysis was performed using GraphPad Prism software. The lowest concentration of standard within the linear range of the assay was defined as the assay's LLOQ = 31.25 pg / mL.

[0274] MPO ELISA MPO concentrations in lung protein extracts were measured using a sandwich ELISA kit (Mouse MPO ELISA Kit, Hycult Biotech) according to the manufacturer's instructions. Briefly, lung homogenates were diluted and incubated on a 96-well plate precoated with anti-MPO capture antibody. Purified mouse MPO was used as a standard. Captured MPO was detected with a biotinylated anti-mouse MPO detection antibody. Biotinylated anti-mouse MPO was detected with purified HRP-conjugated streptavidin. HRP activity was detected with the chromogen 3,3',5,5'-tetramethylbenzidine (TMB). Stop solution was then added, and the optical density at 450 nm (OD ) was measured. 450 ) was measured on a Molecular Devices SpectraMax M5 plate reader. Data analysis was performed using GraphPad Prism software. The lowest concentration of standard within the linear range of the assay was defined as the assay's LLOQ = 156.3 ng / mL.

[0275] Serum collection At the end of the study, whole blood was collected by cardiac puncture into Microtainer tubes. Blood was allowed to clot by standing at room temperature for at least 30 minutes. Clotted blood and cells were pelleted by centrifugation at 18,000 x g for 10 minutes at 4°C. The resulting supernatant, designated serum, was transferred to a clean polypropylene plate and used to determine circulating antibody levels, as described below.

[0276] Determination of SAA levels in serum by ELISA The total SAA concentration in each mouse serum sample was determined using a commercially available immunoassay (Quantikine ELISA, R&D Systems) according to the manufacturer's instructions. Briefly, serum samples were diluted and incubated on a 96-well plate pre-coated with a monoclonal anti-mouse SAA capture antibody. Recombinant mouse SAA was used as a standard. Captured SAA was detected using an HRP-conjugated polyclonal anti-mouse SAA detection antibody. HRP activity was detected using the colorimetric HRP substrate TMB. A stop solution of dilute hydrochloric acid was then added, and the OD 450 was measured on a Molecular Devices SpectraMax M5 plate reader. The concentration of circulating SAA in serum was determined for each sample in ng / mL and graphed as μg / mL. Data analysis was performed using GraphPad Prism software. The lowest concentration of standard within the linear range of the assay was defined as the assay's LLOQ = 31.2 ng / mL.

[0277] Determination of serum IgE concentrations by ELISA The total IgE concentration in each mouse serum sample was determined using a colorimetric sandwich ELISA OPTEIA kit according to the manufacturer's instructions. Briefly, serum samples were diluted and incubated on a 96-well plate pre-coated with anti-IgE capture antibody. Purified mouse IgE was used as a standard. Captured IgE was detected with a biotinylated anti-mouse IgE detection antibody. Biotinylated anti-mouse IgE was detected with purified HRP-conjugated streptavidin. HRP activity was detected with TMB. A stop solution of 2N sulfuric acid was then added, and the OD was measured. 450 was measured using a Molecular Devices SpectraMax M5 plate reader. The concentration of circulating IgE in serum for each sample was expressed as μg / mL. Data analysis was performed using GraphPad Prism software. The lowest concentration of standard within the linear range of the assay was defined as the LLOQ of the assay = 78.15 ng / mL.

[0278] Measurement of serum HDM-specific IgG1 levels by ELISA A colorimetric ELISA assay was developed to determine the level of HDM-specific IgG1 in serum samples. Plates were coated with HDM in phosphate-buffered saline (PBS) at a concentration of 4 μg / mL overnight at 4°C, washed, blocked with a solution of 0.5% BSA in PBS for 1 hour at room temperature, and incubated with serially diluted mouse serum samples. After 1 hour at room temperature, the plates were washed, and the IgG1 antibodies captured on the plates were detected by incubating with a rat anti-mouse IgG1 HRP-conjugated antibody for 1 hour at room temperature. HRP activity was detected using TMB. A stop solution of 2N sulfuric acid was then added, and the OD 450 was measured on a Molecular Devices SpectraMaxM5 plate reader. The relative levels of IgG1 in serum were expressed as titer units. Titer units are calculated by dividing the measured OD 450 background OD 450 More than twice the OD 450 The LLOQ was calculated by multiplying by the dilution factor required to achieve a reading of 100. Data analysis was performed using GraphPad Prism software. The lowest dilution factor used in the assay was defined as the LLOQ=100 of the assay.

[0279] Determination of human target-specific IgG4 antibody levels by ELISA Human antibodies (REGN 3500, dupilumab, or IgG4) in serum samples from each mouse PThe concentrations of purified antibodies (REGN3500, REGN668, and IgG4) were determined using a colorimetric sandwich ELISA developed to detect human IgG4 antibodies. Microtiter wells were coated with antigens specific to the human antibodies being measured, i.e., human IL-33 (REGN3931) for capturing REGN3500, human IL-4Rα (REGN560) for capturing REGN668, and Natural Fel d1 for capturing REGN1945, at a concentration of 2 μg / mL in PBS overnight at 4°C. The wells were washed four times with 0.05% Tween 20 in DPBS, blocked with a solution of 5% BSA in DPBS for 3 hours at room temperature, and incubated with serially diluted mouse serum samples or serially diluted calibration standards. Purified antibodies (REGN3500, REGN668, and IgG4) were analyzed using a colorimetric sandwich ELISA developed to detect human IgG4 antibodies. Microtiter wells were coated with antigens specific to the human antibodies being measured, i.e., human IL-33 (REGN3931) for capturing REGN3500, human IL-4Rα (REGN560) for capturing REGN668, and Natural Fel d1 for capturing REGN1945, at a concentration of 2 μg / mL in PBS overnight at 4°C. The wells were washed four times with 0.05% Tween 20 in DPBS, blocked with a solution of 5% BSA in DPBS for 3 hours at room temperature, and incubated with serially diluted mouse serum samples or serially diluted calibration standards. P The control antibody was used as a standard for calibration and quantification of the respective antibody concentration in serum. After 1 hour at room temperature, the plate was washed seven times, and the captured human IgG4 was detected using a biotinylated mouse anti-human IgG4-specific monoclonal antibody, followed by incubation with a poly-HRP streptavidin conjugate. HRP activity was detected using TMB substrate according to the manufacturer's instructions. After 10 minutes, absorbance was measured at 450 nm using a Molecular Devices SpectraMax multimode plate reader. The lowest concentration (0.002 μg / mL) of the standard used for calibration (REGN3500, REGN668, or IgG4) was used. P The control antibody) was defined as the LLOQ of this assay. Data analysis was performed using GraphPad Prism™ (GraphPad Software, CA). The concentration of human antibody in the serum of each sample was expressed as μg / mL.

