Methods of using il-33 antagonists

Abstract

The present invention relates to methods of using IL-33 antagonists. In particular, the present disclosure relates to an IL-33 antagonist for use in the prevention or treatment of an epithelial physiology abnormality or an EGFR-mediated disease, and corresponding methods of prevention or treatment comprising administering the IL-33 antagonist to a patient in need thereof.

Description

[0001] This application is a divisional application of Chinese patent application 202080076133.0, "Method using IL-33 antagonist", filed on November 3, 2020.

[0002] This application claims priority to European Patent Application No. 19206984.7, filed on 4 November 2019. The entire contents of the above application are incorporated herein by reference. Technical Field

[0003] This disclosure relates to an IL-33 antagonist for the prevention or treatment of epithelial physiological abnormalities or EGFR-mediated diseases, and corresponding prevention or treatment methods, which include administering the IL-33 antagonist to patients in need. Background Technology

[0004] Interleukin-33 (IL-33), also known as IL-1F11, is a member of the IL-1 cytokine family. IL-33 is a 270-amino acid protein composed of two domains: a homology domain and a cytokine (IL-1-like) domain. The homology domain contains the nuclear localization signal (NLS). IL-33 is known to exist in two forms: a reduced form (redIL-33) and an oxidized form (oxIL-33). Previous studies have shown that the reduced form is rapidly oxidized under physiological conditions to form disulfide bonds in at least one oxidized form, and that these two forms may have different binding modes and functions.

[0005] Previously, it was found that the reduced form of IL-33 binds to ST2 and is actually the only known ligand for the ST2 receptor expressed by Th2 cells and mast cells. Reduced IL-33 stimulates target cells by binding to ST2, subsequently activating the NF4B and MAP kinase pathways, thereby producing cytokines and chemokines such as IL-4, IL-5, and IL-13 to promote inflammation. Soluble ST2 (sST2) is considered a decoy receptor that blocks reduced IL-33 signaling.

[0006] Recent findings suggest that the oxidized form of IL-33 also has physiological functions. Oxidized IL-33 was found to not bind to ST2, but instead binds to the receptor for advanced glycation end products (RAGE), and conducts signal transduction through this alternative pathway.

[0007] IL-33 has attracted considerable interest as a therapeutic target, primarily due to the known ability of its reduced form to stimulate ST2 and produce potent inflammatory effects. However, research and interest in the oxidized IL-33 pathway as a therapeutic target are limited, partly due to its later discovery and the fact that RAGE has numerous ligands and its downstream interactions are not yet fully understood.

[0008] This article describes in more detail at least one of these downstream RAGE interactions generated by oxidized IL-33 stimulation. It has been surprisingly found that RAGE complexes with the epidermal growth factor receptor (EGFR) as part of the oxidized IL-33 pathway. Reduced IL-33 is rapidly converted to oxidized IL-33, which then binds to RAGE and complexes with EGFR to stimulate EGFR activity. The significance of this surprising discovery of EGFR involvement lies in the fact that EGFR is a key therapeutic target for a variety of diseases involving abnormalities in epithelial physiology.

[0009] Because of this discovery, it is believed that antagonists that can bind to any form of IL-33 can effectively block the signaling of oxidized IL-33. This can be done directly by binding to oxidized IL-33 itself, or indirectly by inhibiting the conversion of reduced IL-33 to oxidized IL-33, both of which in turn block the stimulation of RAGE and EGFR. This reduction in EGFR stimulation will have therapeutic benefits in any EGFR-mediated disease, but particularly in conditions where EGFR is overstimulated.

[0010] EGFR is known to have multiple homeostatic effects on epithelial physiology. EGFR stimulation increases epithelial cell differentiation, epithelial cell migration, and epithelial mucosal production. It is believed that inhibiting EGFR-mediated signaling will treat or prevent disorders involving epithelial physiology abnormalities, such as abnormal airway epithelial tissue remodeling or excessive mucus production.

[0011] IL-33 has previously been associated with tissue remodeling in the airways (Li et al., JACI, 2014 134: 1422-32; Vannella et al., Sci Transl Med, 337ra65; Allinne et al., JACI, 2019, 144: 1624-37). However, this is thought to occur indirectly through a self-perpetuating amplification loop, through which IL-33 signaling upregulates the expression of both IL-33 and its homologous receptor ST2, thereby inducing chronic ST2 axis signaling. It has not been previously established or proposed that IL-33 itself directly affects airway epithelial biology, as the activity generated by ST2 is mediated by ST2-expressing innate cells, such as macrophages and type 2 innate lymphoid cells.

[0012] As stated above, this disclosure is based on the finding that IL-33 also acts directly to affect epithelial physiology through a different mechanism: the RAGE-EGFR pathway. This new understanding is important because it can be used to expand the therapeutic applications of IL-33 antagonists to treat more diseases, more disease symptoms, and more patients. The therapeutic opportunity to directly control and inhibit IL-33-mediated EGFR-mediated signaling by targeting IL-33 has not been previously realized.

[0013] This application discloses for the first time that the use of IL-33 antagonists can directly affect impaired epithelial repair responses by directly inhibiting RAGE / EGFR-mediated oxIL-33 activity, reducing epithelial goblet cell differentiation and proliferation, reducing mucus production, and improving mucociliary movement in patients with epithelial physiological abnormalities, such as those with COPD or bronchitis. Therefore, the study presented herein supports the therapeutic use of IL-33 antagonists in the direct prevention or treatment of epithelial physiological abnormalities typically caused by EGFR-mediated effects and thus present in EGFR-mediated diseases. Summary of the Invention

[0014] According to the first aspect, an IL-33 antagonist is provided for the prevention or treatment of epithelial physiological abnormalities by modulating or inhibiting RAGE-EGFR-mediated action.

[0015] According to the first aspect of the alternative, a method for preventing or treating epithelial physiological abnormalities in patients is provided, the method comprising: administering an effective amount of an IL-33 antagonist to a patient in need to modulate or inhibit RAGE-EGFR-mediated action.

[0016] According to the first aspect of the alternative, the use of IL-33 antagonists in the preparation of medicaments for the prevention or treatment of epithelial physiological abnormalities is provided.

[0017] According to the second aspect, an IL-33 antagonist is provided for the prevention or treatment of EGFR-mediated diseases.

[0018] According to the second aspect of the alternative, a method for preventing or treating EGFR-mediated disease in a patient is provided, the method comprising: administering an effective amount of an IL-33 antagonist to a patient in need.

[0019] According to the second aspect of the alternative, the use of IL-33 antagonists in the preparation of medicaments for the prevention or treatment of EGFR-mediated diseases is provided.

[0020] According to a third aspect, an IL-33 antagonist is provided for the prevention or treatment of disease by improving epithelial physiology.

[0021] According to the third alternative, a method for preventing or treating respiratory diseases by improving the epithelial physiology of a patient is provided, the method comprising: administering an effective amount of an IL-33 antagonist to a patient in need.

[0022] According to the third alternative, the use of IL-33 antagonists in the preparation of medicaments for the prevention or treatment of respiratory diseases by improving epithelial physiology is provided.

[0023] According to the fourth aspect, an IL-33 antagonist is provided for the prevention or treatment of disease by inhibiting EGFR-mediated effects.

[0024] According to the fourth aspect of the alternative, a method for preventing or treating respiratory diseases by inhibiting the EGFR-mediated effects in a patient is provided, the method comprising: administering an effective amount of an IL-33 antagonist to a patient in need.

[0025] According to the fourth aspect of the alternative, the use of IL-33 antagonists in the preparation of medicaments for the prevention or treatment of respiratory diseases by inhibiting EGFR-mediated effects is provided.

[0026] According to another aspect, an IL-33 antagonist is provided for the prevention or treatment of disease by inhibiting IL-33-mediated EGFR signaling.

[0027] According to another alternative, a method for preventing or treating disease by inhibiting IL-33-mediated EGFR signaling in a patient is provided, the method comprising: administering an effective amount of an IL-33 antagonist to a patient in need.

[0028] According to another alternative, the use of IL-33 antagonists in the preparation of medicaments for the prevention or treatment of diseases by inhibiting IL-33-mediated EGFR signaling is provided.

[0029] Other features and embodiments of the aspects defined above are described in the heading sections below. Each part can be combined with any of the aspects described above in any compatible combination.

[0030] This invention relates to the following solutions: 1. An IL-33 antagonist for the prevention or treatment of epithelial physiological abnormalities by modulating or inhibiting RAGE-EGFR-mediated action.

[0031] 2. An IL-33 antagonist used according to Scheme 1, wherein the epithelial physiological abnormality is a mucociliary physiological abnormality, preferably a mucociliary physiological abnormality of the respiratory epithelium.

[0032] 3. An IL-33 antagonist used according to Scheme 2, wherein the mucosal ciliary physiological abnormality is selected from: abnormal mucus production; abnormal goblet cell differentiation; abnormal goblet cell proliferation; abnormal epithelial thickness; abnormal mucus clearance; and / or abnormal mucus composition.

[0033] 4. An IL-33 antagonist used according to Scheme 3, wherein the mucus production abnormality includes MUC5AC production abnormality; and / or wherein the goblet cell differentiation abnormality includes MUC5AC + goblet cell differentiation abnormality; and / or wherein the goblet cell proliferation abnormality includes MUC5AC + goblet cell proliferation abnormality; and / or wherein the epithelial thickness abnormality includes MUC5AC in the total tissue region of the epithelium. + The number of goblet cells is abnormal.

[0034] 5. An IL-33 antagonist used according to schemes 2, 3 or 4, wherein mucosal ciliary physiological abnormalities include: increased mucus production; increased goblet cell differentiation; increased goblet cell proliferation; increased epithelial thickness; and / or decreased mucus clearance.

[0035] 6. An IL-33 antagonist used according to Scheme 5, wherein increased mucus production includes increased MUC5AC production; and / or wherein increased goblet cell differentiation includes MUC5AC + increased goblet cell differentiation; and / or wherein increased goblet cell proliferation includes MUC5AC + increased goblet cell proliferation; and / or wherein increased epithelial thickness includes MUC5AC in the total tissue region of the epithelium. + The number of goblet cells increases.

[0036] 7. An IL-33 antagonist used according to Scheme 3, wherein the mucus composition abnormality includes an increase or decrease in the ratio of different mucus compounds contained in the mucus; an increase or decrease in one or more mucus compounds; and / or an increase or decrease in mucus concentration or thickness.

[0037] 8. An IL-33 antagonist used according to Scheme 7, wherein the mucus composition aberration includes an increased MUC5AC:MUC5B ratio; and / or wherein the mucus composition aberration includes an increased MUC5AC content in the mucus; and / or wherein the mucus composition aberration includes an increased mucus thickness.

[0038] 9. An IL-33 antagonist used according to Scheme 1, wherein the epithelial physiological abnormality is an epithelial remodeling abnormality.

[0039] 10. An IL-33 antagonist for use according to any of the foregoing schemes, wherein the epithelial physiological abnormality is an epithelial physiological abnormality of the respiratory tract, preferably a mucociliary physiological abnormality of the respiratory tract.

[0040] 11. An IL-33 antagonist used according to Scheme 10, wherein the respiratory tract is the lower respiratory tract, preferably the bronchus.

[0041] 12. An IL-33 antagonist for the prevention or treatment of EGFR-mediated diseases.

[0042] 13. An IL-33 antagonist used according to scheme 12, wherein the EGFR-mediated disease is a RAGE-EGFR-mediated disease.

[0043] 14. An IL-33 antagonist for use according to scheme 12 or 13, wherein the EGFR-mediated disease is characterized by abnormal EGFR activity.

[0044] 15. An IL-33 antagonist used according to any one of schemes 12-14, wherein the EGFR-mediated disease is characterized by epithelial physiological abnormalities.

[0045] 16. An IL-33 antagonist used to prevent or treat disease by improving epithelial physiology.

[0046] 17. An IL-33 antagonist used to prevent or treat disease by inhibiting EGFR-mediated effects.

[0047] 18. An IL-33 antagonist used according to Scheme 17, wherein the EGFR-mediated effect is EGFR signal transduction.

[0048] 19. An IL-33 antagonist used according to scheme 17 or 18, wherein the EGFR-mediated effect is a RAGE-EGFR-mediated effect.

[0049] 20. An IL-33 antagonist for use according to any one of schemes 17-19, wherein the EGFR-mediated effect is RAGE-EGFR-mediated signal transduction.

[0050] 21. An IL-33 antagonist used according to any one of schemes 16-20, wherein the disease is a respiratory disease.

[0051] 22. An IL-33 antagonist used according to scheme 21, wherein the respiratory disease is characterized by epithelial physiological abnormalities and / or abnormal EGFR activity.

[0052] 23. An IL-33 antagonist for use according to scheme 21 or 22, wherein the respiratory disease is a lower respiratory tract disease, preferably a bronchial respiratory disease.

[0053] 24. An IL-33 antagonist used according to any one of schemes 21-23, wherein the respiratory disease is selected from: COPD; bronchitis; emphysema; bronchiectasis, such as CF-bronchodilator or -CF-bronchodilator; asthma; or asthma overlapping with COPD (ACO).

[0054] 25. An IL-33 antagonist used according to any one of schemes 21-24, wherein the respiratory disease is COPD, preferably bronchitis-related COPD.

[0055] 26. An IL-33 antagonist used according to any one of schemes 21-24, wherein the respiratory disease is asthma, preferably bronchitis-related asthma.

[0056] 27. An IL-33 antagonist used according to any one of schemes 16-26, wherein the prevention or treatment improves mucus clearance.

[0057] 28. An IL-33 antagonist used according to any one of schemes 16-27, wherein the prevention or treatment inhibits or reduces mucus production abnormalities.

[0058] 29. An IL-33 antagonist used according to scheme 28, wherein the prevention or treatment inhibits or reduces the production of MUC5AC.

[0059] 30. An IL-33 antagonist used according to any one of schemes 16-29, wherein the prevention or treatment inhibits abnormal mucus composition.

[0060] 31. An IL-33 antagonist used according to Scheme 30, wherein the prevention or treatment inhibits or reduces the MUC5AC: MUC5B ratio; and / or wherein the prevention or treatment inhibits or reduces MUC5AC in mucus; and / or wherein the prevention or treatment reduces the thickness of mucus.

[0061] 32. An IL-33 antagonist used according to any one of schemes 16-31, wherein the prevention or treatment inhibits epithelial remodeling abnormalities.

[0062] 33. An IL-33 antagonist used according to any one of schemes 16-32, wherein the prevention or treatment inhibits abnormal goblet cell differentiation or proliferation.

[0063] 34. An IL-33 antagonist used according to scheme 33, wherein the goblet cell differentiation or proliferation abnormality is MUC5AC + Goblet cell differentiation or proliferation is abnormal.

[0064] 35. An IL-33 antagonist used according to any one of schemes 16-34, wherein the prevention or treatment reduces the thickness of the respiratory epithelium.

[0065] 36. An IL-33 antagonist used according to scheme 35, wherein the prevention or treatment reduces MUC5AC in the total tissue area of ​​the epithelium. + The number of goblet cells.

[0066] 37. An IL-33 antagonist for use according to any of the foregoing schemes, wherein the IL-33 antagonist inhibits the activity of oxidized IL-33.

[0067] 38. An IL-33 antagonist for use according to any of the foregoing schemes, wherein the IL-33 antagonist prevents oxidized IL-33 from binding to RAGE, thereby inhibiting RAGE-EGFR signaling.

[0068] 39. An IL-33 antagonist for use according to any of the foregoing schemes, wherein the IL-33 antagonist downregulates or inhibits RAGE-EGFR-dependent signaling and / or RAGE-EGFR-mediated effects.

[0069] 40. An IL-33 antagonist for use according to any of the foregoing schemes, wherein the IL-33 antagonist is a binding molecule or fragment thereof that binds to IL-33, preferably to reduced IL-33 or oxidized IL-33, preferably to reduced IL-33.

[0070] 41. An IL-33 antagonist for use according to any of the foregoing schemes, wherein the IL-33 antagonist is an antibody or an antigen-binding fragment thereof, preferably an anti-IL-33 antibody or an antigen-binding fragment thereof, and more preferably an anti-reduced IL-33 antibody or an antigen-binding fragment thereof.

[0071] 42. An IL-33 antagonist for use according to any of the foregoing schemes, wherein the IL-33 antagonist is a binding molecule comprising a complementarity-determining region (CDR) of a pair of variable heavy chain domains (VH) and variable light chain domains (VL) selected from Table 1.

[0072] 43. An IL-33 antagonist for use according to any of the foregoing schemes, wherein the IL-33 antagonist is a binding molecule comprising a pair of variable heavy chain domains (VH) and variable light chain domains (VL) selected from Table 1.

[0073] 44. An IL-33 antagonist for use according to any of the foregoing embodiments, wherein the IL-33 antagonist is a binding molecule comprising VHCDR1 having the sequence of SEQ ID NO:37, VHCDR2 having the sequence of SEQ ID NO:38, VHCDR3 having the sequence of SEQ ID NO:39, VLCDR1 having the sequence of SEQ ID NO:40, VLCDR2 having the sequence of SEQ ID NO:41, and VLCDR3 having the sequence of SEQ ID NO:42.

[0074] 45. A method for preventing or treating epithelial physiological abnormalities in a subject, wherein the method is carried out by administering a therapeutically effective amount of an IL-33 antagonist to modulate or inhibit RAGE-EGFR-mediated action.

[0075] 46. ​​The method of Scheme 45, wherein the epithelial physiological abnormality is a mucociliary physiological abnormality, preferably a mucociliary physiological abnormality of the respiratory epithelium.

[0076] 47. The method of Scheme 46, wherein the mucosal ciliary physiological abnormality is selected from: abnormal mucus production; abnormal goblet cell differentiation; abnormal goblet cell proliferation; abnormal epithelial thickness; abnormal mucus clearance; and / or abnormal mucus composition.

[0077] 48. The method of Scheme 47, wherein abnormal mucus production includes abnormal MUC5AC production; and / or wherein abnormal goblet cell differentiation includes MUC5AC + abnormal goblet cell differentiation; and / or wherein abnormal goblet cell proliferation includes MUC5AC + abnormal goblet cell proliferation; and / or wherein the epithelial thickness abnormality includes MUC5AC in the total tissue region of the epithelium. + The number of goblet cells is abnormal.

[0078] 49. The method as described in scheme 47 or 48, wherein the mucociliary physiological abnormalities include: increased mucus production; increased goblet cell differentiation; increased goblet cell proliferation; increased epithelial thickness; and / or decreased mucus clearance.

[0079] 50. The method of Scheme 49, wherein increased mucus production includes increased MUC5AC production; and / or wherein increased goblet cell differentiation includes MUC5AC + increased goblet cell differentiation; and / or wherein increased goblet cell proliferation includes MUC5AC + increased goblet cell proliferation; and / or wherein increased epithelial thickness includes MUC5AC in the total tissue region of the epithelium. + The number of goblet cells increases.

[0080] 51. The method of claim 47, wherein the mucus composition abnormality includes an increase or decrease in the ratio of different mucus compounds contained in the mucus; an increase or decrease in one or more mucus compounds; and / or an increase or decrease in mucus concentration or thickness.

[0081] 52. The method of claim 51, wherein the mucus composition anomaly includes an increased MUC5AC: MUC5B ratio; and / or wherein the mucus composition anomaly includes an increased MUC5AC content in the mucus; and / or wherein the mucus composition anomaly includes an increased mucus thickness.

[0082] 53. The method of scheme 45, wherein the epithelial physiological abnormality is an epithelial remodeling abnormality.

[0083] 54. The method of any one of claims 45-53, wherein the epithelial physiological abnormality is an epithelial physiological abnormality of the respiratory tract, preferably a mucociliary physiological abnormality of the respiratory tract.

[0084] 55. The method of Scheme 54, wherein the airway is the lower respiratory tract, preferably the bronchus.

[0085] 56. A method for preventing or treating EGFR-mediated diseases, wherein the method is performed by administering a therapeutically effective amount of an IL-33 antagonist.

[0086] 57. The method of scheme 56, wherein the EGFR-mediated disease is a RAGE-EGFR-mediated disease.

[0087] 58. The method as described in scheme 56 or 57, wherein the EGFR-mediated disease is characterized by abnormal EGFR activity.

[0088] 59. The method of any one of schemes 56-58, wherein the EGFR-mediated disease is characterized by epithelial physiological abnormalities.

[0089] 60. A method for preventing or treating a disease, wherein the method is carried out by administering a therapeutically effective amount of an IL-33 antagonist to improve epithelial physiology.

[0090] 61. A method for preventing or treating a disease, wherein the method is carried out by administering a therapeutically effective amount of an IL-33 antagonist to inhibit EGFR-mediated action.

[0091] 62. The method as described in Scheme 61, wherein the EGFR-mediated function is EGFR signal transduction.

[0092] 63. The method as described in scheme 61 or 62, wherein the EGFR-mediated effect is a RAGE-EGFR-mediated effect.

[0093] 64. The method of any one of schemes 61-63, wherein the EGFR-mediated function is RAGE-EGFR-mediated signal transduction.

[0094] 65. The method of any one of schemes 60-64, wherein the disease is a respiratory disease.

[0095] 66. The method of scheme 65, wherein the respiratory disease is characterized by epithelial physiology abnormalities and / or abnormal EGFR activity.

[0096] 67. The method as described in Scheme 65 or 66, wherein the respiratory disease is a lower respiratory tract disease, preferably a bronchial respiratory disease.

[0097] 68. The method of any one of schemes 65-67, wherein the respiratory disease is selected from: COPD; bronchitis; emphysema; bronchiectasis, such as CF-bronchiolesion or -CF-bronchiolesion; asthma; or asthma overlapping with COPD (ACO).

[0098] 69. The method of any one of schemes 65-68, wherein the respiratory disease is COPD, preferably bronchitis-related COPD.

[0099] 70. The method of any one of schemes 65-69, wherein the respiratory disease is asthma, preferably bronchitis-related asthma.

[0100] 71. The method of any one of claims 45-70, wherein the method improves mucus removal.

[0101] 72. The method of any one of claims 45-71, wherein the method inhibits or reduces mucus production abnormalities.

[0102] 73. The method as described in scheme 72, wherein the mucus production anomaly is an increase in MUC5AC production.

[0103] 74. The method of any one of claims 45-73, wherein the method inhibits abnormal mucus composition.

[0104] 75. The method of claim 74, wherein the method inhibits or reduces the MUC5AC: MUC5B ratio; and / or wherein the method inhibits or reduces MUC5AC in the mucus; and / or wherein the method reduces the thickness of the mucus.

[0105] 76. The method of any one of claims 45-75, wherein the method inhibits epithelial remodeling abnormalities.

[0106] 77. The method of any one of schemes 45-76, wherein the method inhibits or reduces abnormal goblet cell differentiation or proliferation.

[0107] 78. The method of claim 77, wherein the method inhibits or reduces MUC5AC + Goblet cell differentiation or proliferation is abnormal.

[0108] 79. The method of any one of claims 45-78, wherein the method reduces the thickness of the respiratory epithelium.

[0109] 80. The method of scheme 79, wherein the method reduces MUC5AC in the total tissue area of ​​the respiratory epithelium. + The number of goblet cells.

[0110] 81. The method of any one of claims 45-80, wherein the IL-33 antagonist inhibits the activity of oxidized IL-33.

[0111] 82. The method of any one of claims 45-81, wherein the IL-33 antagonist prevents oxidized IL-33 from binding to RAGE, thereby inhibiting RAGE-EGFR signaling.

