How to Use IL-33 Antagonists

Abstract

IL-33 antagonists for use in the prevention or treatment of abnormal epithelial physiology or EGFR-mediated diseases, and methods of use, are provided. IL-33 antagonists are provided for use in the prevention or treatment of abnormal epithelial physiology by modulating or inhibiting RAGE-EGFR mediated effects.

Description

[Technical Field]

[0001] This application claims priority to European Patent Application No. 19206984.7, filed November 4, 2019, the contents of which are incorporated herein by reference in their entirety.

[0002] The present disclosure relates to IL-33 antagonists for use in the prevention or treatment of abnormal epithelial physiology or EGFR-mediated diseases, and corresponding prophylactic or therapeutic methods comprising administering an IL-33 antagonist to a patient in need thereof. [Background technology]

[0003] Interleukin-33 (IL-33), also known as IL-1F11, is a member of the IL-1 family of cytokines. IL-33 is a 270-amino acid protein consisting of two domains: a homeodomain and a cytokine (IL-1-like) domain. The homeodomain contains a 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 at least one disulfide bond in the oxidized form, and that the two forms may have different binding patterns and effects.

[0004] It was previously discovered that reduced IL-33 binds to ST2 and is, in fact, 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 NFκB and MAP kinase pathways, which leads to the production of cytokines and chemokines, such as IL-4, IL-5, and IL-13, to promote inflammation. Soluble ST2 (sST2) is thought to be a decoy receptor that prevents reduced IL-33 signaling.

[0005] More recently, oxidized IL-33 has also been found to have physiological effects: it does not bind to ST2 but instead binds to the receptor for advanced glycation end products (RAGE) and signals through this alternative pathway.

[0006] There has been much interest in IL-33 as a therapeutic target, primarily due to the ability of what is now known as the reduced form to stimulate ST2 and produce potent inflammatory effects. However, there has been little research and interest in the oxidized IL-33 pathway as a therapeutic target. This is due in part to its later discovery and the fact that RAGE has many ligands and its downstream interactions are not well understood.

[0007] At least one of these downstream RAGE interactions resulting from oxidized IL-33 stimulation is described in more detail herein. Surprisingly, RAGE was found to complex 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 forms a complex with EGFR to stimulate EGFR activity. The surprising discovery of EGFR involvement is significant in that EGFR is an important therapeutic target for many diseases involving aspects of abnormal epithelial physiology.

[0008] As a result of this discovery, it is believed that antagonists capable of binding to either form of IL-33 can effectively prevent oxidized IL-33 signaling. This could be either directly by binding to oxidized IL-33 itself, or indirectly by inhibiting the conversion of reduced IL-33 to oxidized IL-33, thereby preventing both RAGE stimulation and EGFR stimulation. This reduction in EGFR stimulation provides therapeutic benefit in any EGFR-mediated disease, particularly in conditions where EGFR is overstimulated.

[0009] EGFR is known to have various homeostatic effects on epithelial physiology. Stimulation of EGFR increases epithelial cell differentiation, epithelial cell migration, and epithelial mucus production. Inhibition of EGFR-mediated signaling is thought to treat or prevent disorders associated with abnormal epithelial physiology, such as abnormal airway epithelial tissue remodeling or mucus overproduction.

[0010] IL-33 has previously been implicated in airway tissue remodeling (Non-Patent Document 1; Non-Patent Document 2; Non-Patent Document 3). However, this is thought to occur indirectly through a self-perpetuating amplification loop, whereby IL-33 signaling upregulates the expression of both IL-33 and its cognate receptor ST2, resulting in chronic ST2 axis signaling. Because ST2-mediated activity is mediated by innate cells that express ST2, such as macrophages and type 2 innate lymphocytes, it has not previously been established or suggested that IL-33 itself directly influences airway epithelial biological function.

[0011] As noted above, the present disclosure is based on the discovery that IL-33 also acts directly through a different mechanism: the RAGE-EGFR pathway; directly impacting epithelial physiology. This new understanding is important because it broadens the therapeutic applications of IL-33 antagonists, allowing them to be used to treat more diseases, more disease symptoms, and more patients. The therapeutic opportunity to directly regulate and inhibit IL-33-mediated EGFR-mediated signaling by targeting IL-33 has not previously been realized.