[0280] statistical analysis Statistical analysis was performed using GraphPad Prism version 7.0 (GraphPad Software, CA).

[0281] Statistical analysis of data from the characterization of IL-33, IL-4, and IL-4Rα humanized mice in a 4-week HDM exposure model Results were interpreted by two-way analysis of variance (ANOVA) followed by Tukey's post-hoc test for multiple comparisons. Differences were considered statistically significant when p≦0.05.

[0282] Statistical analysis of data from REGN3500 / dupilumab treatment in a 19-week HDM exposure-induced lung inflammation model The normality of the data was assessed using the Shapiro-Wilk test. If the data passed the normality test and the standard deviations of different groups were not statistically different from each other as assessed by the Brown-Forsythe test, the results were interpreted by one-way analysis of variance, followed by Tukey's post hoc test for multiple comparisons. If the data did not pass the normality test or the standard deviations were significantly different, the results were interpreted using the Kruskal-Wallis test, followed by Dunn's multiple comparison test. Differences were considered statistically significant when p ≤ 0.05.

[0283] result Characterization of IL-33, IL-4, and IL-4Rα ectodomain-humanized mice Wild-type mice, Il33 hu / hu - and Il4ra hu / hu Single humanized mouse, Il4ra hu / hu Il4 hu / hu Double humanized mice, and Il4ra hu / hu Il4 hu / hu Il33 hu / hu Triple-humanized mice were exposed to saline or HDM three times per week for 4 weeks. Four days after the last exposure, mice were sacrificed and lungs were harvested to assess lung infiltration by activated eosinophils, identified by high CD11c expression. Individual mouse data and statistical analysis are provided in Figure 2. Triple-humanized Il4ra hu / hu Il4 hu / hu Il33 hu / hu Il4ra mice responded strongly to HDM, similar to wild-type mice, as indicated by a significant increase in the frequency of activated eosinophils in lung tissue after 4 weeks of HDM exposure. hu / hu Il4 hu / hu Il33 hu / huIl4ra and wild-type mice also showed similar frequencies of activated eosinophils in lung tissue in the absence of HDM exposure (saline-exposed control mice). hu / hu With the exception of the single humanized mice, no statistically significant differences were observed comparing HDM-exposed wild-type mice with HDM-exposed mice derived from any of the humanized mouse strains tested. hu / hu The lack of a statistically significant HDM-induced increase in the percentage of activated eosinophilic lung infiltrates in mice is likely due to the fact that mouse IL-4 does not signal through the human IL-4Rα receptor. Human IL-33, on the other hand, has been shown to signal through the mouse receptor complex (REGN3500-MX-16069). Furthermore, no statistically significant differences were observed when comparing saline-exposed wild-type mice with saline-exposed mice derived from any of the humanized mouse strains tested. These findings support the notion that Il4ra hu / hu Il4 hu / hu Il33 hu / hu This validates the use of this mouse strain as a mouse model of HDM exposure-induced lung inflammation.

[0284] Effects of REGN3500 and dupilumab treatment in HDM-exposed mice for 19 weeks Il4ra hu / hu Il4 hu / hu Il33 hu / hu Mice were exposed to saline or HDM IN three times per week for 11 or 19 weeks. Four groups of 19-week HDM-exposed mice received SC injections twice per week between weeks 12 and 19; all other groups received no treatment (none, light gray boxes). Antibodies were administered alone or in combination at a final protein dose of 11 mg / kg as follows: 11 mg / kg isotype control antibody, 1 mg / kg REGN3500 + 10 mg / kg isotype Control antibody, 10 mg / kg dupilumab + 1 mg / kg isotype control antibody, or 1 mg / kg REGN3500 + 10 mg / kg dupilumab. One cohort of mice was sacrificed after 11 weeks of exposure (11-week exposure group) to determine the inflammatory profile at the start of antibody treatment. The other four cohorts were sacrificed on day 134 (19-week exposure group), 4 days after the last exposure and antibody injection. Whole blood was collected by cardiac puncture for serum isolation, and lungs were harvested for further analysis. Unless otherwise noted, all groups consisted of mice from the same strain (Il4ra hu / hu Il4 hu / hu Il33 hu / hu ) was included.

[0285] Analysis of gross lung lesions Relative lung weight was significantly increased in 19-week HDM-exposed mice compared with saline-exposed control mice (Figure 3). This is likely due to increased cellular infiltration, collagen deposition, muscle hypertrophy, and mucus production. In HDM-exposed mice, combined administration of REGN3500 and dupilumab significantly prevented HDM-exposure-induced increases in relative lung weight compared with mice treated with an isotype control antibody (Figure 3). A trend toward decreased relative lung weight was also observed in HDM-exposed mice treated with REGN3500 alone.

[0286] Analysis of lung cellular infiltrates Four days after the last antibody injection, mouse lungs were harvested and the caudal lobe of the right lung was crushed into a single cell suspension for flow cytometry analysis of eosinophils. Eosinophils were identified as intact, single, live CD45 + , F4 / 80 + , Ly6G - , SiglecF + Activated eosinophils were defined as CD11c Hi Pulmonary infiltration by activated eosinophils was reported as the frequency (%) of total lung eosinophils in (A), and total lung eosinophil infiltration was reported as the frequency (%) of total lung eosinophils in live (intact, single, living) cells. Total activated lung eosinophils (Figures 4A and 4B) and lung ST2 + CD4+ T cells (ST2 + CD4 + T cells are intact, single, live CD45 + , CD3 + , CD19 - , CD4 + , CD8 - , ST2 + defined as CD4 + Compared to saline-exposed control mice, 19 weeks of HDM exposure significantly increased cellular lung infiltration, as measured by flow cytometry to detect pulmonary neutrophils (reported as the frequency of T cells) (Figure 5) or by immunoassay to detect lung MPO protein levels as a marker of neutrophils (MPO protein levels were measured by enzyme-linked immunosorbent assay). Lung MPO protein levels are expressed as μg of MPO protein per lung lobe (Figure 6).

[0287] Administration of REGN3500 and dupilumab in combination, but not either antibody alone, significantly reduced the level of lung infiltration by activated eosinophils in 19-week HDM-exposed mice compared with administration of an isotype control antibody. Notably, the level of lung infiltration by activated eosinophils in mice treated with REGN3500 and dupilumab in combination was also significantly reduced compared with the level induced by 11 weeks of HDM exposure, which corresponds to the start of treatment (Figure 4A). Administration of either antibody alone had no significant effect, but a trend toward reduced lung infiltration by activated eosinophils was observed in dupilumab-treated mice. HDM-exposed mice treated with REGN3500 and dupilumab in combination also showed a trend toward reduced HDM-induced lung infiltration by total eosinophils (Figure 4B).