[0112] 83. The method of any one of claims 45-82, wherein the IL-33 antagonist downregulates or inhibits RAGE-EGFR-dependent signaling and / or RAGE-EGFR-mediated effects.

[0113] 84. The method of any one of claims 45-83, wherein the IL-33 antagonist is a binding molecule or fragment thereof that binds to IL-33, preferably reduced IL-33 or oxidized IL-33, preferably proto-IL-33.

[0114] 85. The method of any one of claims 45-84, wherein the IL-33 antagonist is an antibody or an antigen-binding fragment thereof, preferably an anti-IL-33 antibody or an antigen-binding fragment thereof, and more preferably an anti-reduced IL-33 antibody or an antigen-binding fragment thereof.

[0115] 86. The method of any one of claims 45-85, wherein the IL-33 antagonist is a binding molecule comprising a complementarity-determining region (CDR) of a pair of variable heavy chain domains (VH) and variable light chain domains (VL) selected from Table 1.

[0116] 87. The method of any one of claims 45-86, wherein the IL-33 antagonist is a binding molecule comprising a pair of variable heavy chain domains (VH) and variable light chain domains (VL) selected from Table 1.

[0117] 88. The method of any one of claims 45-87, wherein the IL-33 antagonist is a binding molecule comprising VHCDR1 having the sequence of SEQ ID NO:37, VHCDR2 having the sequence of SEQ ID NO:38, VHCDR3 having the sequence of SEQ ID NO:39, VLCDR1 having the sequence of SEQ ID NO:40, VLCDR2 having the sequence of SEQ ID NO:41, and VLCDR3 having the sequence of SEQ ID NO:42.

[0118] 89. The method of any one of schemes 45-88, or an IL-33 antagonist used according to any one of schemes 1-44, wherein the IL-33 antagonist is an anti-IL33 antibody or an antigen-binding fragment thereof, the anti-IL33 antibody or the antigen-binding fragment thereof comprising the VH domain of the sequence of SEQ ID NO:1 and the VL domain of the sequence of SEQ ID NO:19. Detailed Implementation definition As used herein, 'IL-33' protein refers to interleukin 33, particularly mammalian interleukin 33 protein, such as the human protein deposited under UniProt number 095760. However, it is clear that this entity is not a single species, but exists in reduced and oxidized forms. Given that the reduced form is rapidly oxidized in vivo, for example, over a period of 5 to 40 minutes, and in vitro, prior art references to IL-33 may actually refer to the oxidized form. Furthermore, commercial assays may not be able to effectively distinguish between the reduced and oxidized forms. The terms "IL-33" and "IL-33 polypeptide" are used interchangeably. In some embodiments, IL-33 is full-length. In another embodiment, IL-33 is mature truncated IL-33 (amino acids 112-270). Recent studies have shown that full-length IL-33 is active (Cayrol and Girard, Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences] 106(22): 9021-6 (2009); Hayakawa et al., Biochem Biophys Res Commun. [Biochemistry and Biophysics Research Communications] 387(1):218-22 (2009); Talabot-Ayer et al., J Biol Chem.[Journal of Biochemistry] 284(29):19420-6 (2009). However, N-terminal treated or truncated IL-33 (including but not limited to aa 72-270, 79-270, 95-270, 99-270, 107-270, 109-270, 111-270, 112-270) may have enhanced activity (Lefrancais 2012, 2014). In another embodiment, IL-33 may comprise full-length IL-33, fragments thereof, or IL-33 mutant or variant polypeptides, wherein the IL-33 fragment or IL-33 variant polypeptide retains some or all of the functional properties of active IL-33.

[0119] As used herein, 'oxidized IL-33' or 'oxIL-33' refers to the form of IL-33 that binds to RAGE and triggers RAGE-EGFR-mediated signaling. Oxidized IL-33 refers to a protein that is visible as a distinct band, for example, by Western blot analysis under non-reducing conditions, particularly a protein with a mass 4 Da smaller than its corresponding reduced form. Specifically, it refers to a protein having one or two disulfide bonds between cysteine ​​residues independently selected from cysteine ​​208, 227, 232, and 259. In one embodiment, oxidized IL-33 shows no binding to ST2.

[0120] As used herein, 'reduced IL-33' or 'redIL-33' refers to the form of IL-33 that binds to ST2 and triggers ST2-mediated signaling. Specifically, the reduced forms of cysteine ​​residues 208, 227, 232, and 259 are not bound to disulfide bonds. In one embodiment, reduced IL-33 does not bind to RAGE.

[0121] It should be understood that references to “WT IL-33” or “IL-33” may refer to the reduced or oxidized form, or both, unless it is clear from the context in which these forms are used that one of these forms is being referred to.

[0122] As used herein, 'different antigenic forms of IL-33' refers to any form of IL-33 that can act as an antigen and be bound by an antibody or its binding fragment; typically, in the context of this disclosure, this refers to oxidized IL-33, reduced IL-33, and reduced IL-33 / sST2 complexes.

[0123] As used in this paper, 'ST2-mediated signaling / function' refers to the IL-33 / ST2 system in which ST2 recognition of reduced IL-33 promotes dimerization with IL-1RAcP on the cell surface and promotes the recruitment of receptor complex components MyD88, TRAF6, and IRAK1-4 to the intracellular TIR domain. Therefore, ST2-dependent signaling / function can be interrupted or attenuated by perturbing the interaction between IL-33 and ST2, or alternatively by disrupting the interaction with IL-1RAcP.

[0124] As used in this article, "RAGE-EGFR-mediated signaling / function" refers to the oxidized IL-33 / RAGE-EGFR system, in which RAGE recognition of oxidized IL-33 promotes intracellular EGFR recombination. Therefore, RAGE-EGFR-mediated signaling / function can be interrupted or weakened by perturbing the interaction between oxidized IL-33 and RAGE, or by disrupting the conversion of reduced IL-33 to oxidized IL-33.

[0125] As used in this article, 'attenuating the activity of...' means reducing or inhibiting the relevant activity or stopping the relevant activity. Generally, attenuating and inhibiting are used interchangeably in this article.

[0126] It should be noted that the term "a / an" refers to one or more of the entities described; for example, "anti-IL-33 antibody" should be understood to mean one or more anti-IL-33 antibodies. Therefore, the terms "a / an", "one or more", and "at least one" are used interchangeably herein.

[0127] As used herein, the term "treat" refers to therapeutic procedures and preventative or therapeutic measures aimed at preventing or mitigating (alleviating) undesirable physiological changes or impairments. Beneficial or desired clinical outcomes include, but are not limited to, symptom relief, reduction in disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of the disease state, and reduction (whether partial or complete), whether detectable or undetectable. "Treatment" can also mean prolonged survival compared to expected survival without treatment. Those requiring treatment include those who already have a condition or impairment, those who are susceptible to a condition or impairment, or those who intend to prevent a condition or impairment.

[0128] The terms "subject," "individual," "animal," "patient," or "mammal" refer to any subject requiring diagnosis, prognosis, or treatment, particularly a mammalian subject, unless the subject is defined as a 'healthy subject.' Mammal subjects include humans; livestock; farm animals; such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, etc.

[0129] IL-33 antagonists This disclosure relates to the medical use of IL-33 antagonists, particularly to the medical use of IL-33 antagonists for the prevention or treatment of diseases by inhibiting IL-33-mediated EGFR signaling. In certain cases, this disclosure relates to the use of IL-33 antagonists for the prevention or treatment of epithelial physiological abnormalities that may be found in EGFR-mediated diseases.

[0130] As used herein, 'IL-33 antagonist' refers to any agent that weakens the activity of IL-33, such as reduced IL-33 activity, oxidized IL-33 activity, or both. Suitably, the IL-33 antagonist is specific to reduced and / or oxidized IL-33. Suitably, this weakening occurs by binding to the reduced or oxidized form of IL-33. Suitably, in cases where the antagonist weakens both reduced and oxidized IL-33 activity, this weakening occurs by binding to the reduced form of IL-33 (i.e., by binding to reduced IL-33).

[0131] Suitablely, IL-33 antagonists are binding molecules or fragments thereof.

[0132] The term "binding molecule" or "antigen-binding molecule" in this disclosure, in its broadest sense, refers to a molecule that specifically binds to an antigenic determinant. Suitablely, the binding molecule specifically binds to IL-33, particularly reduced or oxidized IL-33.

[0133] Suitablely, the binding molecule may be selected from: antibodies, their antigen-binding fragments, aptamers, at least one heavy or light chain CDR of a reference antibody molecule, and at least six CDRs from one or more reference antibody molecules.

[0134] Suitablely, an IL-33 antagonist is an antibody or a binding fragment thereof. Suitablely, an IL-33 antagonist is an anti-IL-33 antibody or a binding fragment thereof. Suitablely, an anti-IL-33 antibody or a binding fragment thereof specifically binds to IL-33, particularly reduced IL-33 or oxidized IL-33.

[0135] As used in this article, “antibody” refers to immunoglobulin molecules, particularly full-length antibodies or molecules containing full-length antibodies, such as DVD-Ig molecules.

[0136] "Its binding fragment" and "its antigen-binding fragment" are interchangeable and refer to the epitope / antigen-binding fragment of the antibody fragment, which, for example, contains a binding region, particularly containing 6 CDRs, such as 3 CDRs in the heavy chain variable region and 3 CDRs in the light chain variable region.

[0137] Suitablely, the antibody or its binding fragment is selected from: natural, polyclonal, monoclonal, multispecific, mouse, human, humanized, primate-derived, or chimeric antibodies or their binding fragments. Suitablely, the antibody or its binding fragment may be an epitope-binding fragment, such as Fab' and F(ab')2, Fd, Fv, single-chain Fv (scFv), disulfide-linked Fv (sdFv), a fragment containing a VL or VH domain, or a fragment generated from a Fab expression library. Suitablely, the antibody or its binding fragment may be a microantibody, a biantibody, a triantibody, a tetraantibody, or a single-chain antibody. Suitablely, the antibody or its binding fragment is a monoclonal antibody. scFv molecules are known in the art and are described, for example, in U.S. Patent No. 5,892,019.

[0138] The immunoglobulin or antibody molecules disclosed herein can be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, etc.), or subclass of immunoglobulin molecules.

[0139] Suitablely, IL-33 antagonists inhibit the activity of oxidized IL-33 by inhibiting the formation of oxidized IL-33. Suitablely, IL-33 antagonists inhibit the conversion of reduced IL-33 to oxidized IL-33.

[0140] Suitablely, the IL-33 antagonist is a reduced IL-33 antagonist. In other words, the IL-33 antagonist attenuates the activity of reduced IL-33. Suitablely, this attenuation occurs by binding to reduced IL-33. Suitablely, by binding to reduced IL-33, the antagonist also inhibits / attenuates the activity of oxidized IL-33 by preventing the conversion of reduced IL-33 to its oxidized form. Suitablely, inhibition of oxidized IL-33 activity downregulates or shuts down RAGE-dependent signaling and / or RAGE-mediated effects. Suitablely, this inhibition downregulates or shuts down RAGE-EGFR-dependent signaling and / or RAGE-EGFR-mediated effects. Suitablely, this inhibition downregulates or shuts down EGFR-dependent signaling. Suitablely, this inhibition downregulates or shuts down EGFR-mediated effects. In particular, IL33 antagonists that bind to reduced IL-33 have been shown to prevent oxidized IL-33 from binding to RAGE, thereby inhibiting RAGE-EGFR signaling.

[0141] Suitablely, inhibition of oxidized IL-33 activity downregulates or prevents RAGE-EGFR complexation. Suitablely, this inhibition downregulates or prevents EGFR activation, suitablely, RAGE-mediated EGFR activation.

[0142] Suitablely, IL-33 antagonists have all of the above-mentioned inhibitory effects. Suitablely, reduced IL-33 antagonists have all of the above-mentioned inhibitory effects.

[0143] Suitablely, an IL-33 antagonist is a reduced IL-33 binding molecule or a fragment thereof. Suitablely, an IL-33 antagonist is a reduced IL-33 antibody or a binding fragment thereof, and suitablely, an anti-reduced IL-33 antibody or a binding fragment thereof.

[0144] Suitablely, the binding molecule or a fragment thereof binds to redIL-33 with a binding affinity (Kd) of less than 5 x 10-1. -2 M, 10 -2 M, 5 x 10 -3 M, 10 -3 M, 5 x 10 -4 M, 10 -4 M, 5 x 10 -5 M, 10 -5 M, 5 x 10 -6 M, 10 -6 M, 5x 10 -7 M, 10 -7 M, 5 x 10 -8 M, 10 -8 M, 5 x 10 -9 M, 10 -9 M, 5 x 10 -10 M, 10 -10 M, 5 x 10 -11 M, 10 -11 M, 5 x 10 -12 M, 10 -12 M, 5 x 10 -13 M, 10 -13 M, 5 x 10 -14 M, 10 -14 M, 5 x 10 -15 M or 10 -15 M. Suitablely, the binding affinity with redIL-33 is less than 5 x 10⁻⁶. -14 M (i.e., 0.05 pM). Suitablely, a kinetic exclusion assay (KinExA) or BIACORE is used. TMKinExA was suitably used, and binding affinity was measured using a protocol such as that described in WO 2016 / 156440 (see, for example, Example 11), which is incorporated herein by reference in its entirety. The binding molecule that binds to redIL-33 with this binding affinity appears to bind tightly enough to prevent dissociation of the binding molecule / redIL-33 complex within a biologically relevant timeframe. Not wishing to be bound by theory, it is assumed that this binding strength prevents the release of the antigen before the antibody / antigen complex degrades in vivo, thus preventing the release of redIL-33 and the conversion from redIL-33 to oxIL-33. Therefore, when the binding molecule binds to redIL-33 with this binding affinity, it can inhibit or attenuate the activity of oxIL-33 by preventing the formation of oxIL-33, thereby inhibiting RAGE signaling.

[0145] Suitablely, the binding molecules or fragments thereof can be at a ratio of 10 or greater. 3 M -1 sec -1 5 x 10 3 M -1 sec -1 10 4 M - 1 sec -1 Or 5 x 10 4 M -1 sec -1 The binding rate (k(on)) of the molecules specifically binds to redIL-33. For example, the binding molecules disclosed herein can bind at a rate greater than or equal to 10. 5 M -1 sec -1 5 x 10 5 M -1 sec -1 10 6 M -1 sec -1 Or 5 x 10 6 M -1 sec -1 Or 10 7 M - 1 sec -1 The binding rate (k(on)) with redIL-33 or its fragments or variants. Suitablely, the k(on) rate is greater than or equal to 10. 7 M -1 sec -1 .

[0146] Suitablely, the binding molecules or fragments thereof can be less than or equal to 5 x 10-1 sec -1 10 -1 sec -1 5 x 10 - 2 sec -1 10 -2 sec -1 5 x 10 -3 sec -1 Or 10 -3 sec -1 The dissociation rate (k(off)) of the redIL-33-specific binding is such that the molecule can bind at a rate less than or equal to 5 x 10⁻⁶. -4 sec -1 10 -4 sec -1 5 x 10 -5 sec -1 10 - 5 sec -1 5 x 10 -6 sec -1 10 -6 sec -1 5 x 10 -7 sec -1 Or 10 -7 sec -1 The dissociation rate (k(off)) of redIL-33 or its fragments or variants binds to it. Suitablely, the k(off) rate is less than or equal to 10. -3 sec -1 IL-33 is an alarm protein cytokine released rapidly and at high concentrations in response to inflammatory stimuli. Approximately 5–45 minutes after release into the extracellular environment, redIL-33 is converted to its oxidized form. Therefore, to prevent the conversion of redIL-33 to oxIL-33, the binding molecules described herein can bind to redIL-33 at these k(on) and / or k(off) rates. Without being bound by theory, it is assumed that these k(on) / k(off) rates ensure that the binding molecules can rapidly bind to redIL-33 before its conversion to oxIL-33, thereby reducing the formation of oxIL-33, thus attenuating RAGE signaling, suitably attenuating RAGE / EGFR signaling, and thus weakening the RAGE / EGFR-mediated effect.

[0147] Suitablely, IL-33 binding molecules can competitively inhibit the binding of IL-33 to binding molecule 33_640087-7B (as described in WO 2016 / 156440). Suitablely, WO 2016 / 156440 describes 33_640087-7B binding to redIL-33 with a particularly high affinity and attenuating ST-2 and RAGE-dependent IL-33 signaling. Therefore, binding molecules that competitively inhibit the binding of IL-33 to binding molecule 33_640087-7B are highly likely to inhibit both redIL-33 and oxIL-33 signaling, and are therefore particularly suitable for the methods described herein.

[0148] If a binding molecule or fragment thereof binds to a given epitope to a degree that blocks the binding of a reference antibody to that epitope, then the binding molecule or fragment thereof is said to competitively inhibit the binding of the reference antibody to that epitope. Competitive inhibition can be determined by any method known in the art, such as solid-phase assays (e.g., competitive ELISA assays), dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA). ® PerkinElmer (PerkinElmer) and radioligand binding assays. For example, a technician can determine whether a binding molecule or fragment thereof competitively binds to redIL-33 by using an in vitro competitive binding assay, such as a derivative assay of the HTRF assay described in Example 1 of WO 2016 / 156440, which is incorporated herein by reference. For example, a technician can label the recombinant antibody of Table 1 with a donor fluorophore and mix samples of redIL-33 labeled with a fixed concentration of the receptor fluorophore. Subsequently, the fluorescence resonance energy transfer between the donor and receptor fluorophores within each sample can be measured to determine the binding characterization. To elucidate the competitive binding molecule, a technician can first mix various concentrations of the test binding molecule with a fixed concentration of the labeled antibody of Table 1. When the mixture is incubated with labeled IL-33, a reduction in the FRET signal compared to a positive control labeled only with antibody indicates competitive binding to IL-33. It can be stated that the binding molecule or fragment thereof competitively inhibits the binding of the reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0149] In some embodiments, the binding molecule is selected from any of the following anti-IL-33 antibodies: 33_640087-7B (as described in WO 2016 / 156440), ANB020 of Etokimab (as described in WO 2015 / 106080), 9675P (as described in US 2014 / 0271658), A25-3H04 (as described in US 2017 / 0283494), Ab43 (as described in WO 2018 / 081075), IL33-158 (as described in US 2018 / 0037644), 10C12.38.H6.87Y.581lgG4 (as described in WO 2016 / 077381), or a binding fragment thereof, each of which is incorporated herein by reference. These antibodies are all mentioned in Table 1.

[0150] Suitablely, IL-33 antagonists are antibodies or antigen-binding fragments comprising complementarity-determining regions (CDRs) selected from the variable heavy chain domain (VH) and variable light chain domain (VL) pairs listed in Table 1. Pair 1 corresponds to the VH and VL domain sequences of 33_640087-7B as described in WO 2016 / 156440. Pairs 2-7 correspond to the VH and VL domain sequences of the antibody described in US 2014 / 0271658. Pairs 8-12 correspond to the VH and VL domain sequences of the antibody described in US 2017 / 0283494. Pair 13 corresponds to the VH and VL domain sequences of ANB020 as described in WO 2015 / 106080. Pairs 14-16 correspond to the VH and VL domain sequences of the antibody described in WO 2018 / 081075. Pair 17 corresponds to the VH and VL domain sequence of IL33-158 as described in US 2018 / 0037644. Pair 18 corresponds to the VH and VL domain sequence of 10C12.38.H6. 87Y.581 lgG4 as described in WO 2016 / 077381.

[0151] Table 1: Exemplary anti-IL-33 antibodies VH and VL against [various antibodies] .

[0152] Suitablely, IL-33 antagonists are antibodies or antigen-binding fragments comprising: a complementarity-determining region (CDR) of the heavy chain variable region (HCVR) comprising the sequence of SEQ ID NO:1, and a complementarity-determining region (CDR) of the light chain variable region (LCVR) comprising the sequence of SEQ ID NO:19. These CDRs correspond to CDRs derived from 33_640087-7B (as described in WO 2016 / 156440), which bind to reduced IL-33 and inhibit its conversion to oxidized IL-33. 33_640087-7B is fully described in WO 2016 / 156440, which is incorporated herein by reference.

[0153] Suitablely, IL-33 antagonists are antibodies or antigen-binding fragments comprising: complementarity-determining regions (CDRs) of the heavy chain variable region (HCVR) comprising the sequence of SEQ ID NO:7, and complementarity-determining regions (CDRs) of the light chain variable region (LCVR) comprising the sequence of SEQ ID NO:25. These CDRs correspond to the CDRs derived from antibody 9675P. 9675P is fully described in US 2014 / 0271658, which is incorporated herein by reference.

[0154] Suitablely, IL-33 antagonists are antibodies or antigen-binding fragments comprising: a complementarity-determining region (CDR) of the heavy chain variable region (HCVR) comprising the sequence of SEQ ID NO:11, and a complementarity-determining region (CDR) of the light chain variable region (LCVR) comprising the sequence of SEQ ID NO:29. These CDRs correspond to the CDRs derived from antibody A25-3H04. A25-3H04 is fully described in US 2017 / 0283494, which is incorporated herein by reference.

[0155] Suitablely, an IL-33 antagonist is an antibody or antigen-binding fragment comprising: a complementarity-determining region (CDR) of the heavy chain variable region (HCVR) comprising the sequence of SEQ ID NO:13, and a complementarity-determining region (CDR) of the light chain variable region (LCVR) comprising the sequence of SEQ ID NO:31. These CDRs correspond to the CDRs derived from the antibody ANB020, which is fully described in WO 2015 / 106080, and is incorporated herein by reference.

[0156] Suitablely, an IL-33 antagonist is an antibody or antigen-binding fragment comprising: a complementarity-determining region (CDR) of the heavy chain variable region (HCVR) comprising the sequence of SEQ ID NO:16, and a complementarity-determining region (CDR) of the light chain variable region (LCVR) comprising the sequence of SEQ ID NO:34. These CDRs correspond to the CDRs derived from antibody Ab43, which is fully described in WO 2018 / 081075 and is incorporated herein by reference.

[0157] Suitablely, IL-33 antagonists are antibodies or antigen-binding fragments comprising: a complementarity-determining region (CDR) of the heavy chain variable region (HCVR) comprising the sequence of SEQ ID NO:17, and a complementarity-determining region (CDR) of the light chain variable region (LCVR) comprising the sequence of SEQ ID NO:35. These CDRs correspond to the CDRs derived from the antibody IL33-158. IL33-158 is fully described in US 2018 / 0037644, which is incorporated herein by reference.

[0158] Suitablely, the IL-33 binding molecule is an antibody or antigen-binding fragment comprising: a complementarity-determining region (CDR) of the heavy chain variable region (HCVR) containing the sequence of SEQ ID NO:18, and a complementarity-determining region (CDR) of the light chain variable region (LCVR) containing the sequence of SEQ ID NO:36. These CDRs correspond to the CDRs derived from antibody 10C12.38.H6.87Y.581 lgG4. 10C12.38.H6.87Y.581 lgG4 is fully described in WO 2016 / 077381, which is incorporated herein by reference.

[0159] Suitablely, those skilled in the art are familiar with methods available in the art for identifying CDRs within the variable regions of the heavy and light chains of antibodies or their antigen-binding fragments. Suitablely, those skilled in the art can perform, for example, sequence-based annotation. Regions between CDRs are generally highly conserved, and therefore, logical rules can be used to determine CDR locations. Those skilled in the art can use a set of sequence-based rules for routine antibodies (Pantazes and Maranas, Protein Engineering, Design and Selection, 2010), or alternatively, they can refine the rules based on multiple sequence alignments. Alternatively, those skilled in the art can use the BLASTP instructions of BLAST+ to compare the antibody sequence with publicly available databases operated according to the Kabat, Chothia, or IMGT methods to identify the most similar annotated sequence. Each of these methods employs a unique residue numbering scheme according to which the residues in the hypervariable region are numbered, and then the start and end of each of the six CDRs are determined based on certain key positions. After, for example, alignment with the most similar annotated sequence, the CDRs can be extrapolated from the annotated sequence to the non-annotated sequence, thereby identifying the CDRs. Suitable tools / databases include: Kabat database, Kabatman, Scalinger, IMGT, and Abnum.

[0160] Suitablely, an IL-33 antagonist is an antibody or antigen-binding fragment comprising a pair of variable heavy chain domains (VH) and variable light chain domains (VL) selected from Table 1.

[0161] Suitablely, the IL33 antibody or its antigen-binding fragment comprises the VH domain of the sequence of SEQ ID NO:1 and the VL domain of the sequence of SEQ ID NO:19.

[0162] Suitablely, the IL33 antibody or its antigen-binding fragment comprises the VH domain of the sequence of SEQ ID NO:7 and the VL domain of the sequence of SEQ ID NO:25.

[0163] Suitablely, the IL33 antibody or its antigen-binding fragment comprises the VH domain of the sequence of SEQ ID NO:11 and the VL domain of the sequence of SEQ ID NO:29.

[0164] Suitablely, the IL33 antibody or its antigen-binding fragment comprises the VH domain of the sequence of SEQ ID NO:13 and the VL domain of the sequence of SEQ ID NO:31.

[0165] Suitablely, the IL33 antibody or its antigen-binding fragment comprises the VH domain of the sequence of SEQ ID NO:16 and the VL domain of the sequence of SEQ ID NO:34.

[0166] Suitablely, the IL33 antibody or its antigen-binding fragment comprises the VH domain of the sequence of SEQ ID NO:17 and the VL domain of the sequence of SEQ ID NO:35.

[0167] Therefore, suitablely, an IL-33 antagonist is a binding molecule that may contain, for example, three CDRs independently selected from the heavy chain variable regions of SEQ ID NO:1, 7, 11, 13, 16, 17 and 18.

[0168] Suitablely, IL-33 antagonists are binding molecules comprising three CDRs in the heavy chain variable region according to SEQ ID NO:1.

[0169] Suitablely, an IL-33 antagonist is a binding molecule that may contain three CDRs independently selected from the light chain variable regions of SEQ ID NO: 19, 25, 29, 31, 34, 35 and 36.

[0170] Suitablely, IL-33 antagonists are binding molecules comprising three CDRs in the light chain variable region according to SEQ ID NO:19.

[0171] Therefore, suitablely, the IL-33 antagonist is a binding molecule that may comprise, for example, three CDRs independently selected from the heavy chain variable regions of SEQ ID NO:1, 7, 11, 13, 16, 17 and 18, and, for example, three CDRs independently selected from the light chain variable regions of SEQ ID NO:19, 25, 29, 31, 34, 35 and 36.

[0172] Therefore, suitably, the IL-33 antagonist is a binding molecule comprising three CDRs in the heavy chain variable region according to SEQ ID NO:1 and three CDRs in the light chain variable region according to SEQ ID NO:19.

[0173] Therefore, suitablely, IL-33 antagonists are binding molecules that may contain a variable heavy chain domain (VH) and a variable light chain domain (VL), the binding molecule having VH CDRs 1-3 having sequences SEQ ID NO:37, 38 and 39 respectively, wherein one or more VHCDRs have three or fewer single amino acid substitutions, insertions and / or deletions.

[0174] Therefore, suitablely, IL-33 antagonists are binding molecules containing a VH domain comprising VHCDR 1-3 having SEQ ID NO:37, SEQ ID NO:38 and SEQ ID NO:39 respectively.

[0175] Therefore, suitablely, IL-33 antagonists are binding molecules containing a VH domain comprising VHCDR 1-3 composed of SEQ ID NO:37, SEQ ID NO:38 and SEQ ID NO:39, respectively.

[0176] Therefore, suitablely, IL-33 antagonists are binding molecules that may contain a variable heavy chain domain (VH) and a variable light chain domain (VL), the binding molecule having VL CDRs 1-3 having sequences of SEQ ID NO:40, 41 and 42, respectively, wherein one or more VLCDRs have three or fewer single amino acid substitutions, insertions and / or deletions.

[0177] Therefore, suitablely, IL-33 antagonists are binding molecules containing a VL domain comprising VLCDR 1-3 having SEQ ID NO:40, SEQ ID NO:41, and SEQ ID NO:42, respectively.

[0178] Therefore, suitablely, IL-33 antagonists are binding molecules containing a VL domain comprising VLCDR 1-3 composed of SEQ ID NO:40, SEQ ID NO:41 and SEQ ID NO:42, respectively.

[0179] Therefore, suitably, the IL-33 antagonist is a binding molecule that may comprise VHCDR1 having the sequence of SEQ ID NO:37, VHCDR2 having the sequence of SEQ ID NO:38, VHCDR3 having the sequence of SEQ ID NO:39, VLCDR1 having the sequence of SEQ ID NO:40, VLCDR2 having the sequence of SEQ ID NO:41, and VLCDR3 having the sequence of SEQ ID NO:42.

[0180] Therefore, suitably, an IL-33 antagonist is an antibody comprising VH and VL or a binding fragment thereof, wherein VH has an amino acid sequence that is at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% consistent with VH according to SEQ ID NO:1, 7, 11, 13, 16, 17, and 18.

[0181] Therefore, suitably, an IL-33 antagonist is an antibody or a binding fragment thereof comprising VH and VL, wherein VH has an amino acid sequence that is at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to VH according to SEQ ID NO:1.

[0182] Therefore, suitablely, an IL-33 antagonist is an antibody or a binding fragment thereof comprising VH and VL, wherein the VH disclosed above has a sequence in which 1, 2, 3 or 4 amino acids in the frame are deleted, inserted with different amino acids and / or substituted independently.

[0183] Therefore, suitably, an IL-33 antagonist is an antibody comprising VH and VL or a binding fragment thereof, wherein VL has an amino acid sequence that is at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to VL according to SEQ ID NO:19, 25, 29, 31, 34, 35, and 36.

[0184] Therefore, suitably, an IL-33 antagonist is an antibody comprising VH and VL or a binding fragment thereof, wherein VL has an amino acid sequence that is at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to VL according to SEQ ID NO:19.

[0185] Therefore, suitablely, an IL-33 antagonist is an antibody or a binding fragment thereof comprising VH and VL, wherein the VL disclosed above has a sequence in which 1, 2, 3 or 4 amino acids in the frame are independently deleted, inserted and / or substituted with different amino acids.

[0186] Therefore, suitably, an IL-33 antagonist is an antibody comprising VH and VL or a binding fragment thereof, wherein VH has an amino acid sequence that is at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, identical to that of VH according to SEQ ID NO: 1, 7, 11, 13, 16, 17, and 18, and VL has an amino acid sequence that is at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, identical to that of VL according to SEQ ID NO: 19, 25, 29, 31, 34, 35, and 36.

[0187] Therefore, suitably, an IL-33 antagonist is an antibody comprising VH and VL or a binding fragment thereof, wherein VH has an amino acid sequence consisting of SEQ ID NO:1, 7, 11, 13, 16, 17 and 18, and VL has an amino acid sequence consisting of SEQ ID NO:19, 25, 29, 31, 34, 35 and 36.

[0188] Therefore, suitably, an IL-33 antagonist is an antibody or a binding fragment thereof comprising VH and VL, wherein VH has an amino acid sequence consisting of SEQ ID NO:1 and VL has an amino acid sequence consisting of SEQ ID NO:19.

[0189] Composition and application The IL-33 antagonists described in this article for medical uses and methods can be administered to patients in the form of pharmaceutical compositions.

[0190] Suitablely, any reference to "IL-33 antagonist" in this document may also refer to a pharmaceutical composition comprising an IL-33 antagonist. Suitablely, a pharmaceutical composition may comprise one or more IL-33 antagonists.

[0191] Suitablely, IL-33 antagonists may be administered in an effective amount for the in vivo treatment of epithelial physiological abnormalities, EGFR-mediated diseases, or respiratory diseases as defined in the medical uses and methods of treatment described herein.

[0192] Where appropriate, the “pharmacologically effective amount” or “therapeuticly effective amount” of an IL-33 antagonist should be understood as an amount sufficient to achieve effective binding with IL-33 and to provide benefits, such as improving symptoms of a disease or condition as described in the medical uses / methods herein.

[0193] Suitablely, IL-33 antagonists or pharmaceutical compositions thereof may be administered to humans or other animals in an amount sufficient to produce a therapeutic effect, in accordance with the aforementioned treatment methods / medical uses.

[0194] Suitablely, IL-33 antagonists or pharmaceutical compositions thereof can be administered to such humans or other animals in conventional dosage forms prepared by combining an IL-33 antagonist with a conventionally pharmaceutically acceptable carrier or diluent according to known techniques.

[0195] Those skilled in the art will recognize that the form and characteristics of a pharmaceutically acceptable carrier or diluent are determined by the amount of the active ingredient in combination with it, the route of administration, and other well-known variables. Those skilled in the art will further understand that mixtures containing one or more IL-33 antagonists can prove to be particularly effective.

[0196] The amount of IL-33 antagonist that can be combined with a carrier material to produce a single dosage form will vary depending on the subject being treated and the specific route of administration. Suitably, the pharmaceutical composition can be administered as a single dose, multiple doses, or via infusion over a defined period of time. Suitably, the dosing regimen can also be adjusted to provide the best desired response (e.g., a therapeutic or preventative response).

[0197] Suitable IL-33 antagonists are formulated to facilitate the application of IL-33 antagonists and enhance their stability.

[0198] Suitable pharmaceutical compositions are formulated to contain pharmaceutically acceptable, non-toxic, sterile carriers, such as physiological saline, non-toxic buffers, preservatives, etc.

[0199] Suitablely, the pharmaceutical composition may comprise a pharmaceutically acceptable carrier, including, for example, water; an ion exchanger; alumina; aluminum stearate; lecithin; serum proteins, such as human serum albumin; buffering substances, such as phosphates; glycine; sorbic acid; potassium sorbate; a mixture of glycerides of saturated vegetable fatty acids; water, salts, or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts; colloidal silica; magnesium trisilicate; polyvinylpyrrolidone; cellulose-based substances; polyethylene glycol; sodium carboxymethyl cellulose; polyacrylates; waxes; polyethylene-polyoxypropylene block polymers; polyethylene glycol; and lanolin.

[0200] Suitable pharmaceutical compositions may include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include, for example, water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffer media.

[0201] Suitable pharmaceutically acceptable carriers include, but are not limited to, 0.01-0.1 M, preferably 0.05 M, phosphate buffer or 0.8% saline. Other common parenteral carriers include sodium phosphate solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or non-volatile oils. Intravenous carriers include fluids and nutritional supplements, electrolyte supplements, such as those based on Ringer's dextrose. Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, and inert gases, may also be present.

[0202] Suitable pharmaceutical compositions for injectable applications may include sterile aqueous solutions (when soluble in water) or dispersions and sterile powders for the provisional preparation of sterile injectable solutions or dispersions. In such cases, the composition must be sterile and must have a flowability sufficient for easy injection. It should be stable under manufacturing and storage conditions and will be preserved to prevent contamination by microorganisms such as bacteria and fungi. Suitablely, the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Appropriate flowability may be maintained, for example, by using a coating (such as lecithin), by maintaining the desired particle size in the case of a dispersion, and by using a surfactant.

[0203] Suitable formulations for the treatments disclosed herein are described in Remington's Pharmaceutical Sciences (Mack Publishing Co.), 16th edition (1980).

[0204] Suitablely, the inhibition of microbial activity can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, isotonic agents (e.g., sugars), polyols (such as mannitol, sorbitol), or sodium chloride are suitably included in the pharmaceutical composition. Prolonged absorption of the injectable composition can be achieved by including agents that delay absorption (e.g., aluminum monostearate and gelatin) in the composition.

[0205] Suitablely, sterile injectable solutions can be prepared by incorporating a desired amount of an active compound (e.g., an IL-33 antagonist, alone or in combination with other active agents) into a suitable solvent having one or a combination of the components listed herein, followed by filtration sterilization if necessary. Typically, dispersions are prepared by incorporating the active compound into a sterile mediator containing a base dispersion medium and other desired components from the list above. In the case of sterile powders used to prepare sterile injectable solutions, preparation methods can include vacuum drying and freeze-drying, which produce powders of the active ingredient and any other desired components from its previous sterile filtered solution.

[0206] The method of administering an IL-33 antagonist or a pharmaceutical composition thereof to a subject in need is well known to or readily determined by a person skilled in the art.

[0207] Suitablely, the route of administration of an IL-33 antagonist or a pharmaceutical composition thereof may be, for example, oral, parenteral, inhalation, or topical. Suitablely, as used herein, the term parenteral includes, for example, intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration.

[0208] Suitablely, IL-33 antagonists or pharmaceutical compositions thereof may be administered orally in acceptable dosage forms, including, for example, capsules, tablets, aqueous suspensions or solutions.

[0209] Suitablely, IL-33 antagonists or pharmaceutical compositions thereof may be administered via nasal aerosol or inhalation. Such compositions may be formulated into saline solutions using benzyl alcohol or other suitable preservatives, bioavailability enhancers, and / or other conventional solubilizers or dispersants.

[0210] Suitable parenteral preparations may be a single bolus dose, infusion, or loaded bolus dose followed by a maintenance dose. These compositions may be administered at specific fixed or variable intervals, such as once daily, or on an "as needed" basis.

[0211] Suitablely, an IL-33 antagonist or a pharmaceutical composition thereof can be delivered directly to the site of a disease or condition, such as an epithelial physiological abnormality, thereby increasing the exposure of the diseased tissue to the therapeutic agent. Suitablely, an IL-33 antagonist or a pharmaceutical composition thereof can be applied directly to the site of a disease or condition. Therefore, suitablely, an IL-33 antagonist or a pharmaceutical composition thereof can be applied to the site of an epithelial physiological abnormality, an EGFR-mediated disease, or a respiratory disease.

[0212] In one embodiment, the IL-33 antagonist or a pharmaceutical composition thereof is administered to the respiratory tract. Suitablely, it is administered intranasally. Suitablely, it is inhaled intranasally. Suitablely, the IL-33 antagonist or a pharmaceutical composition thereof may be provided in an inhaler device. Suitable inhaler devices are well known in the art.

[0213] In one embodiment, an inhaler comprising an IL-33 antagonist or a pharmaceutical composition thereof is provided for the prevention or treatment of a condition or disease as defined herein.

[0214] Therefore, it is suitable to formulate IL-33 antagonists or pharmaceutical compositions thereof into liquid compositions. Suitablely, they are in the form of liquid compositions that can be atomized.

[0215] In one embodiment, the IL-33 antagonist or a pharmaceutical composition thereof is provided in the form of an aerosol.

[0216] Suitablely, the components described above for preparing the pharmaceutical compositions described herein can be packaged and sold in the form of a kit. Such a kit may suitably have a label or package insert indicating that the relevant pharmaceutical composition may be used to treat subjects who have or are predisposed to have a disease or disorder.

[0217] Suitablely, the liquid formulation is treated with components, filled into containers such as ampoules, bags, bottles, syringes, or vials, and sealed under aseptic conditions according to methods known in the art. Suitablely, the container may be pressurized, and suitablely, it may be an aerosol container. These containers may be included in the above-described kit. Suitablely, the kit may further include an inhaler device. Suitablely, the inhaler device contains the IL-33 antagonist or pharmaceutical composition described herein, or is operable to contain the above-described container that may contain the IL-33 antagonist or pharmaceutical composition described herein.

[0218] Epithelial Physiological Abnormalities This disclosure relates to the medical use of IL-33 antagonists for the prevention or treatment of epithelial physiological abnormalities.

[0219] As used herein, "epithelial physiological abnormalities" refers to any abnormality in the function of the human epithelium. The functions of the human epithelium include: acting as a barrier protecting underlying tissues; regulating and exchanging chemical substances between tissues and cavities; secreting chemicals into cavities; and sensation. Abnormalities in any of these functions can have destructive physiological effects. Epithelium is present in many tissues of the body, including the skin, respiratory tract, gastrointestinal tract, reproductive tract, urinary tract, exocrine glands, and endocrine glands; therefore, abnormalities within the epithelium can be associated with a variety of diseases or conditions. Suitablely, the epithelium is the airway epithelium, and epithelial physiological abnormalities are airway epithelial physiological abnormalities.

[0220] As used herein, “abnormal” refers to a difference in function compared to that in healthy subjects, typically an increase or decrease in function compared to that in healthy subjects.

[0221] Suitable epithelium is selected from squamous, cuboidal, columnar, and pseudostratified epithelium. Suitable epithelium is columnar epithelium.

[0222] Suitablely, the epithelium is ciliated epithelium. Suitablely, the epithelium is ciliated columnar epithelium. Suitablely, the epithelial physiological abnormality is a ciliated columnar epithelial physiological abnormality.

[0223] Suitablely, epithelial physiological abnormalities include abnormal epithelial cell migration. Suitablely, epithelial physiological abnormalities may include reduced epithelial cell migration. Suitablely, epithelial physiological abnormalities may include abnormal epithelial cell proliferation. Suitablely, epithelial physiological abnormalities may include reduced epithelial cell proliferation.

[0224] Suitablely, reduced epithelial cell migration leads to impaired epithelial ability to repair scratches. Suitablely, epithelial physiological abnormalities include impaired scratch repair. Impaired scratch repair may include impaired scratch closure and reduced scratch cell density.

[0225] Suitable treatment for epithelial physiology abnormalities may include increasing or improving epithelial cell migration. Suitable treatment for epithelial physiology abnormalities may include increasing or improving epithelial scratch repair. Suitable treatment for epithelial physiology abnormalities may include increasing or improving scratch closure. Suitable treatment for epithelial physiology abnormalities may include increasing or improving scratch cell density.

[0226] Appropriately, epithelial physiology abnormalities are abnormal mucociliary physiology.

[0227] As used in this article, "mucociliary physiology abnormalities" refer to functional abnormalities of specific mucociliary functions in the epithelium. These abnormalities may be due to dysfunction of the ciliated columnar cells and / or goblet cells, which are crucial for mucociliary function. Suitablely, mucociliary physiology abnormalities are due to dysfunction of goblet cells.

[0228] As used in this article, "mucosal cilia" refers to the function of ciliated columnar cells and goblet cells in the epithelium in secreting and moving mucus. The mucosal function of the epithelium may include: goblet cell proliferation; goblet cell differentiation; mucus secretion; regulation of mucus composition; and / or mucus movement or clearance.

[0229] In one embodiment, an IL-33 antagonist is provided for the prevention or treatment of mucociliary physiology abnormalities, such as epithelial mucociliary physiology abnormalities.

[0230] In one embodiment, a method is provided for preventing or treating mucociliary physiology abnormalities in a patient, such as epithelial mucociliary physiology abnormalities, the method comprising: administering an effective amount of an IL-33 antagonist to a patient in need.

[0231] Mucociliary physiology abnormalities may include any abnormal function of the epithelial ciliated columnar cells or goblet cells. Suitably, mucociliary physiology abnormalities include: abnormal mucus production; abnormal goblet cell differentiation; abnormal goblet cell proliferation; abnormal epithelial thickness; abnormal mucus clearance; and / or abnormal mucus composition.

[0232] Suitablely, abnormal mucus production includes abnormal MUC5AC production. Suitablely, abnormal goblet cell differentiation includes abnormal MUC5AC+ goblet cell differentiation. Suitablely, abnormal goblet cell proliferation includes abnormal MUC5AC+ goblet cell proliferation. Suitablely, abnormal epithelial thickness includes abnormal MUC5AC production in the total tissue area of ​​the epithelium. + The number of goblet cells is abnormal.

[0233] Suitable mucociliary physiology abnormalities include: increased number of goblet cells; increased mucus production; increased goblet cell differentiation; increased epithelial thickness; and / or decreased mucus clearance.

[0234] Suitablely, increased mucus production includes increased MUC5AC production. Suitablely, increased goblet cell differentiation includes increased MUC5AC+ goblet cell differentiation. Suitablely, increased goblet cell proliferation includes increased MUC5AC+ goblet cell proliferation. Suitablely, increased epithelial thickness includes increased MUC5AC production in the total tissue region of the epithelium. + The number of goblet cells increases.

[0235] Suitable, MUC5AC generates an increase from MUC5AC Increased gene expression causes this. Suitablely, mucosal ciliary physiology abnormalities include those in epithelial cells. MUC5AC Increased gene expression. Suitablely, mucosal ciliary physiology abnormalities include goblet cells in the epithelium. MUC5AC The expression "to increase" has increased.

[0236] Suitablely, mucociliary physiology abnormalities include changes in the composition of mucus. Such changes may include an increase or decrease in the ratio of different mucus compounds in the mucus, an increase or decrease in one or more specific mucus compounds, or an increase or decrease in the concentration or thickness of the mucus.

[0237] Changes in mucus composition can include increases or decreases in the ratio of different mucins, such as an increase or decrease in the ratio of mucins MUC5AC and MUC5B.

[0238] Changes in mucus composition can include an increase or decrease in the concentration of mucin. Suitablely, changes in mucus composition include a decrease in the concentration of mucin 5AC. Suitablely, changes in mucus composition include a decrease in the number of goblet cells with upregulated MUC5AC expression.

[0239] Such changes in the mucin contained in mucus can be measured and calculated as described in WO 2018 / 204598, which is incorporated herein by reference.

[0240] Suitablely, mucus compositional anomalies include an increased MUC5AC:MUC5B ratio. Suitablely, mucus compositional anomalies include an increased MUC5AC content in the mucus. Suitablely, mucus compositional anomalies include an increased mucus thickness.

[0241] Mucociliary physiology abnormalities may include any one or a combination of the above symptoms.

[0242] Where appropriate, epithelial physiological abnormalities include abnormalities in tissue remodeling, such as epithelial remodeling abnormalities. Where appropriate, epithelial physiological abnormalities include increased tissue remodeling. Where appropriate, epithelial physiological abnormalities include increased epithelial remodeling.

[0243] Epithelial physiological abnormalities can include any one or a combination of the above symptoms.

[0244] Treatment or prevention of epithelial physiological abnormalities, or treatment or prevention of mucociliary physiological abnormalities, may include: Improve or enhance mucosal ciliary clearance; Reduce or inhibit mucus production; Inhibit abnormal mucus composition; Reduce or inhibit epithelial remodeling; and / or Reduce or inhibit goblet cell differentiation and / or proliferation.

[0245] Suitablely, reducing or inhibiting mucus production includes reducing or inhibiting MUC5AC production. Therefore, suitablely, the treatment or prevention reduces or inhibits MUC5AC production.

[0246] Suitablely, inhibiting abnormal mucus composition may include restoring normal mucus composition. Suitablely, this may include reducing the MUC5AC:MUC5B ratio. Therefore, suitablely, the treatment or prevention reduces the MUC5AC:MUC5B ratio. Suitablely, the prevention or treatment inhibits or reduces MUC5AC in the mucus. Suitablely, the prevention or treatment reduces the thickness of the mucus.