[0288] In 19-week HDM-exposed mice administered REGN3500 alone or in combination with dupilumab, ST2 + CD4 +The level of T cell pulmonary infiltration was significantly reduced compared to that in isotype control-treated mice and compared to that induced by 11 weeks of HDM exposure at the start of treatment (Figure 5). Similar inhibition of infiltration (mean frequency within 1.02-fold) was observed with REGN3500 alone and in combination with dupilumab, indicating that this pathology is primarily driven by IL-33.

[0289] Similar to eosinophil infiltration, the combined administration of REGN3500 and dupilumab was more effective at blocking neutrophil infiltration than either antibody alone. HDM exposure-induced increases in lung protein levels of myeloperoxidase (MPO), a marker of neutrophil infiltration, were significantly blocked by the combined administration of REGN3500 and dupilumab compared with the isotype control (Figure 6). Administration of either antibody alone had no significant effect, but a trend toward decreased lung MPO protein levels was observed in REGN3500-treated mice.

[0290] Lung tissue cytokine level analysis The effects of HDM exposure and antibody treatment on lung protein levels (total protein per lobe) were evaluated for the murine cytokines IL-5, IL-13, IL-6, IL-1β, IL-12p70, TNFα, IFNγ, GROα, and MCP-1, and the human cytokine hIL-4.

[0291] Lungs (anterior and middle lobes of the right lung) were harvested, and protein levels of the indicated mouse cytokines were measured by multiplex immunoassay. Human IL-4 protein levels (hIL-4) were detected using a commercially available ELISA kit. IL-5 (Figure 7A) and IL-6 (Figure 7B) protein levels were measured by multiplex immunoassay. Lung tissue cytokine protein levels were calculated as pg of protein per lung lobe. False-color heat maps (not shown) were generated to show relative cytokines ranging from light yellow to dark blue. A scale of relative lung cytokine levels was created by defining the lowest and highest mean lung protein levels for each cytokine as 0% (light yellow) and 100% (dark blue), respectively. Relative lung cytokine protein levels (%) are indicated by numbers and colors in the heat maps. Statistical significance was determined by one-way Kruskal-Wallis analysis of variance with Dunn's multiple comparison post-hoc test.

[0292] IL-12p70 results were below the lower limit of quantification in all groups and are therefore not reported here.

[0293] Eight cytokines (hIL-4, IL-5, IL-6, IL-13, IL-1β, TNFα, GROα, and MCP-1) showed significant increases in lung protein levels in response to 19 weeks of HDM exposure compared with saline-exposed control mice. Only IFNγ did not show significant increases in lung protein levels in response to 19 weeks of HDM exposure compared with saline-exposed control mice, and IFNγ levels were not affected by treatment with therapeutic antibodies compared with 19 weeks of HDM-exposed mice treated with an isotype control antibody. The HDM-induced increases in lung protein levels of five cytokines (hIL-4, IL-6, TNFα, GROα, and MCP-1) were significantly blocked by the combination of REGN3500 and dupilumab, but not by treatment with either antibody alone, compared with mice treated with an isotype control antibody.

[0294] Two other HDM exposure-responsive cytokines (IL-5 and IL-1β) showed a trend toward inhibition with combined REGN3500 and dupilumab treatment, with 83% and 78% reductions in lung protein levels, respectively, compared with isotype control antibody-treated mice (Figures 7A and 7B). Administration of individual antibodies resulted in a trend toward less pronounced reductions in IL-5 and IL-1β levels.

[0295] Analysis of SAA, a systemic marker of inflammation Four days after the last exposure and antibody injection, whole blood was collected by cardiac puncture and serum was isolated. Circulating SAA protein levels were measured using a commercially available ELISA kit. Circulating SAA protein levels are expressed as μg of SAA protein per mL of serum. Circulating protein levels of systemic inflammatory SAA markers were significantly increased in 19-week HDM-exposed mice compared with saline-exposed control mice (Figure 8).

[0296] The HDM exposure-induced increase in circulating SAA levels was significantly reduced in mice treated with REGN3500 alone or in combination with dupilumab (Figure 8), whereas a trend toward decreased circulating SAA levels was observed in mice treated with dupilumab alone.

[0297] Quantification of humoral allergic responses after HDM exposure Four days after the last challenge and antibody injection, whole blood was collected by cardiac puncture and serum was isolated. Circulating IgE protein levels were measured using a commercially available ELISA kit. Circulating IgE protein levels are expressed as μg IgE protein per mL of serum.

[0298] A humoral allergic response was induced by HDM exposure, as assessed by levels of circulating IgE (Figure 9) and HDM-specific IgG1 (Table 15) at the end of the study (day 134).

[0299] Circulating IgE protein levels were significantly increased in 19-week HDM-exposed mice compared with saline-exposed control mice (Figure 9). Mean titers of circulating HDM-specific IgG1 increased from 1.14E+02 in saline-exposed control mice to levels ranging from 1.37E+06 to 2.43E+06 in 19-week HDM-exposed mice (Table 15). No statistically significant effects of REGN3500, dupilumab, or combination treatment were observed on any of these endpoints, although a trend toward decreased serum IgE levels was observed in mice treated with the combination of REGN3500 and dupilumab. [Table 20]

[0300] Quantification of serum concentrations of human antibodies Human IgG4 P Antibodies (IgG4 P Serum concentrations of isotype control, REGN3500, and dupilumab were determined by target-specific anti-human IgG4 ELISA at the end of the study (day 134), 4 days after the last antibody dose. The mean concentrations of human IgG4 antibodies are summarized in Table 16. [Table 21]

[0301] overview Compared to control mice exposed to saline alone, mice exposed to HDM for 19 weeks had significantly higher levels of IgG4 than mice left untreated or exposed to IgG4 P After administration of the isotype control antibody, all but one of the 14 measured inflammatory pathology markers (pulmonary IFNγ levels) showed an increase.