[0247] Suitable methods for reducing or inhibiting goblet cell differentiation and / or proliferation include reducing or inhibiting MUC5AC. + Goblet cell differentiation or proliferation. Therefore, suitably, this treatment or prevention reduces or inhibits MUC5AC. + Differentiation or proliferation of goblet cells.

[0248] Suitablely, reducing or inhibiting epithelial remodeling involves reducing the thickness of the respiratory epithelium. Therefore, suitablely, the treatment or prevention reduces the thickness of the respiratory epithelium.

[0249] Suitablely, reducing or inhibiting epithelial remodeling involves reducing MUC5AC in the total tissue region of the epithelium. + The amount of goblet cells. Therefore, suitably, this treatment or prevention reduces or inhibits MUC5AC in the total tissue area of ​​the epithelium. + The number of goblet cells.

[0250] Improving or increasing mucociliary clearance involves improving or increasing mucociliary movement. Therefore, suitably, this treatment or prevention improves or increases mucociliary movement.

[0251] Suitablely, the epithelium is the respiratory epithelium. Suitablely, the epithelial physiology abnormality is an epithelial physiology abnormality within the respiratory epithelium.

[0252] In one embodiment, an IL-33 antagonist is provided for treating epithelial physiological abnormalities in respiratory diseases.

[0253] In one embodiment, a method for preventing or treating epithelial physiological abnormalities in a patient with a respiratory disease is provided, the method comprising: administering an effective amount of an IL-33 antagonist to the patient in need.

[0254] The appropriate respiratory diseases are as defined elsewhere in this article.

[0255] Appropriately, epithelial physiology abnormalities are mucociliary physiology abnormalities in the respiratory epithelium.

[0256] In one embodiment, an IL-33 antagonist is provided for the prevention or treatment of mucociliary physiological abnormalities in the respiratory epithelium.

[0257] In one embodiment, a method for preventing or treating mucociliary physiology abnormalities of the respiratory epithelium in a patient is provided, the method comprising: administering an effective amount of an IL-33 antagonist to a patient in need.

[0258] Appropriately, epithelial physiology abnormalities are mucociliary physiology abnormalities in respiratory diseases.

[0259] In one embodiment, an IL-33 antagonist is provided for the prevention or treatment of mucociliary physiology abnormalities in respiratory diseases.

[0260] In one embodiment, a method for preventing or treating mucociliary physiology abnormalities in a patient with a respiratory disease is provided, the method comprising: administering an effective amount of an IL-33 antagonist to the patient in need.

[0261] Suitablely, epithelial physiology abnormalities exist in the respiratory tract. Suitablely, epithelial physiology abnormalities are epithelial physiology abnormalities of the respiratory tract. Suitablely, epithelial physiology abnormalities are mucociliary physiology abnormalities of the respiratory tract.

[0262] The respiratory tract comprises the upper and lower respiratory tracts. Typically, the upper respiratory tract includes the nasal passages, paranasal sinuses, pharynx, and larynx. Typically, the lower respiratory tract includes the trachea, bronchi, bronchioles, alveolar ducts, and alveoli.

[0263] Appropriately, epithelial physiology abnormalities are those of the lower respiratory tract, such as the bronchi.

[0264] Suitablely, epithelial physiology abnormalities are epithelial physiology abnormalities of the lower respiratory tract. Suitablely, epithelial physiology abnormalities are epithelial physiology abnormalities of the bronchi. Suitablely, lower respiratory tract epithelial physiology abnormalities are mucociliary physiology abnormalities of the lower respiratory tract. Suitablely, lower respiratory tract mucociliary physiology abnormalities are mucociliary physiology abnormalities of the bronchi.

[0265] In one embodiment, an IL-33 antagonist is provided for the prevention or treatment of mucociliary physiology abnormalities of the lower respiratory tract.

[0266] In one embodiment, a method for preventing or treating mucociliary physiology abnormalities of the lower respiratory tract in a patient is provided, the method comprising: administering an effective amount of an IL-33 antagonist to the patient in need.

[0267] In one embodiment, an IL-33 antagonist is provided for the prevention or treatment of bronchial mucociliary physiology abnormalities.

[0268] In one embodiment, a method for preventing or treating mucociliary physiology abnormalities of the bronchus in a patient is provided, the method comprising: administering an effective amount of an IL-33 antagonist to the patient in need.

[0269] EGFR signal transduction This disclosure is based on the finding that oxidized IL-33 binds to RAGE, which in turn complexes with EGFR and disrupts epithelial homeostasis. Using IL-33 antagonists can inhibit the signaling of oxidized IL-33, thereby inhibiting RAGE activation and suppressing RAGE-EGFR complexation. The data disclosed herein demonstrate that preventing the formation of the RAGE-EGFR complex blocks IL-33-mediated EGFR signaling and restores normal epithelial physiology.

[0270] Suitablely, IL-33 antagonists inhibit oxidized IL-33 signaling.

[0271] Suitablely, IL-33 antagonists inhibit the binding of oxidized IL-33 to RAGE.

[0272] Suitablely, IL-33 antagonists inhibit the formation of the RAGE-EGFR complex. Suitablely, IL-33 antagonists inhibit the formation of the oxidized IL33-RAGE-EGFR complex.

[0273] Suitablely, IL-33 antagonists inhibit EGFR clustering. Suitablely, IL-33 antagonists inhibit EGFR clustering in the cell membrane. Suitablely, IL-33 antagonists inhibit EGFR internalization. Suitablely, IL-33 antagonists inhibit the co-localization of RAGE and EGFR in the cell membrane. Suitablely, IL-33 antagonists inhibit the internalization of the RAGE-EGFR complex.

[0274] Suitablely, IL-33 antagonists inhibit EGFR activation. Suitablely, IL-33 antagonists inhibit EGFR phosphorylation.

[0275] Suitablely, IL-33 antagonists inhibit RAGE-EGFR-mediated effects. Suitablely, IL-33 antagonists inhibit effects mediated by the RAGE-EGFR complex. Suitablely, IL-33 antagonists inhibit effects mediated by the oxidized IL33-RAGE-EGFR complex.

[0276] Suitablely, IL-33 antagonists inhibit EGFR signaling. Suitablely, IL-33 antagonists inhibit RAGE-EGFR signaling. Suitablely, IL-33 antagonists inhibit oxidized IL33-RAGE-EGFR signaling.

[0277] Suitablely, IL-33 antagonists inhibit the binding of oxidized IL-33 to RAGE, thereby inhibiting RAGE-EGFR complexation and thus inhibiting RAGE-EGFR-mediated effects, such as downstream signaling.

[0278] Suitablely, IL-33 antagonists inhibit IL-33-mediated EGFR activity. Suitablely, IL-33 antagonists inhibit IL-33-mediated EGFR signaling. Suitablely, IL-33 antagonists inhibit oxidized IL-33-mediated EGFR activity. Suitablely, IL-33 antagonists inhibit oxidized IL-33-mediated EGFR signaling. Suitablely, IL-33 antagonists inhibit oxidized IL-33-mediated RAGE-EGFR activity. Suitablely, IL-33 antagonists inhibit oxidized IL-33-mediated RAGE-EGFR signaling.

[0279] Suitablely, the RAGE-EGFR-mediated effect is caused by the RAGE-EGFR complex, and suitablely by the oxidized IL-33-RAGE-EGFR complex.

[0280] Suitablely, such effects may typically include downstream signaling, which may be referred to herein as EGFR signaling or RAGE-EGFR signaling. Suitablely, such EGFR signaling may include phosphorylation and / or chemokine release.

[0281] Suitablely, this EGFR signaling includes phosphorylation of EGFR and subsequent phosphorylation of components in the EGFR pathway such as EGFR, PLC, JNK, MAPK / ERK 1 / 2, p38, and STAT5. Suitablely, EGFR signaling includes phosphorylation of tyrosine kinases such as JNK, MAPK / ERK, and p38.

[0282] Suitablely, EGFR signaling involves an increased release of chemokines such as IL-8.

[0283] Therefore, IL-33 antagonists suitably inhibit EGFR-mediated phosphorylation and / or chemokine release.

[0284] Therefore, suitably, IL-33 antagonists inhibit phosphorylation of components in the EGFR pathway. Suitably, IL-33 antagonists inhibit phosphorylation of any of the following: EGFR, PLC, JNK, MAPK / ERK 1 / 2, p38, and STAT5. Suitably, IL-33 antagonists inhibit EGFR-mediated phosphorylation of any of the following: EGFR, PLC, JNK, MAPK / ERK 1 / 2, p38, and STAT5. Suitably, IL-33 antagonists inhibit phosphorylation of tyrosine kinases. Suitably, IL-33 antagonists inhibit phosphorylation of tyrosine kinases selected from: JNK, MAPK / ERK, and p38. Suitably, IL-33 antagonists inhibit EGFR-mediated phosphorylation of tyrosine kinases selected from: JNK, MAPK / ERK, and p38.

[0285] Therefore, suitably, IL-33 antagonists inhibit the release of chemokines. Suitably, IL-33 antagonists inhibit the release of IL-8. Suitably, IL-33 antagonists inhibit the release of EGFR-mediated chemokines. Suitably, IL-33 antagonists inhibit the release of EGFR-mediated IL-8.

[0286] In one embodiment, an IL-33 antagonist is provided for the prevention or treatment of EGFR-mediated diseases.

[0287] In another embodiment, IL-33 antagonists can be used to prevent or treat respiratory diseases by inhibiting EGFR-mediated effects.

[0288] In addition, IL-33 antagonists can be used to prevent or treat epithelial physiological abnormalities in EGFR-mediated diseases.

[0289] In one embodiment, an IL-33 antagonist is provided for the prevention or treatment of EGFR-mediated diseases by improving epithelial physiological abnormalities.

[0290] Suitablely, EGFR-mediated diseases are RAGE-EGFR-mediated diseases.

[0291] Suitablely, the EGFR-mediated effect is the RAGE-EGFR-mediated effect.

[0292] Suitablely, the role of EGFR-mediated signal transduction is the same as that of RAGE-EGFR-mediated signal transduction.

[0293] Suitablely, IL-33 antagonists inhibit EGFR-mediated effects. Suitablely, IL-33 antagonists treat or prevent diseases or conditions by inhibiting EGFR-mediated effects.

[0294] Suitablely, IL-33 antagonists inhibit RAGE-EGFR-mediated effects. Suitablely, IL-33 antagonists treat or prevent diseases or conditions by inhibiting RAGE-EGFR-mediated effects.

[0295] As used herein, “RAGE-EGFR-mediated effects” refers to any physiological action resulting from the complexation of RAGE and EGFR in the cell membrane and the resulting abnormal EGFR activity. RAGE-EGFR-mediated effects may also include and / or be referred to herein as 'RAGE-EGFR signaling', optionally 'RAGE-EGFR-mediated signaling'. Such RAGE-EGFR-mediated effects are typically observed in the epithelium and exist in the form of epithelial physiological abnormalities. Epithelial physiological abnormalities are defined above but may include negative impacts on: barrier integrity; regulation and exchange of chemical entities between tissues and lumens; secretion of chemical substances into lumens; and sensation.

[0296] Suitablely, RAGE-EGFR-mediated diseases and / or effects are characterized by abnormal EGFR activity. Suitablely, RAGE-EGFR-mediated diseases and / or effects are characterized by abnormal RAGE-EGFR signaling. Suitablely, RAGE-EGFR-mediated effects and / or RAGE-EGFR signaling are characteristic of RAGE-EGFR-mediated diseases.

[0297] Suitablely, RAGE-EGFR-mediated diseases can be those characterized by epithelial physiological abnormalities.

[0298] Suitablely, RAGE-EGFR-mediated diseases can be those characterized by epithelial physiology abnormalities in the respiratory epithelium.

[0299] Suitablely, RAGE-EGFR-mediated diseases can be those characterized by abnormalities in mucociliary physiology.

[0300] Suitablely, RAGE-EGFR-mediated diseases can be those characterized by abnormal mucociliary physiology in the respiratory epithelium.

[0301] Suitable RAGE-EGFR-mediated diseases can be selected from any of the respiratory diseases defined below.

[0302] Respiratory diseases This disclosure relates to the medical use of IL-33 antagonists for the prevention or treatment of respiratory diseases by: improving epithelial physiology or modulating EGFR-mediated effects, suitably, inhibiting EGFR-mediated effects, suitably, inhibiting RAGE / EGFR-mediated effects.

[0303] Suitablely, epithelial physiology abnormalities may be a symptom of respiratory diseases. Therefore, suitablely, IL-33 antagonists may be used to treat or prevent respiratory diseases characterized by epithelial physiology abnormalities.

[0304] As defined in another embodiment, an IL-33 antagonist is provided for the prevention or treatment of respiratory diseases by improving epithelial physiology.

[0305] Epithelial physiological abnormalities are defined elsewhere in this article.

[0306] Where appropriate, improvement of epithelial physiology may include improvement of epithelial physiological abnormalities.

[0307] The above describes suitable methods for improving epithelial physiological abnormalities.

[0308] Suitablely, treatment of respiratory diseases by improving epithelial physiology abnormalities may include: Improve or enhance mucosal ciliary clearance; Reduce or inhibit mucus production; Inhibit abnormal mucus composition; Reduce or inhibit epithelial remodeling abnormalities; and / or Reduce or inhibit the differentiation or proliferation of goblet cells.

[0309] The above provides further details about each of these roles in conjunction with the treatment or prevention of epithelial physiological abnormalities, and these can be combined with the treatment of respiratory diseases.

[0310] Suitablely, abnormal EGFR activity may be a symptom of respiratory diseases. Therefore, suitablely, IL-33 antagonists may be used to treat or prevent respiratory diseases characterized by abnormal EGFR activity.

[0311] As defined in another embodiment, an IL-33 antagonist is provided for the prevention or treatment of respiratory diseases by inhibiting EGFR-mediated effects.

[0312] The role mediated by EGFR is defined elsewhere in this article.

[0313] Appropriately, respiratory diseases are lower respiratory diseases; appropriately, respiratory diseases are diseases affecting the trachea, bronchi, bronchioles, alveolar ducts, and / or alveoli. Appropriately, respiratory diseases are bronchial diseases.

[0314] Appropriately, respiratory diseases may be selected from: COPD; bronchitis; emphysema; bronchiectasis, such as CF-bronchioles or non-CF-bronchioles; asthma; asthma overlap with COPD (ACO).

[0315] Appropriately, the respiratory disease is COPD. Appropriately, the respiratory disease is bronchitis-related COPD. Bronchitis-related COPD is a specific form of COPD in which chronic bronchitis is present in patients with COPD. Due to a faster decline in lung function, increased symptoms, and an increased risk of secondary infections, bronchitis-related COPD has a higher mortality rate than COPD. In particular, patients with bronchitis-related COPD have higher total mucoprotein concentrations and excessive mucus secretion. Therefore, based on the findings of IL-33 antagonists inhibiting EGFR activity and improving mucociliary physiology, patients with bronchitis-related COPD may particularly benefit from treatment with the IL-33 antagonists described herein.

[0316] Appropriately, for the same reason, respiratory diseases can be bronchitis-related asthma.

[0317] In one embodiment, an IL-33 antagonist is provided for the prevention or treatment of bronchitis-related COPD.

[0318] In one embodiment, a method for preventing or treating bronchitis-related COPD in a patient is provided, the method comprising: administering an effective amount of an IL-33 antagonist to the patient in need.

[0319] ST2 signal transmission Although this disclosure relates to the medical use of IL-33 antagonists in inhibiting RAGE-EGFR-mediated signaling and action, it is known that IL-33 antagonists can also inhibit ST2-mediated signaling and action. Therefore, the medical uses described herein contemplate the regulation of both EGFR-mediated and ST2-mediated action.

[0320] Suitablely, IL-33 antagonists are used to prevent and treat epithelial physiological abnormalities by modulating EGFR-mediated and ST2-mediated effects. Suitablely, IL-33 antagonists are used to prevent and treat epithelial physiological abnormalities by inhibiting EGFR-mediated and ST2-mediated effects. Suitablely, IL-33 antagonists are used to prevent and treat epithelial physiological abnormalities by inhibiting RAGE / EGFR-mediated and ST2-mediated effects. Epithelial physiological abnormalities are as defined elsewhere in this document.

[0321] Suitablely, IL-33 antagonists are used to prevent and treat EGFR-mediated diseases by modulating EGFR-mediated and ST2-mediated effects. Suitablely, IL-33 antagonists are used to prevent and treat EGFR-mediated diseases by inhibiting EGFR-mediated and ST2-mediated effects. Suitablely, IL-33 antagonists are used to prevent and treat EGFR-mediated diseases by inhibiting RAGE / EGFR-mediated and ST2-mediated effects. EGFR-mediated diseases are as defined elsewhere in this document.

[0322] Suitable ST2-mediated effects include inflammation. Therefore, suitable IL-33 antagonists are used to prevent and treat ST2-mediated diseases by modulating EGFR-mediated and ST2-mediated effects. Suitable ST2-mediated diseases can include diseases characterized by inflammation. Suitable ST2-mediated diseases can include inflammatory diseases.

[0323] ST2-mediated diseases or inflammatory diseases can include: COPD; allergic disorders such as asthma, chronic rhinosinusitis, food allergies, eczema, and dermatitis; fibrotic diseases such as pulmonary fibrosis; pulmonary eosinophilia; pleural malignancies; rheumatoid arthritis; collagen vascular disease; atherosclerotic vascular disease; urticaria; inflammatory bowel disease; Crohn's disease; celiac disease; systemic lupus erythematosus; progressive systemic sclerosis; Wegner's granulomatosis; septic shock; and Behçet's disease.

[0324] Suitablely, ST2-mediated diseases or inflammatory diseases are respiratory diseases. Suitablely, ST2-mediated diseases or inflammatory diseases exist in the respiratory tract as defined above.

[0325] Suitablely, IL-33 antagonists are also used for the prevention and treatment of inflammation or inflammatory diseases. Suitablely, IL-33 antagonists may also be used for the prevention and treatment of ST2-mediated inflammation or ST2-mediated inflammatory diseases.

[0326] Suitablely, ST2-mediated diseases and EGFR-mediated diseases overlap. In other words, ST2-mediated effects and EGFR-mediated effects, suitablely RAGE-EGFR-mediated effects, contribute to disease pathology. Advantageously, it is believed that the medical use of a single IL-33 antagonist can inhibit both RAGE and ST2 activation by IL-33. Therefore, a single IL-33 antagonist can simultaneously treat both RAGE-EGFR-mediated and ST2-mediated diseases.

[0327] In one embodiment, an IL-33 antagonist is provided for the prevention or treatment of epithelial physiological abnormalities and inflammation. In another embodiment, a method for preventing or treating epithelial physiological abnormalities and inflammation in a patient is provided, the method comprising: administering an effective amount of an IL-33 antagonist.

[0328] Suitablely, epithelial physiology abnormalities and inflammation can be symptoms of respiratory diseases. Therefore, statements regarding the treatment and prevention of these symptoms can be made in the context of respiratory diseases and can suitably include the treatment or prevention of epithelial physiology abnormalities and inflammation in respiratory diseases.

[0329] In one embodiment, an IL-33 antagonist is provided for the prevention or treatment of EGFR-mediated and ST2-mediated diseases.

[0330] In one embodiment, a method for preventing or treating EGFR-mediated disease and ST2-mediated disease in a patient is provided, the method comprising: administering an effective amount of an IL-33 antagonist.

[0331] Suitablely, IL-33 antagonists are reduced IL-33 antagonists. Suitablely, reduced IL-33 antagonists are as defined above.

[0332] Suitablely, IL-33 antagonists are as defined above. Suitablely, IL-33 antagonists are 33_640087-7B.

[0333] Alternatively, different IL-33 antagonists can be used in combination therapy to inhibit both RAGE and ST2 activation by IL-33. Therefore, it is envisioned that combinations of IL-33 antagonists can simultaneously treat both RAGE-EGFR-mediated and ST2-mediated diseases.

[0334] Appropriately, respiratory diseases are defined as described above. Appropriately, respiratory diseases are characterized by abnormal EGFR activity and abnormal ST2 activity.

[0335] Therefore, suitably, in one embodiment, a combination of a first IL-33 antagonist for the prevention or treatment of epithelial physiological abnormalities and a second IL-33 antagonist for the prevention or treatment of inflammation is provided.

[0336] Therefore, suitably, in one embodiment, a method for preventing or treating epithelial physiological abnormalities and inflammation in a patient is provided, the method comprising: administering an effective amount of a combination of a first IL-33 antagonist and an effective amount of a second IL-33 antagonist.

[0337] Therefore, suitably, in one embodiment, a combination of a first IL-33 antagonist for the prevention or treatment of EGFR-mediated diseases and a second IL-33 antagonist for the prevention or treatment of ST2-mediated diseases is provided.

[0338] Therefore, suitably, in one embodiment, a method for preventing or treating EGFR-mediated disease and ST2-mediated disease in a patient is provided, the method comprising: administering an effective amount of a combination of a first IL-33 antagonist and an effective amount of a second IL-33 antagonist.

[0339] Suitablely, first-line IL-33 antagonists are used for the prevention or treatment of epithelial physiological abnormalities and / or EGFR-mediated diseases.

[0340] Suitablely, a second IL-33 antagonist is used for the prevention or treatment of inflammatory and / or ST2-mediated diseases.

[0341] Suitablely, the first and second IL-33 antagonists are different.

[0342] Suitably, the first IL-33 antagonist is as defined above. Suitably, the second IL-33 antagonist can be any other IL-33 antagonist known to inhibit ST2-mediated effects. Suitably, the second IL-33 antagonist is also as defined above.

[0343] Suitably, the first antagonist may be a reduced or oxidized IL-33 antagonist. Suitably, the second IL-33 antagonist is a reduced IL-33 antagonist.

[0344] Suitablely, at least one of the IL-33 antagonists is 33_640087-7B. Suitablely, the first antagonist is 33_640087-7B.

[0345] Suitablely, the first and second IL-33 antagonists can be administered in combination. Suitablely, the first and second IL-33 antagonists can be administered simultaneously or in combination at different times. A suitable dosing regimen can be determined by a medical professional.

[0346] These statements also apply to the medical uses / treatments mentioned above, in which ST-2-mediated diseases can be prevented or treated.

[0347] Alternatively, in other embodiments, an IL-33 antagonist may be administered in combination with an ST2 inhibitor. Suitably, the ST2 inhibitor may not be an IL-33 antagonist, but may otherwise inhibit the ST2 receptor. Suitably, the ST2 inhibitor may serve to treat or prevent ST2-mediated diseases as identified above.

[0348] Therefore, in one embodiment, a combination of an IL-33 antagonist for treating or preventing epithelial physiological abnormalities and an ST2 inhibitor for treating or preventing inflammation is provided.

[0349] In one embodiment, a method for preventing or treating epithelial physiological abnormalities and inflammation in a patient is provided, the method comprising: administering an effective amount of an IL-33 antagonist in combination with an effective amount of an ST2 inhibitor.

[0350] Suitablely, epithelial physiology abnormalities and inflammation can be symptoms of respiratory diseases. Therefore, statements regarding the treatment and prevention of these symptoms can be made in the context of respiratory diseases and can suitably include the treatment or prevention of epithelial physiology abnormalities and inflammation in respiratory diseases.

[0351] Therefore, in one embodiment, a combination of an IL-33 antagonist for treating or preventing EGFR-mediated diseases and an ST2 inhibitor for treating or preventing ST2-mediated diseases is provided.

[0352] In one embodiment, a method is provided for preventing or treating a combination of EGFR-mediated disease and ST2-mediated disease in a patient, the method comprising: administering an effective amount of an IL-33 antagonist in combination with an effective amount of an ST2 inhibitor.

[0353] Suitable IL-33 antagonists are as defined elsewhere in this document. Suitable EGFR-mediated and ST2-mediated diseases are as defined elsewhere in this document.

[0354] Suitablely, the ST2 inhibitor may be any such inhibitor known in the art, such as GSK3772847 (described in WO 2013 / 165894) and RG6149 (WO 2013 / 173761), both of which are incorporated herein by reference.

[0355] Suitablely, IL-33 antagonists and ST2 inhibitors can be administered in combination. Suitablely, IL-33 antagonists and ST2 inhibitors can be administered simultaneously or at different times. A suitable dosing regimen can be determined by a medical professional.

[0356] patient These methods and medical applications are then practiced on patients or subjects. Patients may be those who need to identify, diagnose, or treat physiological conditions or diseases such as epithelial physiology abnormalities, EGFR-mediated diseases, or respiratory diseases.

[0357] Appropriately, the patient can be a human being. The patient can be an individual currently receiving medical care or requiring medical care. Appropriately, the patient can be male or female. Appropriately, the patient can be an adult or a child.

[0358] Suitable patients, in the methods described herein, may be those believed to have epithelial physiological abnormalities, or EGFR-mediated diseases or respiratory diseases. For example, suitable patients may have symptoms consistent with such conditions.

[0359] Alternatively, suitable patients in the context of the methods described herein may be those considered to be at risk of developing epithelial physiology abnormalities, or EGFR-mediated diseases or respiratory diseases. For example, such patients may have been exposed to individuals with such conditions, may have related conditions, or may meet risk factors associated with said conditions, such as smoking, advanced age, allergies, etc.

[0360] Example This disclosure can be characterized by the following embodiments, wherein: Example 1 describes an IL-33 antagonist for the prevention or treatment of disease by inhibiting EGFR-mediated effects.

[0361] Example 2 describes an IL-33 antagonist used according to Example 1, wherein the EGFR-mediated effect is a RAGE-EGFR-mediated effect.

[0362] Example 3 describes an IL-33 antagonist for use according to Example 1 or 2, wherein the EGFR-mediated effect is RAGE-EGFR-mediated signal transduction.

[0363] Example 4 describes an IL-33 antagonist for use according to any one of Examples 1-3, wherein the disease is a respiratory disease.

[0364] Example 5 describes an IL-33 antagonist for use according to any one of Examples 1-4, wherein the disease is characterized by epithelial physiological abnormalities and / or abnormal EGFR activity.

[0365] Example 6 describes an IL-33 antagonist for use according to any one of Examples 1-5, wherein the disease is selected from: COPD; bronchitis; emphysema; bronchiectasis, such as CF-bronchial or -CF-bronchial; asthma; or asthma overlapping with COPD (ACO).

[0366] Example 7 describes an IL-33 antagonist for use according to any one of Examples 4-6, wherein the respiratory disease is bronchitis-related COPD.

[0367] Example 8 describes an IL-33 antagonist for use according to any one of Examples 1-7, wherein the treatment: improves mucus clearance; inhibits mucus production abnormalities; inhibits epithelial remodeling abnormalities; and / or inhibits goblet cell differentiation abnormalities.

[0368] Example 9 describes an IL-33 antagonist for use according to any of the foregoing examples, wherein the IL-33 antagonist inhibits the activity of oxidized IL-33.

[0369] Example 10 describes an IL-33 antagonist for use according to any of the foregoing examples, wherein the IL-33 antagonist prevents oxidized IL-33 from binding to RAGE, thereby inhibiting RAGE-EGFR signaling.

[0370] Example 11 describes an IL-33 antagonist for use according to any of the foregoing examples, wherein the IL-33 antagonist is an anti-IL-33 antibody or its antigen-binding fragment, preferably an anti-reduced IL-33 antibody or antigen and its binding fragment.

[0371] Example 12 describes an IL-33 antagonist for use according to Example 11, wherein the anti-IL-33 antibody or its antigen-binding fragment contains a complementarity-determining region (CDR) selected from the variable heavy chain domain (VH) and variable light chain domain (VL) pairs in Table 1.

[0372] Example 13 describes an IL-33 antagonist for use according to Example 12, wherein the anti-IL-33 antibody or its antigen-binding fragment comprises a variable heavy chain domain (VH) and variable light chain domain (VL) pair selected from Table 1.

[0373] Example 14 describes an IL-33 antagonist for use according to any one of Examples 11-13, wherein the anti-IL-33 antibody or its antigen-binding fragment comprises VHCDR1 having the sequence of SEQ ID NO:37, VHCDR2 having the sequence of SEQ ID NO:38, VHCDR3 having the sequence of SEQ ID NO:39, VLCDR1 having the sequence of SEQ ID NO:40, VLCDR2 having the sequence of SEQ ID NO:41, and VLCDR3 having the sequence of SEQ ID NO:42.

[0374] Example 15 describes an IL-33 antagonist for use according to any one of Examples 11-14, wherein the IL-33 antagonist is an anti-IL33 antibody or an antigen-binding fragment thereof, the anti-IL33 antibody or antigen-binding fragment thereof comprising the VH domain of the sequence of SEQ ID NO:1 and the VL domain of the sequence of SEQ ID NO:19. Attached Figure Description

[0375] The following figure will be used as an example to describe the embodiments, wherein: Figure 1 This image shows a grayscale heatmap of the fold increase in kinase phosphorylation compared to the untreated control for each assay on the MAP kinase phosphorylation antibody array. Reduced IL-33 (IL-33-01 and IL-33-16, respectively) did not induce any signal above baseline. oxidized IL-33 (oxidized IL-33-01) increased phosphorylation of multiple kinases. Figure 2 This image shows the signaling patterns of each stimulus condition on the receptor tyrosine kinase (RTK) activity array. oxIL-33, rather than reduced IL33-01 and IL33-16 respectively, triggered positive signals corresponding to the epidermal growth factor receptor (EGFR) on the RTK array. Dot intensity is associated with receptor tyrosine kinase phosphorylation; Figure 3A This study demonstrated the activity of pEGFR (Tyr1068) in normal human bronchial epithelial (NHBE) cells stimulated with increased concentrations of IL-33 or EGFR ligand. oxIL-33, rather than reduced IL-33 (IL33-01), similarly promotes EGFR phosphorylation as EGF, HB-EGF, and TGFα. Figure 3BThis study demonstrates the activity of pEGFR (Tyr1068) in A549 cells stimulated with increased concentrations of IL-33 or EGFR ligands. OxyIL-33 (oxidized IL-33-01), rather than reduced IL-33 (IL-33-01), promotes EGFR phosphorylation in a pattern similar to that seen in NHBE cells, analogous to EGF, HB-EGF, and TGFα.

[0376] Figure 3C This study demonstrates pEGFR (Tyr1068) activity in A549 cells stimulated with increased concentrations of IL-33, EGFR ligand, or RAGE ligand. oxIL-33, non-wild-type (WT) IL-33 (IL-33-01), C->S mutant (mut) IL-33 (IL-33-16), or RAGE ligand, similar to EGF, promotes EGFR phosphorylation. Figure 4 As shown by Western blot analysis, oxidized IL-33 induces phosphorylation of multiple molecules involved in the EGFR pathway (EGFR, PLC, AKT, JNK, ERK 1 / 2, p38); Figure 5 The results showed that increasing the dose of anti-EGFR antibody reduced STAT5 phosphorylation induced by oxIL-33-01 compared to the isotype control. Figure 6 The study showed that immunoprecipitation with anti-EGFR was performed, followed by Western blotting to detect EGFR, RAGE, or IL-33. After NHBE stimulation with oxIL-33, IL-33 and RAGE co-precipitated with EGFR, indicating the formation of a complex. RAGE appeared to be unique for the oxIL-33 signaling complex compared to EGF. Figure 7A The results show that oxIL-33 binds directly to RAGE. HMGB1 is a known RAGE ligand and was used as a positive control in this study. Figure 7B The results show that oxIL-33 does not bind directly to EGFR (but the known EGFR ligand EGF binds directly to EGFR). However, when RAGE is added to this assay in combination with oxIL-33, EGFR binding is observed. Figure 8 : This shows that after activation with oxIL-33 at specified time points, immunoprecipitation with anti-EGFR or anti-RAGE was performed in wild-type and RAGE-deficient A549 cells, followed by Western blotting of EGFR, RAGE and IL-33. Figure 9The results showed that oxIL-33-01-induced STAT5 phosphorylation was reduced by anti-RAGE antibody but not anti-ST2 antibody. Figure 10 This image shows how EGF and oxidized IL33 (oxIL33) induce EGFR aggregation and internalization in EGFR-GFP A549 cells. Representative images are shown 5 minutes after stimulation. Histograms show EGFR depletion in non-clustered regions of cells treated with EGF and oxIL-33 (leftward shift of the bell peak in the histogram), and an increase in the number of saturated pixels (intensity 255) due to clustering in these cells.

[0377] Figure 11 The results show the fold increase in IL-8 secretion from NHBE and DHBE after 24 h of stimulation with separate culture medium (unstimulated control), 30 ng / ml IL-33-01, 30 ng / ml IL-33-16, 30 ng / mL oxidized IL-33, or 30 ng / mL EGF. Bar graphs show the mean and SEM values ​​from four NHBE and three DHBE donors. Figure 12A The graph shows the relative scratch healing density of A549 cells after treatment with reduced IL-33, oxIL-33, or EGF. The bar charts show the mean and SEM values ​​from six technical replicates for each condition. Figure 12B The graph shows the relative scratch healing density of NHBE cells after treatment with reduced IL-33, oxIL-33, or EGF. The bar charts show the mean and SEM values ​​from six technical replicates for each condition. Figure 13 The bar chart shows the percentage of scratch closure in NHBE cells treated with separate culture medium (unstimulated control), reduced IL-33, oxidized IL-33, or oxidized IL-33 in the presence of anti-ST2, anti-RAGE, or anti-EGFR. The bar chart shows the mean and SEM values ​​from six technical replicates for each condition. Figure 14 : Shows the relative scratch healing density in human bronchial epithelial cells from healthy subjects, smokers, and those with COPD, under stimulation with and without oxidized IL-33; Figure 15 The graph shows the 24-hour scratch closure rate (%) of NHBE cells (n = 5 donors) compared to DHBE COPD cells (n = 5 donors) and DHBE cells treated with IgG1 control, anti-IL-33 (33_640087-7B), anti-RAGE (M4F4), and anti-ST2. The bar graph shows the mean and SEM values ​​for n = 5 independent donors. Figure 16A: This shows representative immunohistochemical staining of basal (p63+; blue), cilia (α-tubulin; purple), and goblet (mucin 5ac + mucin B; yellow) cells from ALI cultures from healthy donors.

[0378] Figure 16B The results show the quantification of immunohistochemistry for various epithelial cell types using HALO software after 7 days of treatment with anti-IL-33 (33_640087-7B) or an isotype control antibody; the data shown are the mean and SEM from n = 2-3 independent donors.

[0379] Figure 16C The data shown are the quantification of goblet cells using HALO software after 7 days of treatment with anti-IL-33 (33_640087-7B) or an isotype control antibody; the data shown are the mean and SEM values ​​from n = 2-3 independent donors.

[0380] Figure 17 Exemplary staining of independent mucins (mucin 5AC and mucin 5B) in ALI cultures derived from healthy individuals (1 donor) or COPD patients (1 donor) is shown, as well as a reduction in mucin staining in COPD cultures after 7 days of treatment with anti-IL-33 (33_640087-7B).

[0381] Figure 18 The figures show tSNE plots illustrating the different proportions of cell subtypes found in COPD ALI cultures from independent donors treated with anti-IL-33 compared to untreated cultures.

[0382] Figure 19 A: This shows a representative flow cytometry contour plot of goblet cells detected in ALI cultures from normal donors. Muc5B is on the x-axis, and Muc5AC is on the y-axis. ALI cultures were treated with protein for 7 days. Treatment with reduced IL-33 (IL-33[C->S]) did not increase the number of goblet cells above baseline. oxIL-33 (oxidized IL-33-01) increased the percentage of goblet cells, as did IL-13. IL-13 is known to increase goblet cells in ALI cultures and was used as a positive control in this study. The numbers in the quadrants show the percentage of the total population: the upper left quadrant contains Muc5AC single-positive goblet cells, the lower right quadrant contains Muc5B single-positive goblet cells, and the upper right quadrant contains Muc5AC and Muc5B double-positive goblet cells.

[0383] Figure 19B: This section shows pooled flow cytometry data from ALI cultures of normal donors (n = 6), illustrating the percentage of goblet cells (pooled Muc5AC single-positive, Muc5B single-positive, and Muc5AC and Muc5B double-positive goblet cells) to the total epithelial population. Reduced IL-33 (IL-33[C->S]) did not increase goblet cell percentage above baseline. oxIL-33 (oxidized IL-33-01) increased the percentage of goblet cells similarly to IL-13. A violin plot shows all data points and the median.

[0384] Figure 19 C: Shows pooled flow cytometry data from ALI cultures of normal donors (n = 6), showing Muc5AC single-positive goblet cells. Reduced IL-33 (IL-33[C->S]) did not increase goblet cells above baseline. oxIL-33 (oxidized IL-33-01) increased the percentage of goblet cells similarly to IL-13. A violin plot shows all data points and the median.

[0385] Figure 19 D: Shows pooled RT-qPCR data from ALI cultures of normal donors (n = 4), which demonstrate... MUC5AC fold change in mRNA. Reduced IL-33 (IL-33 [C->S]) did not increase. MUC5AC mRNA. oxIL-33 (oxidized IL-33-01) causes similar problems to IL-13. MUC5AC mRNA increase. The violin plot shows all data points and the median.

[0386] Figure 20A This image shows representative immunohistochemical staining of basal (p63+; purple), cilia (α-tubulin; dark cyan), and goblet (Muc5ac+Muc5B; yellow) cells from ALI cultures derived from healthy donors. Reduced IL-33 (IL-33[C->S]) failed to visually increase goblet cells. OxyIL-33 (oxidized IL-33-01) caused a visible increase in goblet cells.

[0387] Figure 20B The results show the quantification of the mucin 5ac+mucin 5b region (total epithelial tissue region %) from immunohistochemical images (minimum n = 3 donors per condition) using HALO software. oxIL-33 and IL-13 increased the area stained with mucin compared to untreated and reduced IL-33-treated controls.

[0388] Figure 21AThis image shows a representative flow cytometry contour plot of goblet cells in ALI cultures from COPD donors. As depicted, Muc5B is on the x-axis and Muc5AC is on the y-axis. ALI cultures were treated with antibodies for 7 days. Treatment with anti-IL-33 (33_640087-7B) reduced the total number of goblet cells. The numbers in the quadrants show the percentage of the total population: the upper left quadrant contains Muc5AC single-positive goblet cells, the lower right quadrant contains Muc5B single-positive goblet cells, and the upper right quadrant contains both Muc5AC and Muc5B double-positive goblet cells. Figure 21B The table shows pooled flow cytometry data from ALI cultures from COPD donors (n = 6), illustrating the total goblet cell count (pooled Muc5AC single-positive, Muc5B single-positive, and Muc5AC and Muc5B double-positive goblet cells). ALI cultures were treated with antibody for 7 days. Treatment with anti-IL-33 (33_640087-7B) reduced the total goblet cell count. A violin plot shows all data points and the median.

[0389] Figure 21C The table shows pooled flow cytometry data from ALI cultures from COPD donors (n = 6), displaying Muc5AC single-positive goblet cells. ALI cultures were treated with antibody for 7 days. Anti-IL-33 (33_640087-7B) treatment reduced the number of Muc5AC single-positive goblet cells. A violin plot shows all data points and the median.

[0390] Figure 21D This section displays pooled RT-qPCR data from ALI cultures derived from COPD donors (n = 5), showing... MUC5AC fold change in mRNA. Treatment with anti-IL-33 (33_640087-7B) reduced MUC5AC The violin plot shows all data points and the median.

[0391] Figure 21E This section presents pooled flow cytometry data from ALI cultures of COPD donors (n = 6), showing total viability in therapeutic conditions as determined by LD-negative cell staining.

[0392] Figure 22A This image shows representative immunohistochemical staining of basal (p63+; purple), cilia (α-tubulin; dark cyan), and goblet (Muc5ac+MucB; yellow) cells from ALI cultures derived from COPD donors. Anti-IL-33 (33_640087-7B) treatment for 7 days visibly reduced goblet cells.

[0393] Figure 22B The image shows the quantification of the Muc5ac+Muc5b region (total epithelial tissue region %) from immunohistochemical images (n = 4 donors) using HALO software. Anti-IL-33 (33-640087_7B) reduced the area stained with mucin compared to untreated and human IgG1-treated controls.

[0394] Figure 23 A: This shows the quantification of Muc5AC in the top wash buffer obtained from COPD and healthy ALI cultures. As determined by Muc5AC ELISA, COPD cultures showed higher levels of Muc5AC. Figure 23 B: This shows the quantification of Muc5AC in the top wash buffer obtained from healthy ALI cultures. ALI cultures were treated with reduced IL-33mut16 (IL-33 [C->S]), oxIL-33, and wild-type IL-33, and the results were determined by Muc5AC ELISA.

[0395] Figure 23 C: Shows the quantification of Muc5AC in the top wash buffer obtained from COPD ALI cultures. Cells were treated with human and mouse IgG1 controls (hIgG1 and mIgG1), 33-640087_7B, or anti-ST2 antibody. As determined by Muc5AC ELISA, treatment with anti-IL-33 (33-640087_7B) reduced Muc5AC levels.

[0396] Example Example 1 - Oxidized IL-33 drives the formation of the signal transduction complex between RAGE and EGFR In Cohen, ES et al., Nat. Commun. [Nature Communications] 6:8327 (2015), the applicants described the discovery of the oxidized disulfide bonded form of IL-33 (DSB IL-33) and showed that this form does not bind to ST2 and does not activate ST2-dependent signaling. Subsequently (see WO 2016156440A1), the applicants showed that oxIL-33 binds to the receptor for advanced glycation end products (RAGE) and signals in a RAGE-dependent manner to activate STAT5 and affect epithelial migration.

[0397] To further explore the function of oxIL-33, epithelial cells were stimulated with reduced or oxidized forms of IL-33, and signal transduction pathways were investigated. In this paper, the inventors demonstrate that oxIL-33 is a novel ligand for a complex of the receptor for advanced glycation end products (RAGE) and the receptor for epidermal growth factor (EGFR), thereby having a profound impact on epithelial function.

[0398] 1. Cloning and expression of human maturation and cysteine ​​mutant variants of IL33 cDNA molecules encoding the mature human IL-33 fraction (112-270) (accession number (UniProt) 095760) (also known as IL33-01 or IL-33) and a variant with four cysteine ​​residues mutated to serine (also known as IL33-16 or IL-33[C->S]) were synthesized by primer extension PCR and cloned into pJexpress 411 (DNA 2.0). The wild-type (WT) and mutant IL-33 coding sequences were modified to contain 10xHis, Avitag, and factor Xa protease cleavage sites (MHHHHHHHHHHAAGLNDIFEAQKIEWHEAAIEGR SEQ ID NO:43) at the N-terminus of the protein. N-terminal tagged His10 / Avitag IL33-01 (WT, SEQ ID NO:44) and N-terminal tagged His10 / Avitag IL33-16 (WT, SEQ ID NO:45) were generated by transforming *E. coli* BL21(DE3) cells. Transformed cells were cultured for 18 hours at 37°C in autologous induction medium (OvernightExpress™ Autologous Induction System 1, Merck Millipore, 71300-4), followed by cell harvesting by centrifugation and storage at -20°C. Cells were resuspended in 2x DPBS containing a completely EDTA-free protease inhibitor mixture tablet (Roche, 11697498001) and 50 U / ml Benzonase (Merck Millipore, 70746-3) and lysed by sonication. Cell lysates were clarified by centrifugation at 50,000 xg for 30 min at 4°C. IL-33 protein was purified from the supernatant by immobilized metal affinity chromatography at 5 ml / min using a HisTrap excel column (GE Healthcare, 17371205) equilibrated with 2x DPBS and 1 mM DTT. The column was washed with 2x DPBS, 1 mM DTT, and 20 mM imidazole (pH 7.4) to remove impurities, followed by washing with 2x DPBS and 0.1% Triton X-114 to remove immobilized endotoxin protein. After further washing with 2x DPBS, 1 mM DTT, and 20 mM imidazole (pH 7.4), the sample was eluted with 2x DPBS, 1 mM DTT, and 400 mM imidazole (pH 7.4). IL-33 was further purified by size exclusion chromatography using a HiLoad Superdex 75 26 / 600 pg column (GE Healthcare, 28989334) in 2x DPBS at 2.5 ml / min.Peak fractions were analyzed by SDS-PAGE. Fractions containing pure IL-33 were combined, and their concentrations were determined by absorbance at 280 nm. The final sample was analyzed by SDS-PAGE.

[0399] To generate untagged IL-33, N-terminal tagged His10 / Avitag IL33 was incubated with 10 units of factor Xa per mg protein (GE Healthcare, 27084901) in 2x DPBS buffer at room temperature for 1 hour. Untagged IL-33 was then purified in 2x DPBS using SEC chromatography at a flow rate of 1 ml / min on a HiLoad 16 / 600 Superdex 75 pg column (GE Healthcare, 28989333).

[0400] 2. Production and purification of oxidized IL-33 (oxIL-33) Reduced IL33-01 was oxidized by diluting to a final concentration of 0.5 mg / ml in 60% IMDM medium (phenol red-free) and 40% DPBS, followed by incubation at 37°C for 18 h. Aggregates generated during oxidation were removed from the samples by loading them onto a HiTrap Capto Q ImpRes anion exchange column (GE Healthcare, 17547055). Prior to loading, the samples were modified by adding 1 M Tris (pH 9.0) until the pH reached 8.3 and then adding 5 M NaCl to a final concentration of 125 mM—under these loading conditions, the aggregates bound to the column, and the monomeric oxIL-33 flowed through without binding and was collected. Tag cleavage from oxIL-33 was achieved by incubation at 22°C for 120 min with Factor Xa (NEB, P8010L) at a final concentration of 1 µg Factor Xa per 50 µg oxIL-33. To consume any remaining reduced IL-33 in the sample, the soluble human ST2S extracellular domain fused to human IgG1 Fc-His6 was incubated with the sample at 22°C for 30 min to bind reduced IL-33. The sample was concentrated in a centrifuge with a 3,000 Da cutoff and loaded onto a HiLoadSuperdex 75 26 / 600 pg column (GE Healthcare, 28989334) at a flow rate of 2 ml / min to separate monomeric oxIL-33 from other sample components. Fractions containing pure oxIL-33 were combined and concentrated, and the final concentration of the sample was determined by UV absorption spectroscopy at 280 nm. The quality of the final product was evaluated by SDS-PAGE, HP-SEC, and RP-HPLC.