[0302] The combination of REGN3500 and dupilumab significantly improved 10 of 13 HDM exposure-response endpoints tested (relative lung weight, activated eosinophils, neutrophil [MPO levels], and ST2) compared with an isotype control antibody. + CD4 +It significantly inhibited pulmonary infiltration by T cells, protein levels of cytokines IL-4, IL-6, TNFα, GROα, and MCP-1, and serum levels of SAA. Furthermore, it significantly inhibited activated eosinophils and ST2 + CD4 + The level of lung infiltration by T cells was significantly reduced to levels below those observed in mice exposed to HDM for 11 weeks, corresponding to the initiation of antibody treatment. Administration of dupilumab alone did not significantly inhibit any of the 13 tested HDM exposure-responsive endpoints in this model, whereas administration of REGN3500 alone inhibited two test endpoints: ST2. + CD4 + REGN3500 significantly inhibited T cell pulmonary infiltration and circulating SAA levels. For these two endpoints, the inhibition mediated by REGN3500 alone was similar to that mediated by REGN3500 in combination with dupilumab, suggesting that these pathological markers are primarily driven by IL-33.

[0303] Combined administration of REGN3500 and dupilumab showed a trend toward blocking three additional HDM exposure-responsive endpoints (pulmonary infiltration by eosinophils [total], lung protein concentrations of the cytokines IL-5 and IL-1β, and serum protein levels of IgE) without reaching statistical significance. Individual antibody treatment with REGN3500 or dupilumab generally resulted in weaker reductions in these markers than combination treatment.

[0304] All antibody treatment groups were associated with detectable serum levels of target-specific human IgG4 antibodies at the end of the study. Therapeutic antibodies, either alone or in combination, were administered twice weekly for 8 days. In mice treated weekly, mean serum concentrations of REGN3500 were 11.4±10.1 and 12.7±8.8 μg / mL, and mean serum concentrations of dupilumab were 8.0±13.5 and 48.9±27.9 μg / mL, respectively, at the end of the study.

[0305] In conclusion, Il4ra hu / hu Il4hu / hu Il33 hu / hu In a 19-week HDM-exposure-induced lung inflammation model in mice, combined treatment with REGN3500 and dupilumab resulted in more significant improvements in nearly all lung pathology and inflammatory markers tested compared with treatment with either antibody alone.

[0306] conclusion Il4ra hu / hu Il4 hu / hu Il33 hu / hu In a 19-week HDM-exposure-induced lung inflammation model in mice, combined treatment with REGN3500 and dupilumab resulted in more significant improvements in nearly all lung pathology and inflammatory markers tested compared with treatment with either antibody alone.

[0307] Example 7: Evaluation of SAR440340 / REGN3500 or Dupilumab when used alone and as combination therapy in patients with moderate to severe COPD research design This study is a randomized, double-blind, placebo-controlled, parallel-group, 24-week proof-of-concept study to evaluate the efficacy, safety, and tolerability of an IL-33 monoclonal antibody (SAR440340 / REGN3500) and an IL-4R monoclonal antibody (dupilumab, also known as DUPIXENT®), when used alone or in combination, in patients with moderate to severe chronic obstructive pulmonary disease (COPD).

[0308] A total of 832 subjects will participate in the study, which consists of four treatment arms: arm 1 is patients who receive an anti-IL-33 monoclonal antibody (SAR440340 / REGN3500) administered subcutaneously (SC) alone; arm 2 is patients who receive an anti-IL-4R monoclonal antibody (dupilumab) administered subcutaneously alone; arm 3 is patients who receive both SAR440340 / REGN3500 and dupilumab co-administered subcutaneously; and arm 4 is placebo.

[0309] Patients in arm 1 will receive two SC injections of SAR440340 / REGN3500 every two weeks for 24 weeks, co-administered with dupilumab placebo as one SC injection every two weeks for 24 weeks. Patients in arm 2 will receive one SC injection of dupilumab every two weeks for 24 weeks, co-administered with SAR440340 / REGN3500 placebo as two SC injections every two weeks for 24 weeks. Patients in arm 3 will receive two SC injections of SAR440340 / REGN3500 every two weeks for 24 weeks, co-administered with dupilumab as one SC injection every two weeks for 24 weeks. Patients in arm 4 will receive matching placebo doses of SAR440340 / REGN3500 and dupilumab, administered as two and one SC injections, respectively, every two weeks for 24 weeks.

[0310] Research purpose The primary objective of this study was to evaluate the efficacy of interleukin-33 antibody (SAR440340 / REGN3500), interleukin-33 antibody, on improving respiratory function in patients with moderate to severe chronic obstructive pulmonary disease (COPD) treated with background therapy (dual or triple therapy) of inhaled corticosteroids (ICS), and / or long-acting beta-2 adrenergic agonists (LABA), and / or long-acting muscarinic antagonists (LAMA). To determine and compare the effects of dupilumab, a monoclonal antibody to the leukin-4 receptor (LER) and co-administration of both, compared with placebo, as assessed by post-bronchodilator forced expiratory volume in 1 second (FEV1) over 24 weeks.

[0311] A secondary objective is to evaluate the effect of SAR440340 / REGN3500, dupilumab, and the co-administration of both, individually compared with placebo, on the incidence of moderate-to-severe acute exacerbations of COPD (AECOPD) over 24 weeks of treatment.

[0312] Secondary objectives include evaluating the effects of SAR440340 / REGN3500, dupilumab, and co-administration of both, each compared with placebo, on pre-bronchodilator forced expiratory volume in one second over 24 weeks; time from baseline to first moderate or severe AECOPD event over 24 weeks; assessment of clinical symptoms of COPD; and safety and tolerability.

[0313] Inclusion criteria Inclusion criteria for the study were as follows: (1) patients with moderate to severe chronic obstructive pulmonary disease (COPD) (post-bronchodilator forced vital capacity (FVC) in 1 second (FEV1) less than 70% and post-bronchodilator FEV1% greater than 80% but less than 30% predicted); (2) patients with a COPD Assessment Test (CAT) score of 10 or greater at Screening Visit 1 and Visit 2 / randomization; (3) patients with a history of signs and symptoms of chronic bronchitis (chronic productive cough for 3 months in the year prior to screening in patients who excluded other causes of chronic cough (e.g., gastroesophageal reflux disease, chronic rhinosinusitis, bronchiectasis)); (4) patients with a history of chronic productive cough during the 3 months prior to screening in patients who excluded other causes of chronic cough (e.g., gastroesophageal reflux disease, chronic rhinosinusitis, bronchiectasis) at screening; (5) Patients had a documented medical history of two or more moderate exacerbations or one or more severe exacerbations within the year prior to screening; (6) Patients had standard background therapy in the 3 months prior to Visit 2 / randomization, including stable medication for at least 1 month prior to Screening Visit 1 (including dual therapy: long-acting beta-agonist (LABA) + long-acting muscarinic antagonist (LAMA), or inhaled corticosteroid (ICS) + LABA, or ICS + LAMA, or triple therapy: ICS + LABA + LAMA); (7) Patients had signed a written informed consent form; and (8) Patients were current or former smokers with a smoking history of 10 pack years or more.