[0401] 3. Cloning, expression and purification of human ST2 ECD The naturally occurring ST2S soluble isoform (UniProt accession number Q01638-2) encoding ST2 without the endogenous signal peptide (amino acid residues 19-328) was amplified by PCR using primers that encoded a Gibson assembly-compatible extension sequence and a CD33 signal peptide fused to the N-terminus of the ST2S coding sequence. Similarly, the coding sequence for human IgG1 Fc with a C-terminal His6-tag was amplified. The ST2S cDNA and IgG1 Fc-His6 cDNA were assembled using pDEST12.2 OriP, a mammalian CMV promoter-driven expression vector with an OriP replication origin from EBV, enabling episomal maintenance in cell lines expressing EBNA-1 protein. For protein expression, the plasmid was transiently transformed into CHO cell suspension cultures overexpressing EBNA-1 using polyethyleneimine as the transfection reagent. Seven days post-transfection, conditioned medium containing the secreted ST2S-Fc-His6 fusion protein was collected and loaded at 2 ml / min onto a HiTrap MabSelect SuRe (Protein A, GE Healthcare, 11-0034-95) affinity column. The column was washed with 2x DPBS and eluted with 25 mM sodium acetate (pH 3.6). Fractions containing ST2S-Fc-His6 were pooled and loaded at 2 ml / min onto a HiLoad Superdex 200 26 / 600 pg column (GE Healthcare, 28989336) equilibrated in 2x DPBS. Fractions containing purified ST2S-Fc-His6 protein were pooled, and concentrations were determined by absorbance at 280 nm. The final samples were analyzed by SDS-PAGE.

[0402] 4. Cloning, expression and purification of human desialyl glycoprotein receptor (ASGPR) ECD Geneart chemically synthesized a cDNA encoding the extracellular domain (ECD) of the desialylate glycoprotein receptor (UniProt accession number P07306), which contains a cytoplasmic and transmembrane domain (amino acid residues 62-291). This cDNA possessed a His10_Avi tag sequence fused to the N-terminus of the ECD domain following the CD33 signal peptide. The construct was directly cloned into pDEST12.2 OriP, a mammalian CMV promoter-driven expression vector with an OriP replication origin from EBV, enabling cell-free gene maintenance in cell lines expressing EBNA-1 protein. For protein expression, the plasmid was transiently transformed into suspension cultures of HEK Freestyle 293F cells using 293Fectin as a transfection reagent. Seven days post-transfection, conditioned medium containing the secreted HisAVi_hASGPR ECD fusion protein was collected by immobilized metal affinity chromatography at 4 ml / min onto a HisTrap Excel column (GE Healthcare, 17371205) equilibrated in 2x DPBS. The column was washed with 2x DPBS and 40 mM imidazole (pH 7.4) to remove impurities, and the sample was eluted with 2x DPBS and 400 mM imidazole (pH 7.4). Human ASGPR ECD was further purified by size exclusion chromatography using a HiLoad Superdex 75 16 / 600 pg column (GE Healthcare, 28-9893-33) in 2x DPBS at 1 ml / min. Peak fractions were analyzed by SDS-PAGE. Fractions containing pure monomeric ASGPR were combined, and concentrations were determined by absorbance at 280 nm. The final sample was analyzed by SDS-PAGE.

[0403] 5. The oxidized form of IL-33 activates the MAP kinase pathway. Normal human bronchial epithelial (NHBE) cells (CC-2540) were obtained from Lonza and maintained in complete BEGM medium (Lonza) according to the manufacturer's protocol. NHBEs were harvested using an acute enzyme (PAA, #L1 1-007) and cultured at 1 x 10⁻⁶ cells / mL. 6Cells were seeded at 2 ml / well in 6-well culture dishes (Corning Costa, 3516) using BEGM (Lonza CC-3171) and a supplement kit (Lonza CC-4175). Cells were incubated at 37°C and 5% CO2 for 18–24 hours. Afterward, the culture medium was aspirated, and the cells were washed twice with 1 ml PBS, followed by the addition of starvation medium (BEGM (Lonza CC-3171) supplemented with 1% penicillin / streptomycin). The plates were then incubated again at 37°C and 5% CO2 for 18–24 hours before stimulation.

[0404] The MAP kinase phosphorylation antibody array kit (ab211061) was purchased from Abcam and the experiments were performed according to the manufacturer's instructions. NHBE cells in 6-well culture dishes that had been starved for 18–24 h were left untreated or treated with 30 ng / ml reduced IL-33, IL-33-16, or oxidized IL-33, followed by incubation at 37°C, 5% CO2 for 10 min (see Table 2 for activators used in this assay). The plates were removed from the incubator, and the cells were washed with ice-cold PBS, followed by the addition of 100 µl / well of 1x lysis buffer provided in the kit. Protein extracts were transferred to 1.5 ml tubes and incubated at 4°C and 14,000 rpm until clear. Protein concentrations were determined using BCA technology (Thermo, 23225), with 250 µg of total protein used per array membrane. All subsequent steps were performed according to the manufacturer's instructions. The membranes were visualized on LiCor C-digit and quantified using Image Lite studio.

[0405] Table 2 agonists identifier Reconstructed from Final concentration (µg / ml) Unlabeled oxidized IL33-01 RD15 PBS 100 Unlabeled IL33-01 07 / 24 / 2015 PBS 100 Unlabeled IL33-16 11 / 12 / 2015 PBS 100 EGF 236-EG-200 PBS 100

[0406] Compared to wild-type (IL-33) and the C->S (IL-33 [C->S]) reduced forms of IL-33 (IL33-01 and IL33-16, respectively), oxidized IL33-01 (oxIL-33) activates several key signaling molecules consistent with the pathways involved in receptor tyrosine kinases (RTKs). Figure 1 ).

[0407] 6. The oxidized form of IL-33 activates the epidermal growth factor receptor (EGFR). To investigate and identify receptor tyrosine kinases (RTKs) activated by oxIL-33, a 71 RTK array was used for screening. The RTK phosphorylation antibody array kit (ab193662) was purchased from Abogen Biosciences and experiments were performed according to the manufacturer's instructions. NHBEs were cultured and screened at 1 x 10-16 Cells were seeded at 2 ml / well in 6-well plates (Corning Costar, 3516) using medium [BEGM (Lonza CC-3171) and supplement kit (Lonza CC-4175)]. Cells were incubated at 37°C, 5% CO2 for 18–24 h. Afterward, the medium was aspirated, and the cells were washed twice with 1 ml PBS, followed by the addition of starvation medium (BEGM (Lonza CC-3171) without supplement kit). The plates were then incubated again at 37°C, 5% CO2 for 18–24 h before stimulation. Cells were activated (using the activators in Table 2), lysed, and 250 µg of total protein was used per array membrane, following the same procedure described previously for MAP kinase arrays. All subsequent steps were performed according to the manufacturer's instructions. The membranes were visualized on LiCorC-digit and quantified using Image Lite studio. No response was detected to reduced wild-type (IL-33) or C->S (IL-33[C->S]) IL-33 (IL33-01 and IL33-16, respectively). However, oxIL-33 (oxidized IL-33-01) triggered a positive signal corresponding to the epidermal growth factor receptor (EGFR) on the RTK array. Figure 2 ).

[0408] The ability of oxIL-33 (oxidized IL-33-01) to stimulate EGFR signaling was confirmed by other methods. Upon activation, EGFR was phosphorylated at Tyr1068, and this phosphorylated EGFR could be detected using homogeneous FRET (fluorescence resonance energy transfer) HTRF® (homogeneous time-resolved fluorescence, Cisbio International) (Cisbio kit #64EG1PEH). In short, NHBE was 5 x 10⁻⁶ 5Cells were seeded at 100 µl per cell in 96-well plates (Corning Costar, 3598) using BEGM (Lonza CC-3171) and supplementary kit (Lonza CC-4175). The plates were incubated at 37°C and 5% CO2 for 18–24 hours. Afterward, the medium was aspirated, and the cells were washed twice with 0.2 ml PBS, followed by the addition of starvation medium (BEGM (Lonza CC-3171) without supplementary kit). The plates were then incubated at 37°C and 5% CO2 for another 18–24 hours, followed by stimulation with increased concentrations of IL-33-01, IL-33-16, and oxIL-33 (oxidized IL-33-01), as well as EGFR ligands (Tables 2 and 3). The plates were then returned to the incubator at 37°C and 5% CO2 for 10 min. Aspirate the culture medium and replace each well with 50 µl of lysis buffer (Ziesborg, 64EG1PEH). Then perform assays according to the manufacturer's instructions (Ziesborg, 64EG1PEH). Time-resolved fluorescence was read at emission wavelengths of 620 nm and 665 nm using an EnVision plate reader (PerkinElmer). Data were analyzed by calculating the 665 / 620 nm ratio and determining the EC50 value using curve fitting with a four-parameter logarithmic equation via GraphPad Prism software.

[0409] Table 3 agonists supplier identifier Reconstructed from Final concentration (µg / ml) TGFα R&D Systems Company 239-A-100 10 mM acetic acid 100 HB-EGF R&D Systems Company 259-HE-050 / CF PBS 100 AREG (ambidextrin) R&D Systems Company 262-AR-100 / CF PBS 100 β-cytokinin / BTC R&D Systems Company 261-CE-010 / CF PBS 100 Epithelial regulatory proteins R&D Systems Company 1195-EP-025 / CF PBS 100 Epithelial cell mitotic proteins R&D Systems Company 6629-EP-025 / CF PBS 100 HMGB1 R&D Systems Company 1690-HMB-050 PBS 200 S100A8 / A9 R&D Systems Company 8226-S8-050 PBS 500 S100A12 R&D Systems Company 1052-ER-050 PBS 200 S100B R&D Systems Company 1820-SB-050 PBS 200

[0410] Similarly, EGFR phosphorylation in the epithelial cell line A549 was assessed using the HTRF assay as previously mentioned in this section. Briefly, A549 cells were obtained from ATCC and cultured in RPMI GlutaMax medium supplemented with 1% penicillin / streptomycin and 10% FBS. Cells were harvested using an acute enzyme (PAA, #L1 1-007) at 5 x 10⁻⁶ cells / year. 5Cells were seeded at 100 µl per well in 96-well plates and incubated at 37°C, 5% CO2 for 18–24 h. The wells were then washed twice with 100 µl of PBS, followed by the addition of 100 µl of starvation medium (RPMI GlutaMax medium supplemented with 1% penicillin / streptomycin) and incubated at 37°C, 5% CO2 for 18–24 h. Cells were stimulated with increased concentrations of IL-33-01, IL-33-16, and oxIL-33-01 (synonyms for oxidized IL-33-01), EGFR ligands, and RAGE ligands (Tables 2 and 3), and then returned to the incubator at 37°C, 5% CO2 for 10 min. The medium was aspirated, and each well was replaced with 50 µl of lysis buffer (Ziesborg, 64EG1PEH). Assays were then performed according to the manufacturer's instructions (Ziesborg, 64EG1PEH). Time-resolved fluorescence was read at emission wavelengths of 620 nm and 665 nm using an EnVision plate reader (PerkinElmer). Data were analyzed by calculating the 665 / 620 nm ratio and determining the EC50 value using curve fitting of a four-parameter logarithmic equation with GraphPad Prism software.

[0411] In both NHBE and A549 cells, oxIL-33 promoted EGFR phosphorylation similarly to the true agonist EGF (Figure 3). Other RAGE ligands were not tested.

[0412] 7. Western blot of signal transduction components Western blot experiments were performed to further investigate which elements of the EGFR signaling complex are activated in response to oxIL-33 (oxidized IL33-01). NHBE cells were cultured and seeded in 6-well dishes as described above in Section 5. After serum starvation, cells were stimulated with oxIL-33 (30 ng / ml) for 5 to 240 minutes. The culture medium was then aspirated, and the cells were washed with ice-cold PBS, followed by the addition of 150 µl of lysis buffer [1x LDS sample buffer (Thermo Fisher Scientific, NP0008), 10 mM MgCl2 (VWR, 7786-30-3), 2.5% β-mercaptoethanol (Sigma, M6250), and 0.4 µg / ml totipotent nuclease (Millipore, 70746)]. Cells were incubated on ice for 10 min, and the lysate was then transferred to 1.5 ml tubes and heated to 90°C for 5 min. Transfer the solution to a new 1.5 ml tube and run 10 µl of sample and 5 µl of protein ladder (BioRad, 1610374) in MES run buffer (B0002) on a 4–12% SDS-PAGE gel (Thermo Fisher Scientific, NW04127BOX). Transfer the gel to a PVDF membrane (BioRad, 1704156) using a Transblot Turbo (BioRad). Block the PVDF membrane in PBS-Tween solution containing 5% skim milk powder (Marvel) for 10 min. Then incubate the membrane with the primary antibody in PBS-Tween solution containing 5% BSA overnight at 4°C. Wash the membrane five times with PBS-Tween and then incubate it with HRP-tagged secondary antibody in PBS-Tween solution containing 5% skim milk powder for 1 h at room temperature. The membrane was then washed five times with PBS-Tween, followed by the addition of ECL (Bio-Rad Laboratories, 1705062) and visualization using Licor C-digit.

[0413] The results showed that oxIL-33-01 activated several EGFR signal transduction components ( Figure 4 ) 8. Ox-IL-33 induces STAT-5 phosphorylation, which is blocked by EGFR-neutralizing Ab. Next, we attempted to determine whether oxIL33-mediated STAT5 activation could be inhibited by blocking binding to EGFR. Briefly, A549 cells were cultured in RPMI GlutaMax medium supplemented with 1% penicillin / streptomycin and 10% FBS. Cells were harvested using an acute enzyme at 5 x 10⁻⁶ cells / year. 5Inoculate 100 µl of each antibody into a 96-well plate and incubate at 37°C, 5% CO2 for 18–24 hours. Then wash the wells twice with 100 µl of PBS, followed by adding 100 µl of starvation medium (RPMI GlutaMax medium supplemented with 1% penicillin / streptomycin), and incubate at 37°C, 5% CO2 for 18–24 hours. Add anti-EGFR antibody (clone LA1 (05-101, Millipore) or isotype control (MAB002, R&D Systems) to the wells in a dose-dependent manner, and incubate the plate back in the incubator for 30 min. Stimulate the plate with oxidized IL-33 (30 ng / ml) for 30 min, then lyse and develop the ELISA using phosphate-STAT5 ELISA kit lysis buffer (85-86112-11, ThermoFischer Scientific) according to the manufacturer's instructions, and read the absorbance at 450 nM. Figure 5 As shown, cells activated with oxIL-33-01 exhibit STAT5 phosphorylation, which is reduced in the presence of anti-EGFR antibodies. Figure 5 ).

[0414] Example 2 - Oxidized IL-33 induces the formation of a complex between EGFR and RAGE 9. oxIL-33 induces the formation of a complex between EGFR and RAGE. To understand how RAGE and EGFR are involved in promoting oxIL-33 signaling, immunoprecipitation experiments were performed to explore the signaling complex. First, anti-EGFR antibody was covalently conjugated with Dynabead. Two 100 µg vials of anti-EGFR antibody (R&D Systems, AF231) were incubated with 40 mg Dynabead (Thermo Fisher Scientific, 14311D) and covalently conjugated according to the manufacturer's instructions. After successful conjugation, the beads were resuspended in PBS at 30 mg / ml and maintained at 4°C.

[0415] Obtain NHBE (CC-2540) from Lonza, and place frozen vials at 1 x 10 cm⁻¹ per culture dish. 6Cells were directly seeded into 15 cm culture dishes (Thermo Fisher Scientific, 157150). Following the manufacturer's protocol, NHBE cells were maintained in complete BEGM medium (Lonza) for one month, with the medium changed every three days until cell confluence was achieved. These plates were incubated at 37°C and 5% CO2 for this period. The day before stimulation, the plates were washed twice with 20 ml PBS, followed by the addition of 15 ml starvation medium (BEGM without supplementation kit (Lonza CC-3171)). The plates were then incubated at 37°C and 5% CO2 for another 18–24 hours, followed by stimulation with separate medium (unstimulated control), 30 ng / ml reduced IL-33-01, 30 ng / ml oxidized IL-33-01, or 30 ng / ml EGF, and then returned to 37°C and 5% CO2 for 10 min. The culture medium was aspirated, and the plate was washed twice with ice-cold PBS. Then, 1 ml of lysis buffer (Eppendorf, ab152163) containing phosphatase and protease inhibitors (Thermo Fisher Scientific, 78440) was added per 15 cm culture dish. Cells were scraped into the lysis buffer and then transferred to 2 ml protein LoBind tubes (Eppendorf, Z666513) and clarified by centrifugation at 14,000 rpm at 4°C. Protein concentrations were determined using a BCA kit (Thermo Fisher Scientific, 23225), and all protein extracts were normalized to 3 mg / ml with lysis buffer. 6 mg of total protein extract was incubated in 2 ml clean LoBind tubes containing 100 µl of anti-EGFR Dynabead (as described above). The tubes were then placed on a vertical cylindrical mixer at 4°C for 5 h. Dynabeads were immobilized using a magnet (Bio-Rad Laboratories, 1614916), and the protein extract was aspirated and replaced with 2 ml of Wash Buffer 1 (50 mM Tris-HCl pH 7.5 (Thermo Fisher Scientific, 15567027), 0.5% Triton X 100 (Sigma-Aldrich, X100), 0.3 M NaCl). This was repeated four times. The beads were then washed ten more times in the same manner with Wash Buffer 2 (50 mM Tris-HCl pH 7.5). After the final wash, 50 mM Tris-HCl pH 8.0 containing 50 µl of 1% Rapid (w / v) (Waters, 186001861) was added to the beads, and the mixture was heated at 60°C for 10 min. The supernatant was then transferred to a new LoBind 2 ml tube. Add 100 µl of 50 mM Tris-HCl pH 8.0 to the resin and mix, then combine it with the first eluent.Then, TCEP (Sigma-Aldrich, 646547) was added to a final concentration of 5 mM, and the sample was heated at 60 °C for 10 min. The eluent was then alkylated for 20 min by adding iodoacetamide (Sigma-Aldrich, 16125) to 10 mM in the dark at room temperature. Alkylation was quenched by adding DTT (Sigma-Aldrich, D5545) to 10 mM. Tris-HCl buffer (50 mM, pH 8.0) was then added to obtain a final sample volume of 500 µl. 0.5 µg of trypsin (Promega, V5111) was added to each tube, and the sample was digested overnight at 30 °C on a shaking platform at 400 rpm. The sample was then acidified with trifluoroacetic acid (Sigma-Aldrich, 302031) to a final concentration of 2.0% (v / v) and incubated at 37 °C for 1 h. The sample was then centrifuged at 14,000 rpm for 30 min, and the supernatant was transferred to a new 2 mL LoBind tube. The sample was then processed using a C18 column (Thermo Fisher Scientific, 87784) according to the manufacturer's instructions. The sample was then dried using Speed-Vac and subsequently stored at -20°C. The sample was then analyzed by peptide mass fingerprinting mass spectrometry (PMF-LC-MS). Results were analyzed using Scaffold software.

[0416] EGFR was similarly detected under all four conditions, indicating that immunoprecipitation performed well in all samples. RAGE and IL-33 were detected in samples treated with oxIL-33 compared to samples treated with IL33-01 (IL-33) or EGF, suggesting that oxIL-33 and RAGE are associated with EGFR during signal transduction. Consistent with previous observations of EGFR activation in these cells using oxIL-33 and EGF, proteins previously reported to be involved in EGFR signaling and endocytosis, but not reduced IL33-01, were detected after stimulation with these ligands (Table 4).

[0417] Table 4 shows the LCMS analysis of NHBE stimulated with reduced IL-33-01 (IL-33), oxIL-33 (oxidized IL-33-01), or EGF. After stimulation with oxIL-33, IL-33 and RAGE complexed with EGFR were detected, but not after stimulation with reduced IL-33-01 (IL-33) or EGF. Parentheses indicate the number of unique peptides identified for each protein.

[0418] Table 4 Unstimulated IL-33 oxIL-33 EGF EGFR (63) EGFR (62) EGFR (60) EGFR (57) - - IL-33 (11) - - - RAGE (11) - - - AP-2α1 (20) AP-2α1 (14) - - AP-2α2 (16) AP-2α2(10) - - AP-2β(15) AP-2β(16) - - AP-2µ(20) AP-2µ(20) - - AP-2σ(10) AP-2σ(11) - - CBL-B(5) CBL-B(4)

[0419] To confirm these observations, the cell lysates prepared according to the above protocol were subjected to immunoprecipitation and Western blotting. After determining the concentration of the NHBE protein extract, 3 mg of total protein was incubated with 6 µg of anti-EGFR antibody (R&D Systems, AF231) in 1.5 ml tubes and placed on an end-over-end mixer at 4°C for 2.5 h. Then, 1.5 mg of protein A / G magnetic beads (Thermo Fisher Scientific, 88802) were added to each tube, and the tubes were returned to 4°C and maintained under mixing for another 1 h. The beads were then collected using a magnet (Bio-Rad Laboratories, 1614916) and washed three times with 500 µl of 50 mM Tris (pH 7.5), 1% Triton X, and 0.25 M NaCl, and once with 500 µl of 10 mM Tris (pH 7.5). The protein was then released from the magnetic beads using 35 µl of LDS sample buffer (Thermo Fisher Scientific, NP0008) containing a reducing agent (Thermo Fisher Scientific, NP0004) and heated at 95 °C for 5 min. The solution was transferred to a new 1.5 ml tube and run on a 4–12% SDS-PAGE gel (Thermo Fisher Scientific, NW04127BOX) with 10 µl of sample and 5 µl of protein ladder (BioRad, 1610374) in MES run buffer (B0002). The gel was transferred to a PVDF membrane (BioRad, 1704156) using a Transblot Turbo (BioRad). The PVDF membrane was then blocked in PBS-Tween solution containing 5% skim milk powder (Marvel) for 10 min. The membrane was then incubated overnight at 4°C with primary antibodies (anti-EGFR (CellSignaling Technology, 2232), anti-RAGE (CellSignaling Technology, 6996), or anti-IL-33 (R&D Systems, AF3625)) in PBS-Tween containing 5% BSA at 4°C. The membrane was then washed five times with PBS-Tween and incubated for 1 hour at room temperature in PBS-Tween containing 5% skim milk with either a secondary antibody against rabbit HRP-tagged antibodies (CellSignaling Technology, 7074) or an anti-goat HRP-tagged secondary antibody (R&D Systems, HAF109). The membrane was then washed five times with PBS-Tween, followed by the addition of ECL (Bio-Rad Laboratories, 1705062) and visualization using Licor C-digit. Western blot analysis confirmed that RAGE and EGFR co-precipitated in the presence of oxIL-33, while RAGE was not detected upon EGF stimulation. Figure 6 These findings reveal that RAGE and EGFR are functional components of the oxidized IL-33 signaling complex.

[0420] 10. oxIL-33 requires the formation of a complex between RAGE and EGFR The experiments described above demonstrate that oxIL-33 is a ligand for the EGF receptor (EGFR) complex that induces downstream signal transduction. The experiments in this section are designed to determine whether oxIL-33 is a direct binding ligand for RAGE or EGFR. To further understand the formation of the signal transduction complex and assess whether oxIL-33 directly interacts with EGFR, ELISA was used to explore the binding of oxIL-33 to RAGE, ST2-Fc, and EGFR.

[0421] Proteins and Modifications: Proteins containing the Avitag sequence motif (GLNDIFEAQKIEWHE SEQ ID NO:46) were biotinylated using biotin ligase (BirA) (Avidty, Bulk BirA) following the manufacturer's protocol. All Avitag-free proteins used herein were biotinylated via free amines using EZ-linked sulfonyl-NHS-LC-biotin (Thermo / Pierce, 21335) following the manufacturer's protocol. Table 5 is a list of the biotinylated proteins used.

[0422] Table 5. Reagents Biotinylated EGF (Thermo) Avitag-human ASGPR Avitag_IL-33-01 (reduced IL-33) Avitag_IL-33-01 (oxidized IL-33) Avitag_IL-33-16 HMGB1

[0423] Spread 100 µl / well of biotinylated antigen (10 µg / ml, in PBS) onto a streptavidin-containing plate (Thermo Scientific, AB-1226) at room temperature and incubate for 1 hour. Wash the plate three times with 200 µl PBS-T (PBS + 1% (v / v) Tween-20) and block with 300 µl / well of blocking buffer (PBS containing 1% BSA (Sigma, A9576)) for 1 hour. Wash the plate three times with PBS-T. Dilute RAGE-Fc (R&D Systems #1145-RG) or ST2-Fc (R&D Systems #523-ST) to 10 µg / mL in blocking buffer containing PBS, add to the relevant wells, and incubate at room temperature for 1 hour. Alternatively, in the presence or absence of PBS containing 10 µg / mL unlabeled RAGE (Sino Biological, 11629-HCCH), 100 µl of PBS containing 10 µg / mL EGFR-Fc (R&D Systems #344-ER-050) was added and maintained for 1 hour. The plate was washed three times with 200 µl of PBS-T. Then, RAGE-Fc, ST2-Fc, and EGFR-Fc were detected at room temperature for 1 hour with 100 µl / well of anti-human IgG HRP (Sigma AO170, 5.1 mg / mL) diluted 1:10000 in blocking buffer. The plate was washed three times with PBS-T and developed with 100 µl / well of TMB (Sigma, T0440). The reaction was quenched with 50 µl / well of 0.1 M H2SO4. Absorbance was read at 450 nm using a Cytation Gen5 or similar device. The results showed that oxIL-33 exhibited a significant interaction with RAGE ( Figure 7A ), while the direct binding of oxIL-33 to EGFR is negligible ( Figure 7B EGFR binding to oxIL-33 was observed simply by adding sRAGE to this assay. Figure 7B This cannot be reproduced if the true RAGE agonist HMGB1 is replaced with oxIL-33. Figure 7B ).

[0424] The use of RAGE-deficient cell lines further confirmed the requirement of RAGE in oxIL-33-triggered EGFR signaling. The following is an example of generating an A549 cell line with RAGE knocked out: A mammalian plasmid containing an expression vector for red fluorescent protein (RFP), a guide RNA targeting exon 3 of AGER (TGAGGGGATTTTCCGGTGC SEQ ID NO:47), and a Cas9 endonuclease was generated. A549 conditioned medium was prepared by growing A549 cells in T-175 flasks in F12K nut mix (Gibco, supplemented with 10% FBS and 1% penicillin / streptomycin) for two days. The consumed medium was removed from the A549 cells, filtered, and diluted 5-fold with fresh Gibco F12K nut mix (supplemented with 20% FBS and 1% penicillin / streptomycin). A549 cells were cultured at 2 x 10⁻⁶ cells / year. 5Cells / ml were seeded into three T-75 flasks, totaling 15 ml, and incubated overnight at 37°C with 5% CO2. A transfection mixture was prepared using 1.6 ml of F12K nut mix (supplemented with 1% penicillin / streptomycin), 8 µg of AGER guide RNA plasmid, and 22.5 µg of PEI (Polysciences, 23966-2). The mixture was then vortexed for 10 seconds and incubated at room temperature for 15 min. 0.75 ml of the transfection mixture was then added to each T-75 flask. The flasks were returned to the incubator and maintained for two days. A549 cells were then isolated using an acute enzyme, transferred to PBS containing 1% FBS, and single cells were sorted using an Aria cell sorter (BD) based on RFP expression in 96-well dishes. Cells were fed with conditioned medium every 3–5 days. Once cells reached over 50% confluence, they were transferred to 24-well plates and allowed to grow. This grading process continued until each successful clone was aliquoted into a T15 flask. Cells were then aliquoted into 12-well plates and grown until more than 50% confluence was achieved, followed by analysis for successful genomic PCR knockout. Cells in each well were lysed in 100 µl of DNA lysis buffer (Viagen Bitoech, 301-C, supplemented with 0.3 µg / ml proteinase K). These samples were incubated at 55°C for 4 h, followed by incubation at 85°C for 15 min. RAGE PCR was performed using forward and reverse primers with the following sequences: forward -gttgcagcctcccaacttc (SEQ ID NO:48), reverse -aatgaggccagtggaagtca (SEQ ID NO:49). The reaction and cycling settings were as follows: The reaction volume was 50 µl [25 µl Q5 polymerase mixture, 2.5 µl forward primer (10 µM stock solution), 2.5 µl reverse primer (10 µM stock solution), 2 µl template DNA lysate, and 18 µl nuclease-free water]. PCR was performed with an initial denaturation of 30 seconds at 98 °C, followed by 35 cycles of the following: 5 seconds at 98 °C, 10 seconds at 57 °C, and 20 seconds at 72 °C, with a final step at 72 °C for 2 minutes. 4 µl of PCR product was mixed with 6 µl of nuclease-free water and 2 µl of 6x DNA loading buffer (Thermo Scientific, R0611). The sample was run on a 1% agarose gel (1:10000 SYBR safe) at 90 V for 1 hour, and then visualized on a Versadoc imager. Then, following the manufacturer's instructions, the remaining PCR products were cleaned using the QIAquick PCR Purification Kit (Qiagen, 28104). The DNA-50 concentration was measured using nanodrop.Several clones (selected from the results) were sent for internal sequencing. The results showed that stop codons were successfully inserted in clones RAGE09 and RAGE10.

[0425] To determine the necessity of RAGE for oxIL-33-mediated EGFR signaling, immunoprecipitation and Western blotting were performed on A549 and RAGE-deficient A549 cells. Briefly, cell lines were activated with oxIL-33-01 at various time points (0–15 min). Immunoprecipitation for EGFR or RAGE was then performed, followed by Western blotting with anti-RAGE, anti-EGFR, and anti-IL-33 agents, following the experimental protocol detailed in Section 9. The results showed a crucial role for RAGE in the formation of a complex with oxIL-33 and EGFR. Figure 8 ) 11. Oxidized IL-33 induces STAT5 phosphorylation, which is blocked by RAGE but not by ST2 neutralizing antibody block To confirm that RAGE is more important than ST2 in oxIL-33 signaling, blocking antibodies were tested. Briefly, A549 cells were cultured in RPMI GlutaMax medium supplemented with 1% penicillin / streptomycin and 10% FBS. Cells were harvested using an acute enzyme at 5 x 10⁻⁶ cells / year. 5 Inoculate 100 µl of each sample into 96-well plates and incubate at 37°C and 5% CO2 for 18–24 hours. Then wash the wells twice with 100 µl of PBS, followed by adding 100 µl of starvation medium (RPMI GlutaMax medium supplemented with 1% penicillin / streptomycin) and incubating at 37°C and 5% CO2 for 18–24 hours. Add anti-RAGE (M4F4; WO 2008137552); anti-ST2 (AF532; RnD Systems) or allotype control (MAB002, R&D Systems) to the wells in a dose-dependent manner, and incubate the plates back in the incubator for 30 min. The plate was then stimulated with oxidized IL-33 (30 ng / ml) for 30 min, followed by lysis and development using phosphate-STAT5 ELISA kit lysis buffer (85-86112-11, Thermo Fisher Scientific) according to the manufacturer's instructions. The absorbance was then read at 450 nM. Figure 9 As shown, cells activated with oxIL-33-01 exhibit STAT5 phosphorylation, which is reduced in the presence of anti-RAGE antibody but not anti-ST2 antibody. Figure 9 ).

[0426] Example 3 - oxIL-33 triggers internalization of EGFR in epithelial cells The next step was to investigate whether oxIL-33 induces EGFR kinetic changes compared to EGF.

[0427] 12. Confocal experiment of EGF internalization This experiment aimed to investigate the dynamics of EGFR in epithelial cells after stimulation with reduced or oxidized forms of EGF and IL-33 using confocal imaging. EGFR-GFP A549 epithelial cell line (Sigma, CLL1141-1VL) was seeded at a concentration of 20,000 cells / ml (RPMI medium + 10% FCS + Pen / Strep) in 1 ml of 24-well glass plates (Greiner, 662892). The EGF receptor linked to green fluorescent protein (GFP) could be used to track EGFR membrane dynamics and internalization. Cells were washed once with PBS and incubated in RPMI medium (FCS-free). After 24 hours of starvation, cells were washed with RPMI and incubated with 0.5 ml of RPMI medium and CellMask (Invitrogen C10046) Deep Red at a 1:5000 dilution. Cells were briefly stained with CellMask prior to membrane labeling treatment and immediately imaged in real-time at 1 frame / min during confocal processing to record EGFR-GFP kinetics. Cells were stained at 37°C for 5 min, washed once with PBS, and stimulated with 0.5 ml serum-free RPMI / well using 200 ng / ml oxIL-33 (oxidized IL-33-01) or IL-33-16. Confocal images were immediately captured with a 40x oil objective at 1 min / frame, 5 stacks at 2 µm intervals for 25 min (approximately 30 min after protein addition). The interruption of the GFP signal (dashed line) pattern indicates receptor clustering and internalization on the membrane. Pixel intensity histograms were generated at different time points during real-time imaging for membrane regions (masked by CellMask) and intracellular regions (masked by an inverted CellMask, not shown), showing EGFR depletion in non-clustered regions (leftward shift of the histogram bell peak) and an increase in the number of saturated pixels (intensity 255) caused by clustering. oxIL-33 induces clustering and internalization of EGF receptors, but EGF stimulation elicits the most pronounced EGFR clustering. Conversely, the reduced form of IL-33 (IL-33-16) did not show significant changes in EGFR cell distribution. Figure 10 ).

[0428] Example 4 - Similar to EGF, oxIL-33 induces the secretion of IL-8 in epithelial cells 13. Selective secretion of IL-8 by oxIL-33 According to the manufacturer's protocol, human bronchial epithelial cells from healthy subjects (NHBE; Lonza CC-2540) and chronic obstructive pulmonary disease (COPD) (DHBE; Lonza 00195275) were maintained in complete BEGM medium (Lonza) for one month, with the medium changed every three days until the cells reached confluence. Cells were harvested using an acute enzyme and cultured at 5 x 10⁻⁶ cells / day. 5 Cells were seeded at 100 µl per cell in 96-well plates (Corning 3596). These plates were incubated at 37°C, 5% CO2 for 18–24 hours. Afterward, the medium was aspirated, and the cells were washed twice with 100 µl of PBS, followed by the addition of starvation medium (BEGM (Lonza CC-3171) without supplementation kit containing 1% penicillin / streptomycin). The plates were then incubated at 37°C, 5% CO2 for another 18–24 hours, followed by stimulation with separate medium (unstimulated control), 30 ng / mL reduced IL-33-01, 30 ng / mL IL-33-16, 30 ng / mL oxidized IL-33-01, or 30 ng / mL EGF, and returned to 37°C, 5% CO2. 24 hours post-stimulation, the supernatant was collected and chemokine production was evaluated using a multiplex assay (Mesoscale Discovery K15047D-2). Figure 11 As shown, IL-8 secretion in NHBE and DHBE increased fourfold after activation with oxIL-33 compared to unstimulated cells (culture medium alone). No significant differences were observed in other chemokines (TARC, MIP-1a, MIP1b, MCP4, MCP1, IP10, eosinophil activation chemokine (Eotaxin), eosinophil activation chemokine-3, MDC - data not shown).

[0429] Example 5 - oxIL-33 impairs the scratch repair response in deep monolayer epithelial cultures 14. In contrast to EGF, oxIL-33 impairs scratch closure in A549 and NHBE cells A549 cells were obtained from ATCC and cultured in RPMI GlutaMax medium supplemented with 1% penicillin / streptomycin and 10% FBS. Cells were harvested with an acute enzyme (PAA, #L1 1-007) at 5 x 10⁻⁶ cells / year. 5Cells were seeded at 100 µl per cell in 96-well plates and incubated at 37°C, 5% CO2 for 18–24 hours. The wells were then washed twice with 100 µl of PBS, followed by the addition of 100 µl of starvation medium (RPMI GlutaMax medium supplemented with 1% penicillin / streptomycin) and incubated at 37°C, 5% CO2 for 18–24 hours. Cells were then dissected using a WoundMaker™ (Essen Bioscience), followed by washing twice with 200 µl of PBS. The wells were then incubated with RPMI GlutaMax medium supplemented with 0.1% FBS (v / v) and 1% (v / v) penicillin / streptomycin containing the specified stimulus, or with a separate medium (unstimulated control), 30 ng / mL reduced IL-33-01, 30 ng / mL oxidized IL-33-01, or 30 ng / mL EGF, and returned to 37°C, 5% CO2. The plate was placed in IncucyteZoom for scratch healing imaging and analysis over 48 hours. Relative scratch density was calculated using the scratch healing algorithm in the Incucyte Zoom software.

[0430] NHBE (CC-2540) was obtained from Lonza and maintained in complete BEGM medium [BEGM (Lonza CC-3171) and supplement kit (Lonza CC-4175)] according to the manufacturer's protocol. Cells were harvested with an acute enzyme and cultured at 5 x 10⁻⁶ cells / year. 5Cells were seeded at 100 µl per cell in 96-well ImageLock plates (Sartorius, 4379). These plates were incubated at 37°C and 5% CO2 for 18–24 hours. Afterward, the medium was aspirated, and the cells were washed twice with 100 µl of PBS, followed by the addition of starvation medium (BEGM (Lonza CC-3171) without supplementation kit containing 1% penicillin / streptomycin). The plates were then incubated again at 37°C and 5% CO2 for 18–24 hours before scratch formation. Cells were scratched using WoundMaker™ (Essen Biosciences), and wells were washed twice with 200 µl PBS. Then, BEBM medium (Lonza) supplemented with 0.1% FBS (v / v) and 1% (v / v) penicillin / streptomycin containing the specified stimulus was added, along with either BEBM medium (Lonza) or a separate medium (unstimulated control), 30 ng / mL reduced IL-33-01, 30 ng / mL oxidized IL-33-01, or 30 ng / mL EGF, and the plates were incubated at 37°C, 5% CO2. Plates were placed in Incucyte Zoom for scratch healing imaging and analysis within 48 hours. Relative scratch density was calculated using the scratch healing algorithm in Incucyte Zoom software. As shown in Figure 12, oxIL-33 inhibited A549 cells (… Figure 12A ) and NHBE cells ( Figure 12B Scratch healing in deep cultures of EGF showed an opposite effect to the observed increase in scratch cell density.

[0431] 15. The damage of oxidized IL-33 to scratch closure can be blocked by antibodies that neutralize RAGE or EGFR rather than ST2 damage To investigate whether these functional roles of oxIL-33 are mediated through RAGE / EGFR, as described in Section 14, scratch assays were performed in NHBE cells in the presence of antibodies neutralizing different receptor components. NHBE cells were treated with separate culture medium (unstimulated control), reduced IL-33, or oxidized IL-33 in the presence of 10 µg / mL anti-ST2 (AF532, R&D Systems), anti-RAGE (M4F4, WO 2008137552), or anti-EGFR (clone LA1, 05-101 Millipore). oxIL-33, but not reduced IL-33, inhibited scratch closure. This effect of oxIL-33 was reversed by anti-RAGE and anti-EGFR but not anti-ST2, further demonstrating that RAGE and EGFR are essential receptors involved in the oxidized IL-33 signaling pathway. Figure 13 ).

[0432] Example 6 - Anti-IL-33 improves the phenotype of COPD cells in deep cultures 16. oxIL-33 can drive a COPD-like response in healthy NHBE in a scratch closure assay Next, the effects of oxidized IL-33 in bronchial epithelial cells from healthy individuals, smokers, and those with COPD were investigated. NHBE (CC-2540), NHBE (CC-2540) from smokers, and DHBE (COPD, 00195275) were obtained from Lonza and maintained in complete BEGM medium (Lonza) according to the manufacturer's protocol. Scratch assays were performed as described in Section 14. Cells were treated with either a separate medium (unstimulated control) or 30 ng / mL oxidized IL-33. Compared to cells from healthy subjects, bronchial epithelial cells from smokers or those with COPD showed impaired scratch closure ability, similar to the damage observed after treatment of healthy cells with oxIL-33. Figure 14 Compared to healthy cells, oxIL-33 did not further impair the scratch closure response in smokers and COPD HBE cells. Figure 14 ).

[0433] 17. Blocking endogenous IL-33 through the RAGE / EGFR pathway can improve the impaired scratch repair phenotype of COPD basal cells .

[0434] Since epithelial cells are known to produce IL-33, autocrine IL-33 secretion may lead to impaired scratch repair phenotypes observed in COPD cells. To investigate this, scratch closure assays were performed in bronchial epithelial cells from COPD in and in the presence of IL-33. NHBE (Lonza CC-2540) and DHBE (Lonza COPD 00195275) were maintained in complete BEGM medium (Lonza) according to the manufacturer's protocol. Cells were harvested with an acute enzyme and cultured at 5 x 10⁻⁶ cells / mL. 5Cells were seeded at 100 µl per cell in 96-well ImageLock plates (Sartorius, 4379). These plates were incubated at 37°C and 5% CO2 for 18–24 hours. Afterward, the medium was aspirated, and the cells were washed twice with 100 µl of PBS, followed by the addition of starvation medium (BEGM (Lonza CC-3171) without supplementation kit containing 1% penicillin / streptomycin). The plates were then incubated again at 37°C and 5% CO2 for 18–24 hours before scratch formation. Cells were scratched using WoundMaker™ (Essen Biosciences), and wells were washed twice with 200 µl PBS. Then, BEBM medium (Lonza) supplemented with 0.1% FBS (v / v) and 1% (v / v) penicillin / streptomycin was added, containing 10 µg / mL anti-IL-33 (33_640087-7B, described in WO2016 / 156440), anti-ST2 (AF532, R&D Systems), anti-RAGE (M4F4, WO 2008137552), or NIP228 (IgG1 isotype control). The plates were then incubated at 37°C, 5% CO2. Plates were placed in Incucyte Zoom for scratch healing imaging and analysis within 48 hours. Relative scratch density was calculated using the scratch healing algorithm in Incucyte Zoom software. As previously observed, scratch closure response was impaired in COPD cells compared to cells derived from healthy subjects. Anti-IL-33 and anti-RAGE, but not anti-ST2, improved the scratch closure response of COPD cells to a level similar to that of healthy cells. Figure 15 This demonstrates that epithelial cells produce autocrine IL-33, which transduces signals via the RAGE / EGFR pathway. Figure 15 ).

[0435] Example 7 - Anti-IL-33 reduces goblet cells in 3D epithelial cultures 18. Airway basal cell air-liquid interface (ALI) culture Next, the inventors sought to determine the correlation of IL-33 signaling in air-liquid interface cell cultures (“ALI cultures”). ALI culture is a method of basal cell growth in which the basal surface of these basal cells is in contact with a culture medium, and the top (apical) cell layer is exposed to air. ALI culture can generate, in vitro, three-dimensional cellular structures with a pseudostratified epithelial mucociliary phenotype similar to tracheal epithelium. Therefore, ALI cultures can be used to study fundamental aspects of respiratory epithelium, such as intercellular signaling, disease modeling, and respiratory regeneration.

[0436] Frozen vials containing frozen lung basal cells from healthy controls or COPD patients were received from the University of North Carolina and the University of Pittsburgh. Cells were thawed and plated in T-75 flasks coated with Purecol type I bovine collagen (Advanced BioMatrix, San Diego, CA) diluted 1:70 in 1X PBS (Gibco, Waltham, MA) and grown in Epix medium (Propagenix, Rockville, MD 276-201). Once confluence was reached, these cells were aliquoted into appropriate numbers of T-75 flasks and subsequently harvested for ALI culture. Transwells for ALI culture containing 12 mm 0.4 µM polyester membrane inserts (Costar, Corning Incorporated, NY) were prepared by coating the inserts with a 1:70 Purecol solution and incubating at 37°C for 1–16 hours. The Purecol solution was removed, and the Transwells were placed under UV light for 30 minutes, followed by washing with PBS. Basal cells in T-75 flasks were isolated using 4 ml of trypsin solution (Thermo Fisher Scientific, 15400054). The cell suspension was added to 50 ml tubes containing 5 ml of FBS, counted on a ViCell counter (Beckman Coulter, Brea, CA), and centrifuged at 1,000 RPM for 5 minutes. Cells were then cultured at 3.57 x 10⁻⁶ cells / mL. 5 Resuspend the cells at a density of cells / ml in Pneumacult ALI medium (Stemcell Tech, Vancouver, BC) and dispense 700 µl onto each Transwell. Add 1 mL of ALI medium to the space beneath the insert. Allow the cells to remain deep in the ALI medium until confluence and tight junctions form (typically 7 days), at which point remove the medium from the top side to allow cell differentiation for 2 weeks, during which the medium is changed every other day on the basal side. Fully differentiated cultures are treated antibody-free by including a treatment in the medium supplied to the basal side of the culture, either with 1 µg / ml anti-IL-33 (33_640087-7B) or 1 µg / ml NIP228 (IgG1 isotype control) for 7 days. The medium (containing the relevant treatment) is changed every other day.

[0437] 19. IHC triple staining (basal, goblet, and ciliated) and quantification ALI cultures from COPD donors were generated and processed as described in Section 18. ALI epithelial cultures were fixed in 10% neutral-buffered formalin for 24 hours and embedded in paraffin. Paraffin sections (4 μm) were mounted on positively charged slides and stained on Ventana Discovery Ultra by sequential triplet staining. Antigen retrieval was performed with cell regulator 1 (CC1) (catalog number 5424569001, Roche) and endogenous peroxidase was blocked for 12 min with the Discovery inhibitor (catalog number 7017944001, Roche). Anti-p63 (clone 4A4) (catalog number 790-4509, Roche, Basel, Switzerland) was applied at 36°C for 24 min, visualized with mouse anti-HQ (12 min) (catalog number 7017782001, Roche) and anti-HQ HRP (12 min) (catalog number 7017936001, Roche), and incubated in Teal substrate (catalog number 8254338001, Roche) for 12 min. The slides were treated with cell regulator 2 (CC2) (catalog number 5424542001, Roche) for 16 min by an antibody denaturation step (100 °C, 24 min), followed by treatment with 0.01 µg / ml anti-microtubule protein (catalog number ab24610, Abogen Biosciences, Cambridge, UK) diluted with Dako antibody diluent (catalog number S3022), detected with mouse OmniMap-HRP (catalog number 5269652001, Roche) for 8 min, and visualized with Discovery Purple substrate (catalog number 7053983001, Roche) for 16 min. The slides were denatured with antibody again using CC2, then treated with a mixture of 1.1 µg / ml rabbit anti-adhesion 5AC and 7 µg / ml rabbit anti-adhesion 5B (catalog numbers ab198294 and ab87376, respectively, Abogen Biosciences) for 20 min. Visualization was then performed using anti-rabbit NP (4 min, catalog number 7425317001, Roche) and anti-NP-AP (8 min, catalog number 7425325001, Roche), followed by Discovery Yellow (catalog number 7698445001, Roche) for 20 min. The stained slides were rinsed with Dawn detergent, counterstained with hematoxylin (catalog number 5277965001, Roche), rinsed, dehydrated with fractionated ethanol and xylene, and mounted with mounting media. Quantitative analysis using HALO software showed a reduction in goblet cells in ALI cultures derived from healthy donors treated with anti-IL-33 (Figure 16).