[0314] Exclusion criteria The exclusion criteria for this study were as follows: (1) age ≤40 or >75 years, (2) body mass index (BMI) <16, (3) COPD diagnosed within 6 months prior to random selection, (4) current asthma diagnosis according to the Global Initiative in Asthma (GINA) guidelines, and (5) significant lung disease other than COPD (e.g., pulmonary fibrosis, sarcoidosis, interstitial lung disease, pulmonary hypertension, bronchiectasis, eosinophilic granulomatosis with polyangiitis, bilevel positive airway pressure). (5) have another diagnosed pulmonary or systemic disease associated with elevated peripheral eosinophil counts (e.g., significant sleep apnea due to chronic obstructive pulmonary disease (COPD) or elevated peripheral eosinophil counts); (6) have a diagnosis of alpha-1 antitrypsin deficiency; (7) have advanced COPD requiring long-term (>15 hours / day) supplemental oxygen; (8) have had an acute exacerbation of a moderate or severe COPD event within 4 weeks prior to screening; (9) have experienced an upper or lower respiratory tract infection within 4 weeks prior to Screening / Visit 1 or during the screening period; (10) have a history or planned lung resection or lung volume reduction surgery; (11) have a history of systemic hypersensitivity reactions to biologic agents.

[0315] Example 8. Evaluation of SAR440340 / REGN3500 or Dupilumab When Used Alone and as Combination Therapy in Patients with Moderate to Severe Asthma research design This study is a randomized, double-blind, placebo-controlled, parallel-group, 12-week proof-of-concept study to evaluate the efficacy, safety, and tolerability of SAR440340 / REGN3500, dupilumab (also known as DUPIXENT®), and co-administration of SAR440340 and dupilumab in patients with moderate to severe asthma not well controlled with inhaled corticosteroids (ICS) and long-acting beta-2 adrenergic agonist (LABA) therapy.

[0316] A total of 800 subjects will participate in the study, which consists of four treatment arms: arm 1 receives an anti-IL-33 monoclonal antibody (SAR440340 / REGN3500) administered subcutaneously (SC) alone; arm 2 receives an anti-IL-4R monoclonal antibody (dupilumab) administered subcutaneously alone; arm 3 receives both SAR440340 / REGN3500 and dupilumab co-administered subcutaneously; and arm 4 receives a placebo.

[0317] Patients in treatment arm 1 will receive SAR440340 / REGN3500 administered as two subcutaneous (SC) injections every two weeks for 12 weeks, co-administered with dupilumab placebo as one SC injection every two weeks for 12 weeks. Patients in treatment arm 2 will receive dupilumab administered as one SC injection every two weeks for 12 weeks, co-administered with SAR440340 / REGN3500 placebo as two SC injections every two weeks for 12 weeks. Patients in treatment arm 3 will receive SAR440340 / REGN3500 administered as two SC injections every two weeks for 12 weeks, co-administered with dupilumab administered as one SC injection every two weeks for 12 weeks. Patients in treatment arm 4 will receive matching placebo doses of SAR440340 / REGN3500 and dupilumab administered as two and one SC injections, respectively, every two weeks for 12 weeks.

[0318] Research purpose The primary study objective is to evaluate the effect of SAR440340 / REGN3500, with or without dupilumab, compared with placebo on reducing the incidence of "loss of asthma control" (LOAC) events.

[0319] Secondary study objectives include: to evaluate the effect of SAR440340 / REGN3500 and co-administration of SAR440340 / REGN3500 with dupilumab compared to placebo on forced expiratory volume in 1 second (FEV1); to evaluate the effect of co-administration of SAR440340 / REGN3500 with dupilumab compared to SAR440340 / REGN3500 and compared to dupilumab on FEV1; to evaluate the effect of co-administration of SAR440340 / REGN3500 with dupilumab compared to SAR440340 / REGN3500 alone and dupilumab alone on the reduction of LOAC; and to evaluate the safety and tolerability of SAR440340 / REGN3500 alone and co-administered with dupilumab.

[0320] Inclusion criteria The following inclusion criteria were used for this study: (1) Adult patients (≥18 years of age) with a physician-diagnosed asthma for at least 12 months based on the Global Indicators of Asthma (GINA) 2016 guidelines, whose asthma was partially or uncontrolled with ICS / LABA combination therapy with the following criteria: medium- to high-dose inhaled corticosteroids (ICS) (≥250 mcg / day twice daily (BID) of fluticasone propionate, or an equipotent ICS daily dose of up to 2000 mcg / day of fluticasone propionate, or a clinically comparable ICS dose); (1) a combination of a long-acting beta-agonist (LABA) as a second controller and a stable current medication for at least 3 months for at least 1 month prior to Visit 1; (2) a pre-bronchodilator forced expiratory volume in 1 second (FEV1) of 50% or greater but 85% or less of predicted normal at Visit 2 / baseline; (3) a reversibility of at least 12% and 200 mL in FEV1 after administration of 2-4 puffs (200-400 mcg) of albuterol / salbutamol or levalbuterol / levosalbutamol during screening (up to 12 puffs of reliever medication on no more than 3 occasions during the same visit, if tolerated by the patient). (3) Patients must have had at least one of the following events within the year prior to Visit 1: treatment with systemic corticosteroids (oral or parenteral) that worsened asthma, or hospitalization or emergency medical care that worsened asthma; and (4) signed an informed consent form.

[0321] Exclusion criteria Exclusion criteria for this study were as follows: (1) patients under 18 years of age or over 70 years of age (i.e., reaching 71 years of age at the time of the screening visit); (2) patients with a body mass index (BMI) less than 16; (3) chronic lung disease that may impair lung function (e.g., chronic obstructive pulmonary disease [COPD] or idiopathic pulmonary fibrosis [IPF]); (4) a history of life-threatening asthma (i.e., severe exacerbations requiring intubation); (5) comorbidities that may interfere with the assessment of IMP; (6) patients with any of the following events within the 4 weeks prior to Screening Visit 1: treatment with one or more systemic (oral or parenteral) steroid bursts that worsened asthma, or hospitalization or emergency medical care that worsened asthma; and (7) an Asthma Control Questionnaire 5-question version (ACQ-5) score less than 1.25 or greater than 3.0 at Visit 2 / randomization. (8) have received anti-immunoglobulin E (IgE) therapy (e.g., omalizumab [Xolair®]) within 130 days prior to Visit 1, or have received other biological therapy within 2 months or 5 half-lives (whichever is longer) prior to Visit 1 to treat inflammatory or autoimmune diseases (e.g., rheumatoid arthritis, inflammatory bowel disease, primary biliary cirrhosis, systemic lupus erythematosus, multiple sclerosis, etc.) and other diseases; (9) Patients with a history of systemic hypersensitivity to biologic agents; (10) Patients who had undergone or started bronchial thermoplasty within 2 years prior to Visit 1 or who planned to start therapy during the screening or randomized treatment period; (11) Current smokers or those who had quit smoking within 6 months prior to Visit 1; (12) Former smokers with a smoking history of more than 10 pack-years.