[0438] Example 8 - Anti-IL-33 regulates mucins in 3D epithelial cultures from COPD and improves mucus motility 20. IHC double IF staining (mucin 5B + mucin 5AC) ALI cultures from COPD donors were generated and processed as described in Section 18. ALI epithelial cultures were fixed in 10% neutral-buffered formalin for 24 hours and embedded in paraffin. Paraffin sections (4 μm) were mounted on positively charged slides and stained on Ventana Discovery Ultra by sequential double immunofluorescence assays. Antigen retrieval was performed with cell regulator 1 (CC1), followed by blocking of endogenous peroxidase for 12 min with a Discovery inhibitor and then for 8 min with Roche Diagnostics' S Block (RUO) (catalog number 760-4212). The mixture was incubated at 36°C for 24 min in 7 µg / ml anti-adhesion protein 5B diluted with Dako Ab diluent S3022, and detected with anti-rabbit HQ (Roche Diagnostics catalog number 760-4815) for 4 min and anti-HQ-HRP (Roche Diagnostics catalog number 760-4820) for 8 min. The samples were then incubated for 8 min with the tyrosamide conjugate Discovery FITC (Roche Diagnostics catalog 760-232). Duplex sequences were selected in the Discovery Ultra program, and the samples were treated with Cell Regulator 2 (CC2) via an antibody denaturation step (100°C, 24 min), followed by neutralization with a Discovery inhibitor (40°C, 24 min), and then subjected to anti-mucin 5AC at 36°C, 1.1 µg / ml, for 20 min. Mucin 5AC was detected with anti-rabbit-HQ (Roche Diagnostics catalog 760-4815) for 4 min and anti-HQ-HRP (Roche Diagnostics catalog 760-4820) for 8 min, and visualized with the tyrosamide conjugate Discovery Red610 for 8 min. After this step, the stained slides were removed from the Discovery Ultra automated staining system, rinsed with Dawn detergent, and then rinsed with deionized water. The sample was incubated for 2 min in 1 µg / ml of 4',6-diamidinyl-2-phenylindole dihydrochloride (DAPI nucleic acid dye) diluted in deionized water (Thermo Fisher Scientific, catalog number D1306). The sample was rinsed with deionized water and covered with a coverslip in the presence of ProLong Gold Antifade mounting medium (Thermo Fisher Scientific, catalog number P36930), and stored in an opaque slide box. The stained slides were imaged using a Zeiss LSM 880 confocal microscope (Carl Zeiss Microscopy, LLC, White Plains, NY). Figure 17Treatment of ALI cultures with anti-IL-33 may induce downregulation of different mucins in COPD cultures.

[0439] 21. Anti-IL-33 reverses the impaired mucociliary clearance observed in COPD ALI cultures ALI cultures from COPD donors were generated and treated as described in Section 18. Then, 30 µl of 0.2 µM FluoSpheres (Thermo Fisher Scientific, F8811) diluted 1:33 in PBS was added to the top surface, and short films of FluoSphere movement were captured using a Zeiss LSM800 microscope, showing increased mucociliary movement after treatment with anti-IL-33 (33_640087-7B) rather than the control antibody.

[0440] Example 9 - Single-cell RNA analysis of ALI cultures shows changes in goblet cells after treatment with anti-IL-33 ALI cultures from COPD donors were generated and processed as described in Section 18. To obtain a single-cell suspension, the filter insert was incubated with 0.25% trypsin at 37°C for 5 min. Epithelial cells were gently separated from the filter by washing with PBS and then transferred to 15 ml Falcon tubes. The cells were centrifuged at 1000 RPM for 5 min at 4°C. After removing the supernatant, the cells were resuspended in PBS containing 0.4% BSA and the cell concentration was adjusted to 1000 cells / µl for sequencing. The cell suspension was loaded into the Chromium Single Cell 3' Kit according to the standard protocol to capture 5000 to 10,000 cells / channel. Version 2 chemistry was used. Single-cell 3' libraries for Illumina sequencing were obtained following the manufacturer's protocol (Chromium™ Single Cell 3' Kit, v2 chemistry). Library quality was assessed (TapeStation 4200, Agilent), and sequencing was performed on a NextSeq 500 or NovaSeq 6000 instrument (Illumina). Initial data processing was performed using the Cell Ranger version 2.0 procedure (10xGenomics). Post-processing was performed using the Seurat package to exclude low-quality cells and normalize. Each sample was analyzed as independent data to capture intrasample heterogeneity (cell subtypes). Clustering and visualization were performed using t-Distributed Stochastic Neighbor Embedding (tSNE). Identification of cell populations in COPD was guided by marker genes. For other samples, the initial populations were manually examined, and then Seurat's label transfer algorithm was applied for subtype identification. MUC5AC and MUC5B gene expression analysis was performed on each population from COPD ALI cultures that underwent and did not undergo anti-IL-33 treatment as described in Section 18. Use Seurat to generate heatmaps and tSNE plots. Figure 18 tSNE plots are shown, illustrating the different proportions of cell subtypes found in ALI cultures treated with anti-IL-33 (33_640087-7B) compared to untreated cultures. Figure 18 As shown, a reduction in MUC5B cells was observed after anti-IL-33 treatment.

[0441] Example 10 - Anti-IL-33 reduces goblet cells in COPD 3D epithelial cultures 22. Airway basal cell air-liquid interface (ALI) culture To quantify and inquire about the role of oxIL-33 in physiologically relevant air-liquid interface (ALI) culture systems, a flow cytometry assay was developed to differentiate goblet cell types (MUC5ac vs. MUC5b) from the remaining epithelial population (mucin-negative).

[0442] Frozen vials containing frozen lung basal cells from healthy (CC-2540) controls or COPD (195275) patients were received from Lonza. Each donor vial was thawed and plated in four T-175 flasks of Epix medium (276-201, Propagini, Rockville, MD). After confluence, these cells were frozen at P2 at 1e6 cells per vial. Cells at P2 were freshly added to two T-75 flasks of Epix medium and grown until 80% confluence. ALI culture using Transwell with 12 mm or 6.5 mm 0.4 µM polyester membrane inserts (Costar, Corning Incorporated, NY) was prepared by coating the inserts with 1x collagen I solution (Celladhere™ Collagen I-Stem Cells #07001, prepared in dH2O) and incubating at 37°C for 1–16 hours. Remove collagen I solution and wash Transwell cells with PBS. Wash basal cells in T-75 flasks with PBS and separate them using 6 ml trypsin solution (Lonza Trypsin Subculture Pack - #CC-5034). Neutralize trypsin with 6 ml trypsin neutralization solution (Lonza Trypsin Subculture Pack - #CC-5034), add cell suspension to 15 ml tubes, count, and centrifuge at 1200 RPM for 5 min. Then, collect cells at 8 x 10⁻⁶ cells / mL. 5Cells were resuspended at a density of cells / ml in Pneumacult ALI medium (Stem Cell Technologies, Vancouver, British Columbia), with 0.5 ml allocated to each 12 mm transwell and 0.25 ml to each 6.5 mm transwell. 1 ml of ALI medium was added to the space beneath the 12 ml inserts, and 0.5 ml of ALI medium was added to the space beneath the 6.5 mm inserts. Cells were allowed to remain in the deep layer of ALI medium until confluence and tight junctions formed (typically 7 days), at which point the medium was removed from the top side, allowing cells to differentiate for 3 weeks, with the medium changed on the basal side every Monday, Wednesday, and Friday. Fully differentiated normal cultures were left untreated, or treated for 7 days with reduced or oxidized unlabeled IL33-01 (30 ng / ml), unlabeled IL33-16 (30 ng / ml), IL-13 (10 ng / ml), EGF (30 ng / ml), or HMGB1 (30 ng / ml) in the medium supplied to the basal side of the culture. Fully differentiated COPD cultures were left untreated, or treated for 7 days with 1 µg / ml anti-IL-33 (33_640087-7B), 1 µg / ml NIP228 (IgG1 isotype control), 10 µg / ml mNIP228, 10 µg / ml anti-ST2, 1 µg / ml anti-RAGE, or 1 µg / ml anti-EGFR in the medium supplied to the basal side of the culture. The medium (containing the relevant treatment) was changed every Monday, Wednesday, and Friday.

[0443] Table 6 Antibody Identifier hIgG1 NIP228_SP14-266 Anti-IL-33 (33_640087-7B) SP15-124 mIgG1 mNIP228_SP14-108 Anti-ST2 Ab1440361 Anti-RAGE M4F4 Anti-EGFR LA1 (Merck, 05-101)

[0444] 23. FACS analysis of goblet cells in ALI cultures Seven days after treatment (Table 6), 4-week-old normal control or COPD ALI cultures on 6.5 mm inserts were analyzed by flow cytometry. 200 µl of 37°C PBS was added to the top region (transwell surface) of each Transwell and incubated for 30 min. The top wash was stored at -80°C for mucin analysis. 150 µl of trypsin (Lonza Trypsin Subculture Pack - #CC-5034) was added to the top and outer base compartments (below the Transwell). The Transwell was returned to the incubator and maintained for 30 min. ALI was isolated by gently aspirating the trypsin. 150 µl of trypsin neutralization solution (Lonza Trypsin Subculture Pack - #CC-5034) was added to each top cavity and mixed. The cell suspension was transferred to U-shaped 90-well plates, cells were counted, and centrifuged at 1200 RPM for 5 min at 4°C. Remove trypsin / TNS and add 200 µl of live / dead dye (eBioscience™ immobilizable viable dye eFluor™ 780, Thermo Fisher Scientific 65-0865-14, diluted 1:2000 in PBS) to each well. Resuspend cells and incubate on ice in the dark for 10 min. Centrifuge the plate at 1200 RPM for 5 min at 4 °C to remove live / dead dye and add 200 µl of PBS to each well. Centrifuge the plate at 1200 RPM for 5 min at 4 °C to remove PBS and replace with 200 µl of fixation / permeation solution (Thermo Fisher Scientific 00-5123 and 00-5223). Incubate the plate on ice in the dark for 40 min. Centrifuge the plate at 1200 RPM for 5 min at 4 °C and remove the solution. Resuspend cells in 300 µl of 1x permeation solution (Thermo Fisher Scientific 00-8333). Add 5e4 cells from each well to a new 96-well U-plate, centrifuged at 1200 RPM for 5 min at 4 °C, and resuspend the cells in 50 µl of 1x permeation solution. Add 50 µl of antibody staining mixture (1:400 anti-Muc5AC and 1:800 anti-Muc5B) or an equivalent dilution of the same type of staining mixture. Incubate the plate on ice in the dark for 30 min. Centrifuge the plate at 1200 RPM for 5 min at 4 °C and remove the solution. Wash the cells with PBS, centrifuge, and resuspend in 150 µl of PBS. Data were then acquired on BDFACSymphony™ and analyzed using FlowJo software.

[0445] Table 7 name identifier supplier Fluorescent clusters Cell markers Anti-Muc5AC ab3649 Abogen Corporation Coupled to AF488 (Expedeon, 332-0005). cup-shaped Anti-Muc5B ab105460 Abogen Corporation Coupled to AF647 (Expedeon, 336-0005). cup-shaped AF488 type 400109 BioLegend FITC Same type AF647 type 400130 BioLegend Company AF647 Same type

[0446] 24. qPCR / batch RNA sequencing analysis of ALI cultures.

[0447] Seven days after treatment (Table 6), 4-week-old normal control or COPD ALI cultures were lysed onto 6.5 mm inserts for RNA analysis. First, 200 µl of 37°C PBS was added to the top surface of each ALI, and the plate was returned to the incubator for 30 min. The top wash buffer was stored at -80°C for mucin analysis. ALI cultures were lysed and RNA extracted using the MagMAX™-96 Total RNA Isolation Kit (Thermo Scientific, AM1830). cDNA was then synthesized from the RNA using the High-Capacity RNA-to-cDNA™ Kit (Thermo Scientific, 4388950). 9 µl of each RNA sample was incubated with 10 µl of 2X RT buffer mixture and 1 µl of 20X RT enzyme mixture in a PCR tube (Thermo Scientific, AM12230) on a Thermo cycler at 37°C for 60 min. The reaction was terminated by heating to 95°C, maintaining for 5 minutes, and then holding at 4°C. 60 µl of nuclease-free water (Thermo Fisher Scientific, 750024) was added to each tube containing 20 µl cDNA. For RT-qPCR, 4 µl cDNA was added to a barcode-coded MicroAmp™ EnduraPlate™ optical 384-well clear plate (Thermo Fisher Scientific, 4483273) along with 5 µl TaqMan Rapid Advanced Premix (Thermo Fisher Scientific, 4444557), 0.5 µl Muc5AC FAM probe (Thermo Fisher Scientific, Hs01365616_m1), and 0.5 µl GAPDH VIC probe (Thermo Fisher Scientific, Hs02786624_g1). The plate was sealed and briefly centrifuged, then analyzed using a QuantStudio™ 7Flex Real-Time PCR System (Thermo Fisher Scientific). Δ-Δ-ct was then calculated by normalizing the data against an untreated normal control.

[0448] oxIL-33, rather than reduced IL-33, increases the number of goblet cells, especially the MUC5AC+ goblet cell subset. Figure 19 (A to 19C). Accordingly, as determined by qPCR, treatment with oxIL-33, MUC5AC Increase in mRNA copy number ( Figure 19 D).

[0449] 25. IHC triple staining (basal, cup, and ciliary) and quantification Next, quantitative imaging analysis from ALI immunohistochemistry was evaluated. ALI cultures from COPD donors were generated and processed as described in Section 22: Air-Liquid Interface (ALI) Culture of Airway Basal Cells. ALI epithelial cultures were fixed in 10% neutral-buffered formalin for 24 hours and embedded in paraffin. Paraffin sections (4 μm) were mounted on positively charged slides and stained on Ventana Discovery Ultra by sequential triplet staining. Antigen retrieval was performed with cell regulator 1 (Ultra CC1) (catalog number 5424569001, Roche) and endogenous peroxidase was blocked for 12 min with Discovery inhibitor (catalog number 7017944001, Roche). Anti-p63 (clone 4A4) (catalog number 790-4509, Roche, Basel, Switzerland) was applied for 24 min, and then visualized with anti-mouse HQ (12 min) (catalog number 7017782001, Roche) and anti-HQ HRP (12 min) (catalog number 7017936001, Roche), and incubated for 12 min with the Discovery Purple kit (catalog number 07053983001, Roche). The slides were denatured (92°C, 24 min) with cell regulator 2 (Ultra CC2) (catalog number 5424542001, Roche), and then treated with anti-microtubule protein (catalog number ab24610, Abogen Biosciences, Cambridge, UK) diluted with Dako antibody diluent (catalog number S3022) (concentration on slide: 0.003 µg / ml) for 16 min. The slides were then detected with mouse OmniMap-HRP (catalog number 5269652001, Roche) (8 min) and visualized using the Discovery Teal HRP kit (catalog number 82544338001, Roche). The slide was denatured with CC2 again, and then a mixture of 1.1 µg / ml (dispenser concentration) rabbit anti-adhesion 5AC and 7 µg / ml (dispenser concentration) rabbit anti-adhesion 5B (catalog numbers ab198294 and ab87376, respectively, Abogen) was applied for 20 min. Visualization was performed using anti-rabbit NP (4 min) (catalog number 7425317001, Roche) and anti-NP-AP (8 min) (catalog number 7425325001, Roche). Visualization was then performed using the Discovery Yellow Kit (catalog number 7698445001, Roche) for 20 min.The stained slides were counterstained with hematoxylin II (8 min) (catalog number 5277965001, Roche) and bluing reagent (4 min) (catalog number 5266769001, Roche), rinsed with dish soap, dehydrated with graded ethanol and xylene series, and mounted with permanent mounting medium.

[0450] IHC images were analyzed in HALO v3.1 (Indica Labs), where they were first manually annotated to exclude focal and tissue damage areas. A random forest classifier was learned to identify epithelium and separate it from the transmembrane and slide background. For cilia region quantification, another random forest classifier was learned to coarsely detect tubulin staining, followed by fine detection using the Area Quantification algorithm v2.1.7. For mucin region quantification, Area Quantification v2.1.7 was used directly to detect staining. For basal (p63+) cell counting, the CytoNuclear algorithm 2.0.9 was used to segment cells based on nuclear staining, and basal cells were further detected by counting p63-positive nuclei. All quantitative methods were human-verified and achieved an accuracy exceeding 90%.

[0451] Consistent with previous findings, oxIL-33 profoundly affects the number of goblet cells (MUC5ac+b). Figure 20A and 20B ).

[0452] In summary, these studies demonstrate that oxIL-33 plays a role in promoting goblet cell differentiation in lung epithelium. This suggests that epithelium exposed to ox-IL33 over a long period develops a goblet hyperplasia phenotype, which negatively impacts lung function.

[0453] 26. Reversing the cupid phenotype of COPD with blocking agents A key hallmark of COPD is excessive mucus production due to goblet cells and increased mucus secretion (Gohy et al., 2019 Sci Rep [Science Communications] 9:17963). To investigate whether oxidized IL-33 can play a direct role in the goblet COPD phenotype, ALI cultures from COPD donors were established, with readings described in Sections 22–25.

[0454] COPD ALI was cultured in the presence of anti-IL-33 (33-640087_7B), anti-RAGE, or anti-EGFR neutralizing antibodies. All three treatments reduced the number of MUC5AC+ goblet cells. Figures 21A to 21D No treatment affected the viability of ALI cultures. Figure 21EThis confirms that the treatment phenomenon is not a spurious sign or a result of antibody toxicity. Consistent with previous results, anti-ST2 treatment did not cause a decrease in goblet cell count, further demonstrating that this is a disease phenotype directly mediated by IL-33, primarily ox-IL-33, via the oxIL-33-RAGE-EGFR pathway. Immunohistochemical analysis further confirmed the effect of the anti-IL-33 antibody (33-640087_7B) on COPD ALI, where, in paired analysis, blocking IL-33 reduced the number of goblet cells (…). Figure 22A and 22B After treatment with anti-IL-33 antibody (33-640087_7B), the epithelium of COPD ALI cultures resembled that of healthy epithelium, such as... Figure 20A As shown.

[0455] Finally, the release of MUC5AC and MUC5B from the apical mucus of healthy and COPD ALI cultures was measured using ELISA. To quantify the mucin released from ALI cultures, the level of MUC5AC in the apical supernatant was analyzed by immunoassay (Novus NBP2-76703) according to the manufacturer's protocol. Samples were diluted 1:2000 in sample diluent, and concentrations were extrapolated from the recombinant MUC5AC protein standard curve. Figure 23 As shown, compared with ALI from healthy donors, ALI cultures from COPD patients released increased levels of MUC5AC (…). Figure 23 A). Treatment with exogenous oxIL-33 increased mucin secretion in healthy ALI cultures. Figure 23 B). In COPD ALI donors, the increase in mucin levels was reduced by blocking with anti-IL-33 (33_640087_7B), which inhibited the release of MUC5AC protein levels from ALI cultures, but not by anti-ST2 or isotype mAb controls. Figure 23 C).

[0456] Overall, this case highlights the role of oxidized IL-33 in the dysregulation of epithelial cell differentiation in the lungs. The results suggest that, in an uncontrolled state, oxidized IL-33 may be the cause of goblet cell hyperplasia and excessive mucus production seen in some COPD phenotypes. Therefore, treatment with oxIL-33 signaling axis antagonists, such as anti-IL-33, anti-RAGE, or anti-EGFR binding molecules, may offer significant therapeutic benefits to COPD patients by restoring normal epithelial physiology, for example, by reducing goblet cell numbers and decreasing excessive mucus production.

[0457] Other sequences In addition to the sequences listed in Table 1, we also provide the following other CDR sequences: SEQ ID NO 37: SYAMS SEQ ID NO 38:GISAIDQSTYYADSVKG SEQ ID NO 39: QKFMQLWGGGLRYPFGY SEQ ID NO 40: SGEGMGDKYAA SEQ ID NO 41: RDTKRPS SEQ ID NO 42: GVIQDNTGV N-terminal His10 / Avitag / Factor Xa protease cleavage site SEQ ID NO 43:MHHHHHHHHHHAAGLNDIFEAQKIEWHEAAIEGR IL-33-01 SEQ ID NO 44: IL-33-16 SEQ ID NO 45: Avitag sequence motif SEQ ID NO 46:GLNDIFEAQKIEWHE gRNA vectors targeting RAGE exon 3 SEQ ID NO 47: TGAGGGGATTTTCCGGTGC RAGE forward primers SEQ ID NO 48:gttgcagcctcccaacttc RAGE reverse primer SEQ ID NO 49: aatgaggccagtggaagtca Human ST2S (signal peptide with underline) SEQ ID NO 50: Human ST2S-huIgG1 Fc-His6 (signal peptide is underlined) SEQ ID NO 51: SEQ ID NO 52: His10 / Avitag human ASGPR ECD (signal peptide is underlined, and the tag is double-underlined)

Claims

1. An IL-33 antagonist for the prevention or treatment of epithelial physiological abnormalities by modulating or inhibiting RAGE-EGFR-mediated action.

2. The IL-33 antagonist used according to claim 1, wherein the epithelial physiological abnormality is a mucociliary physiological abnormality, preferably a mucociliary physiological abnormality of the respiratory epithelium.

3. The IL-33 antagonist used according to claim 2, wherein the mucosal ciliary physiological abnormality is selected from: abnormal mucus production; abnormal goblet cell differentiation; abnormal goblet cell proliferation; abnormal epithelial thickness; abnormal mucus clearance; and / or abnormal mucus composition.

4. The IL-33 antagonist used according to claim 3, wherein the mucus production abnormality includes MUC5AC production abnormality; and / or wherein the goblet cell differentiation abnormality includes MUC5AC + goblet cell differentiation abnormality; and / or wherein the goblet cell proliferation abnormality includes MUC5AC + goblet cell proliferation abnormality; and / or wherein the epithelial thickness abnormality includes MUC5AC in the total tissue region of the epithelium. + The number of goblet cells is abnormal.

5. An IL-33 antagonist used according to claim 2, 3, or 4, wherein the mucociliary physiological abnormality includes: Increased mucus production; increased goblet cell differentiation; increased goblet cell proliferation; increased epithelial thickness; and / or decreased mucus clearance.

6. An IL-33 antagonist for use according to claim 5, wherein increased mucus production comprises increased MUC5AC production; and / or wherein increased goblet cell differentiation comprises MUC5AC + increased goblet cell differentiation; and / or wherein increased goblet cell proliferation comprises MUC5AC + increased goblet cell proliferation; and / or wherein increased epithelial thickness comprises MUC5AC in the total tissue region of the epithelium. + The number of goblet cells increases.

7. The IL-33 antagonist used according to claim 3, wherein the mucus composition abnormality includes an increase or decrease in the ratio of different mucus compounds contained in the mucus; an increase or decrease in one or more mucus compounds; and / or an increase or decrease in mucus concentration or thickness.

8. The IL-33 antagonist used according to claim 7, wherein the mucus composition anomaly includes an increased MUC5AC:MUC5B ratio; and / or wherein the mucus composition anomaly includes an increased MUC5AC content in the mucus; and / or wherein the mucus composition anomaly includes an increased mucus thickness.

9. The IL-33 antagonist used according to claim 1, wherein the epithelial physiological abnormality is an epithelial remodeling abnormality.

10. An IL-33 antagonist for use according to any of the preceding claims, wherein the epithelial physiological abnormality is an epithelial physiological abnormality of the respiratory tract, preferably a mucociliary physiological abnormality of the respiratory tract.

Images