Claims

1. 1. A method of treating an inflammatory disease or disorder or at least one symptom associated with said inflammatory disease or disorder, comprising administering to a subject in need thereof one or more doses of a therapeutically effective amount of an interleukin-33 (IL-33) antagonist in combination with one or more doses of a therapeutically effective amount of an interleukin-4 receptor (IL-4R) antagonist, wherein said administration of said combination results in enhanced therapeutic efficacy compared to the therapeutic efficacy observed with administration of said IL-33 antagonist alone or said IL-4R antagonist alone.

2. 10. The method of claim 1, wherein the inflammatory disease or disorder is alleviated or reduced in severity, duration, or frequency of occurrence, or at least one symptom associated with the inflammatory disease or disorder is alleviated or reduced in severity, duration, or frequency of occurrence.

3. 3. The method of claim 1 or 2, wherein the inflammatory disease or disorder is selected from the group consisting of asthma, chronic obstructive pulmonary disease (COPD), asthma and COPD overlap syndrome (ACOS), atopic dermatitis, allergic reactions, chronic bronchitis, emphysema, chronic rhinosinusitis with or without nasal polyps, inflammatory bowel disease, Crohn's disease, ulcerative colitis, hypersensitivity pneumonitis, multiple sclerosis, arthritis (osteoarthritis, rheumatoid arthritis, and psoriatic arthritis), allergic rhinitis, fibrosis, eosinophilic esophagitis, vasculitis, urticaria, Churg-Strauss syndrome, inflammatory pain, and psoriasis.

4. 4. The method of claim 3, wherein the chronic obstructive pulmonary disease is exacerbated by one or more of asthma, a viral disease, a bacterial infection, exposure to an allergen, exposure to a chemical or chemical fumes, or exposure to an environmental irritant or air pollution.

5. 4. The method of claim 3, wherein the asthma is exacerbated by one or more of a viral illness, a bacterial infection, exposure to an allergen, exposure to a chemical or chemical fumes, or exposure to an environmental irritant or air pollution.

6. 6. The method of claim 3 or 5, wherein the asthma is eosinophilic asthma, noneosinophilic asthma, steroid-resistant asthma, or steroid-sensitive asthma.

7. 5. The method of claim 3 or 4, wherein the chronic obstructive pulmonary disease is caused by or exacerbated in part by tobacco smoke.

8. 8. The method of any one of claims 1 to 7, further comprising administering an effective amount of one or more additional therapeutic agents useful for alleviating the inflammatory disease or disorder or at least one symptom of the inflammatory disease or disorder.

9. The one or more additional therapeutic agents are selected from the group consisting of a nonsteroidal anti-inflammatory drug (NSAID), a corticosteroid, a bronchodilator, an antihistamine, epinephrine, a decongestant, a thymic stromal lymphopoietin (TSLP) antagonist, an IL-1 antagonist, an IL-8 antagonist, an IL-13 antagonist, a different IL-4 antagonist, an IL-4 / IL-13 dual antagonist, an IL-33 antagonist, an IL-4 / IL-13 ...33 antagonist, an IL-4 / IL-13 antagonist, an IL-33 antagonist, an IL-33 antagonist, an IL-4 / IL-13 antagonist, an IL-33 antagonist, an IL-33 antagonist, an IL-4 antagonist, an IL-33 antagonist, an IL-3 / IL-13 dual antagonist, IL-5 antagonist, IL-6 antagonist, IL-12 / 23 antagonist, IL-22 antagonist, IL-25 antagonist, IL-17 antagonist, IL-31 antagonist, TNF inhibitor, IgE inhibitor, leukotriene inhibitor, oral PDE4 inhibitor, methylxanthine, nedocromil sodium, cromolyn sodium, long-term 9. The method of claim 8, wherein the therapeutic agent is selected from the group consisting of a long acting beta 2 agonist (LABA), a long acting muscarinic antagonist (LAMA), an inhaled corticosteroid (ICS), and another IL-33 antagonist.

10. 10. The method of claim 9, wherein the additional IL-33 antagonist is selected from the group consisting of a different antibody to IL-33, a different IL-33 receptor-based trap, an ST2 antibody, a soluble ST2 receptor, an antagonist to an IL-33 receptor other than ST2, an IL-1RAcP antagonist, and an antibody that interacts with the IL-33 / ST2 complex.

11. 1. A method for treating a fibrotic disease or disorder or at least one symptom associated with said fibrotic disease or disorder, comprising administering to a subject in need thereof one or more doses of a therapeutically effective amount of an interleukin-33 (IL-33) antagonist in combination with one or more doses of a therapeutically effective amount of an interleukin-4 receptor (IL-4R) antagonist, wherein said administration of said combination results in enhanced therapeutic efficacy compared to the therapeutic efficacy observed with administration of said IL-33 antagonist alone or said IL-4R antagonist alone.

12. 12. The method of claim 11, wherein the fibrotic disease or disorder is alleviated or reduced in severity, duration, or frequency of occurrence, or at least one symptom associated with the fibrotic disease or disorder is alleviated or reduced in severity, duration, or frequency of occurrence.

13. 13. The method of claim 11 or 12, wherein the fibrotic disease or disorder is a pulmonary fibrotic disease or disorder.

14. 14. The method of claim 13, wherein the pulmonary fibrotic disease or disorder is selected from the group consisting of idiopathic pulmonary fibrosis, fibrosis associated with acute lung injury or acute respiratory distress, silicosis, radiation-induced fibrosis, bleomycin-induced pulmonary fibrosis, asbestos-induced pulmonary fibrosis, and bronchiolitis obliterans syndrome.

15. 1. A method for preventing or reducing the severity of an allergic reaction in a subject in need thereof, comprising administering one or more doses of a therapeutically effective amount of an IL-33 antagonist in combination with one or more doses of a therapeutically effective amount of an IL-4R antagonist, wherein said administration of said combination results in enhanced therapeutic efficacy for preventing or reducing the severity of an allergic reaction compared to the therapeutic efficacy observed with administration of the IL-33 antagonist alone or the IL-4R antagonist alone.

16. 16. The method of any one of claims 1, 11, or 15, wherein the enhanced therapeutic efficacy is measured by any one or more of the following parameters: a) a decrease in the frequency of one or more of eosinophils, activated B cells, activated CD8 T cells, or the CD4 / CD8 T cell ratio in a tissue sample; b) a decrease in one or more of interleukin-1 beta (IL-1β), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-13 (IL-13), monocyte chemoattractant protein-1 (MCP-1), or tumor necrosis factor alpha (TNFα) levels in a tissue sample; or c) A decrease in the gene expression levels of one or more of Il4, Il5, Il6, Il9, Il13, Il1rl1, Il13ra2, tnf, Tgfb1, Ccl2, Ccl11, Ccl24, Col15a1, and / or Col24a1 in the tissue sample.

17. The enhanced therapeutic efficacy is further measured by any one or more of the following parameters: The method of claim 16, wherein: d) a decrease in serum IgE levels; e) reduced goblet cell metaplasia in the lungs; f) improvement in lung consolidation, or g) Reduction of subepithelial fibrosis in the lungs.

18. 17. The method of claim 16, wherein the tissue sample is obtained from lung, liver, kidney, heart, or whole blood.

19. 19. The method of any one of claims 1 to 18, wherein administration of the IL-33 antagonist in combination with the IL-4R antagonist results in an increase in type 1 immune response and / or a decrease in type 2 immune response induced by the disease or by a causative agent of the disease or allergy.

20. The method of any one of claims 1 to 19, wherein the IL-4R antagonist is an antibody or antigen-binding fragment thereof that binds to IL-4Rα and prevents the interaction of IL-4 and / or IL-13 with type 1 or type 2 IL-4α receptor.

21. The method of claim 20, wherein the antibody or antigen-binding fragment thereof that binds to IL-4Rα prevents the interaction of IL-4 and / or IL-13 with both type 1 and type 2 IL-4α receptors.

22. The method of claim 20 or 21, wherein the IL-4Rα antibody or antigen-binding fragment thereof comprises a heavy chain complementarity-determining region (HCDR) of a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 335 or 337, and a light chain complementarity-determining region (LCDR) of a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 336 or 338.

23. The method of any one of claims 20 to 22, wherein the IL-4Rα antibody or antigen-binding fragment thereof comprises three HCDRs (HCDR1, HCDR2, and HCDR3) and three LCDRs (LCDR1, LCDR2, and LCDR3), wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 339, the HCDR2 comprises the amino acid sequence of SEQ ID NO: 340, the HCDR3 comprises the amino acid sequence of SEQ ID NO: 341, the LCDR1 comprises the amino acid sequence of SEQ ID NO: 342, the LCDR2 comprises the amino acid sequence of SEQ ID NO: 343, and the LCDR3 comprises the amino acid sequence of SEQ ID NO:

344.

24. The method of any one of claims 20 to 23, wherein the IL-4Rα antibody or antigen-binding fragment thereof comprises an HCVR comprising the amino acid sequence of SEQ ID NO: 335 or SEQ ID NO: 337, and an LCVR comprising the amino acid sequence of SEQ ID NO: 336 or SEQ ID NO:

338.

25. The method of any one of claims 20 to 24, wherein the IL-4Rα antibody or antigen-binding fragment thereof comprises the HCVR / LCVR amino acid sequence pair of either SEQ ID NOs: 335 / 336 or 337 / 338.

26. The method of any one of claims 1 to 25, wherein the IL-4Rα antagonist is dupilumab or a biological equivalent thereof.

27. The method of any one of claims 1 to 19, wherein the IL-33 antagonist is an antibody or an antigen-binding fragment thereof that specifically binds to IL-33 and blocks the interaction between IL-33 and ST2.

28. The antibody or antigen-binding fragment thereof that specifically binds to IL-33 comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, and 308. and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, and 316.

29. 29. The method of any one of claims 1-19 or 27-28, wherein the antibody or antigen-binding fragment thereof that specifically binds to IL-33 comprises a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 82, 98, 114, 130, 146, 162, 178, 194, 210, 226, 242, 258, 274, 290, and 308.

30. 30. The method of any one of claims 1-19 or 27-29, wherein the antibody or antigen-binding fragment thereof that specifically binds to IL-33 comprises a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 74, 90, 106, 122, 138, 154, 170, 186, 202, 218, 234, 250, 266, 282, 298, and 316.

31. The antibody or antigen-binding fragment thereof that specifically binds to IL-33 is (a) an HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 228, 244, 260, 276, 292, and 310; (b) an HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214, 230, 246, 262, 278, 294, and 312; (c) an HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216, 232, 248, 264, 280, 296, and 314; (d) an LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 220, 236, 252, 268, 284, and 318; (e) an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 206, 222, 238, 254, 270, 286, and 320; (f) an LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 208, 224, 240, 256, 272, 288, and 322.

32. Any of claims 1 to 19 or 27 to 31, wherein the antibody or antigen-binding fragment thereof that specifically binds to IL-33 comprises an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 106, 114 / 122, 130 / 138, 146 / 154, 162 / 170, 178 / 186, 194 / 202, 210 / 218, 226 / 234, 242 / 250, 258 / 266, 274 / 282, 290 / 298, and 308 / 316.

10. The method according to claim 1.

33. 33. The method of any one of claims 1 to 19 or 27 to 32, wherein the IL-33 antibody or antigen-binding fragment thereof interacts with an amino acid sequence ranging from about position 1 to about position 12 of SEQ ID NO: 349 and / or an amino acid sequence ranging from about position 50 to about position 94 of SEQ ID NO: 349 as determined by hydrogen / deuterium exchange.

34. The method of any one of claims 1 to 19 or 27 to 33, wherein the IL-33 antibody or antigen-binding fragment thereof interacts with an amino acid sequence ranging from about position 112 to about position 123 of SEQ ID NO: 348 and / or an amino acid sequence ranging from about position 161 to about position 205 of SEQ ID NO: 348 as determined by hydrogen / deuterium exchange.

35. 35. The method of any one of claims 1-19 or 27-34, wherein the IL-33 antibody or antigen-binding fragment thereof interacts with either the amino acid sequence of SEQ ID NO: 350, or the amino acid sequence of SEQ ID NO: 351, or both SEQ ID NOs: 350 and 351, as determined by hydrogen / deuterium exchange.

36. 36. The method of any one of claims 1-19 or 27-35, wherein the IL-33 antibody or antigen-binding fragment comprises a heavy chain complementarity determining region (HCDR) and a light chain complementarity determining region (LCDR) of the heavy chain variable region / light chain variable region (HCVR / LCVR) amino acid sequence pair of SEQ ID NOs: 274 / 282.

37. 37. The method of any one of claims 1-19 or 27-36, wherein the IL-33 antibody or antigen-binding fragment comprises the HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 domains of SEQ ID NOs: 276-278-280-284-286-288, respectively.

38. 38. The method of any one of claims 1-19 or 27-37, wherein the antibody or antigen-binding fragment that specifically binds to human interleukin-33 (IL-33) comprises (a) a heavy chain variable region (HCVR) having the amino acid sequence of SEQ ID NO:274, and (b) a light chain variable region (LCVR) having the amino acid sequence of SEQ ID NO:

282.

39. 39. The method of any one of claims 1-19 or 27-38, wherein the IL-33 antibody or antigen-binding fragment thereof competes for binding to IL-33 with a reference antibody comprising the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 274 / 282.

40. 40. The method of any one of claims 1-19 or 27-39, wherein the IL-33 antibody or antigen-binding fragment thereof binds to the same epitope on IL-33 as a reference antibody comprising the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 274 / 282.

41. 20. The method of any one of claims 1 to 19, wherein the IL-33 antagonist is an IL-33 trap comprising a first IL-33 binding domain (D1) linked to a multimerization domain (M), wherein D1 comprises the IL-33 binding portion of an ST2 protein.

42. 42. The method of claim 41, wherein the IL-33 antagonist is an IL-33 trap further comprising a second IL-33 binding domain (D2) bound to D1 and / or M, wherein D2 comprises the extracellular portion of the IL-1RAcP protein.

43. 42. The method of claim 41, wherein D1 is attached to the N-terminus of M.

44. 42. The method of claim 41, wherein D1 is attached to the C-terminus of M.

45. 43. The method of claim 42, wherein D2 is attached to the N-terminus of M.

46. 43. The method of claim 42, wherein D2 is attached to the C-terminus of M.

47. 43. The method of claim 42, wherein D1 is attached to the N-terminus of D2 and D2 is attached to the N-terminus of M.

48. 42. The method of claim 41, wherein D1 comprises the amino acid sequence of SEQ ID NO: 328 or 329, or an amino acid sequence having at least 90% identity thereto.

49. 43. The method of claim 42, wherein D2 comprises the amino acid sequence of SEQ ID NO: 330 or 331, or an amino acid sequence having at least 90% identity thereto.

50. 20. The method of any one of claims 1 to 19, wherein the IL-33 antagonist is an IL-33 trap comprising a first IL-33 binding domain (D1) linked to a first multimerization domain (M1) and a second IL-33 binding domain (D2) linked to a second multimerization domain (M2), wherein the D1 and / or D2 domains comprise an IL-33 binding portion of a receptor selected from the group consisting of ST2 and IL-1RAcP.

51. 51. The method of claim 50, wherein the IL-33 antagonist comprises a third IL-33 binding domain (D3) linked to either D1 or M1, wherein D3 comprises an IL-33 binding portion of a receptor selected from the group consisting of ST2 and IL-1RAcP.

52. 52. The method of claim 51, wherein the IL-33 antagonist comprises a fourth IL-33 binding domain (D4) linked to either D2 or M2, wherein D4 comprises an IL-33 binding portion of a receptor selected from the group consisting of ST2 and IL-1RAcP.

53. 51. The method of claim 50, wherein D1 is attached to the N-terminus of M1 and D2 is attached to the N-terminus of M2.

54. 52. The method of claim 51, wherein D3 is attached to the N-terminus of D1.

55. 52. The method of claim 51, wherein D3 is attached to the C-terminus of M1.

56. 52. The method of claim 51, wherein D4 is attached to the N-terminus of D2.

57. 53. The method of claim 52, wherein D4 is attached to the C-terminus of M2.

58. 53. The method of claim 52, wherein D3 is attached to the N-terminus of D1, D1 is attached to the N-terminus of M1, D4 is attached to the N-terminus of D2, and D2 is attached to the N-terminus of M2.

59. 59. The method of claim 58, wherein D3 is the same as or substantially the same as D4 and D1 is the same as or substantially the same as D2.

60. 60. The method of claim 59, wherein D3 and D4 each comprise the IL-33 binding portion of the ST2 protein, and D1 and D2 each comprise the extracellular portion of the IL-1RAcP protein.

61. 20. The method of any one of claims 1 to 19, wherein the IL-33 antagonist is an IL-33 trap comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 323, 324, 325, 326, and 327.

62. 62. The method of any one of claims 1-19 or 61, wherein the IL-33 antagonist is an IL-33 trap comprising the amino acid sequence of SEQ ID NO:

327.

63. 63. The method of any one of claims 1 to 62, wherein the IL-33 antagonist and the IL-4R antagonist are administered to a patient in need thereof in separate formulations.

64. 63. The method of any one of claims 1-62, wherein the IL-33 antagonist and the IL-4R antagonist are co-formulated prior to administration to a patient in need thereof.

65. 65. The method of any one of claims 1 to 64, wherein the IL-33 antagonist and the IL-4R antagonist are administered to the subject subcutaneously, intravenously, intramuscularly, or intranasally.

66. 66. The method of any one of claims 1 to 65, wherein the IL-33 antagonist is a monoclonal antibody comprising three heavy chain complementarity determining regions (HCDRs) contained within the heavy chain variable region sequence of SEQ ID NO: 274 and three light chain complementarity determining regions (LCDRs) contained within the light chain variable region (LCVR) sequence of SEQ ID NO: 282, and the IL-4R antagonist is a monoclonal antibody comprising three heavy chain complementarity determining regions (HCDRs) contained within the heavy chain variable region sequence of SEQ ID NO: 337 and three light chain complementarity determining regions (LCDRs) contained within the light chain variable region (LCVR) sequence of SEQ ID NO:

338.

67. The method of any one of claims 1 to 66, wherein the IL-33 antagonist is a monoclonal antibody comprising the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 274 / 282, and the IL-4R antagonist is a monoclonal antibody comprising the HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 337 / 338.

68. The method of any one of claims 1 to 67, wherein the IL-33 antagonist is a monoclonal antibody comprising three HCDRs (HCDR1-HCDR2-HCDR3) having the amino acid sequences of SEQ ID NOs: 276-278-280, respectively, and three LCDRs (LCDR1-LCDR2-LCDR3) having the amino acid sequences of SEQ ID NOs: 284-286-288, respectively, and the IL-4R antagonist is a monoclonal antibody comprising three HCDRs (HCDR1-HCDR2-HCDR3) having the amino acid sequences of SEQ ID NOs: 339-340-341, respectively, and three LCDRs (LCDR1-LCDR2-LCDR3) having the amino acid sequences of SEQ ID NOs: 342-343-344, respectively.

69. 69. The method of any one of claims 1-68, wherein the IL-33 antagonist is REGN3500 and the IL-4R antagonist is dupilumab.

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