[0012] The present disclosure demonstrates for the first time that the use of IL-33 antagonists can directly affect impaired epithelial repair responses, reduce epithelial goblet cell differentiation and proliferation, decrease mucus production, and improve mucociliary motility in patients with abnormal epithelial physiology, such as those with COPD or bronchitis, through direct inhibition of RAGE / EGFR-mediated oxIL-33 activity. Thus, the studies presented herein support the therapeutic use of IL-33 antagonists in the direct prevention or treatment of abnormal epithelial physiology, which is typically due to EGFR-mediated effects and thereby present in EGFR-mediated diseases. [Prior art documents] [Non-patent literature]

[0013] [Non-Patent Document 1] Li et al JACI,2014 134:1422-32 [Non-patent document 2] Vannella et al Sci Transl Med,337ra65 [Non-patent document 3] Allinne et al JACI,2019,144:1624-37 Summary of the Invention [Means for solving the problem]

[0014] According to a first aspect, there is provided an IL-33 antagonist for use in the prevention or treatment of abnormal epithelial physiology by modulating or inhibiting RAGE-EGFR mediated effects.

[0015] According to an alternative first aspect, there is provided a method of preventing or treating abnormal epithelial physiology in a patient, the method comprising: administering to a patient in need thereof an effective amount of an IL-33 antagonist to modulate or inhibit RAGE-EGFR mediated effects.

[0016] According to an alternative first aspect there is provided the use of an IL-33 antagonist in the manufacture of a medicament for the prevention or treatment of abnormal epithelial physiology.

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

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

[0019] According to an alternative second aspect there is provided the use of an IL-33 antagonist in the manufacture of a medicament for the prevention or treatment of an EGFR mediated disease.

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

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

[0022] According to an alternative third aspect there is provided the use of an IL-33 antagonist in the manufacture of a medicament for the prevention or treatment of respiratory disease by improving epithelial physiology.

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

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

[0025] According to an alternative fourth aspect there is provided the use of an IL-33 antagonist in the manufacture of a medicament for the prevention or treatment of a respiratory disease by inhibiting an EGFR mediated effect.

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

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

[0028] According to an alternative further aspect, there is provided the use of an IL-33 antagonist in the manufacture of a medicament for the prevention or treatment of a disease by inhibiting IL-33-mediated EGFR signaling.

[0029] Further features and embodiments of the above-defined aspects are described in the following heading sections, each of which may be combined with any of the above aspects in any compatible combination. DETAILED DESCRIPTION OF THE INVENTION

[0030] definition As used herein, "IL-33" protein refers to interleukin-33, particularly mammalian interleukin-33 protein, e.g., the human protein deposited under UniProt No. 095760. However, it is clear that this entity is not a single species, but rather exists as reduced and oxidized forms. Given that the reduced form oxidizes rapidly in vivo (e.g., within a time period of 5 to 40 minutes) and in vitro, prior art references to IL-33 actually refer to the oxidized form. Furthermore, commercially available assays may not effectively distinguish between the reduced and oxidized forms. The terms "IL-33" and "IL-33 polypeptide" are used interchangeably. In certain embodiments, IL-33 is full-length. In another embodiment, IL-33 is mature truncated IL-33 (amino acids 112-270). Recent studies suggest that full-length IL-33 is active (Cayrol and Girard, Proc Natl Acad Sci USA 106(22):9021-6(2009); Hayakawa et al., Biochem Biophys Res Commun. 387(1):218-22(2009); Talabot-Ayer et al., J Biol Chem. 284(29):19420-6(2009)). However, N-terminally processed or truncated IL-33, including but not limited to aa 72-270, 79-270, 95-270, 99-270, 107-270, 109-270, 111-270, and 112-270, may have enhanced activity (Lefrancais 2012, 2014). In another embodiment, IL-33 may include full-length IL-33, a fragment thereof, or an IL-33 mutant or variant polypeptide, where the fragment of IL-33 or IL-33 variant polypeptide retains some or all of the functional properties of active IL-33.

[0031] "Oxidized IL-33" or "oxIL-33," as used herein, refers to a form of IL-33 that binds to RAGE and initiates RAGE-EGFR-mediated signaling. Oxidized IL-33 refers to a protein that appears as a characteristic band, for example, by Western blot analysis under non-reducing conditions, and in particular has a mass 4 Da less than the corresponding reduced form. Specifically, it refers to a protein that has one or two disulfide bonds between cysteines independently selected from cysteines 208, 227, 232, and 259. In one embodiment, oxidized IL-33 does not exhibit binding to ST2.

[0032] "Reduced IL-33" or "redIL-33," as used herein, refers to a form of IL-33 that binds to ST2 and initiates ST2-mediated signaling. In particular, cysteines 208, 227, 232, and 259 in the reduced form are not disulfide bonded. In one embodiment, reduced IL-33 does not exhibit binding to RAGE. It should be understood that reference to "WT IL-33" or "IL-33" can refer to either the reduced or oxidized form, or both, unless it is clear from the context in which it is used that one of the forms is meant.

[0033] As used herein, "antigenically distinct forms of IL-33" refers to any form of IL-33 that can act as an antigen and be bound by an antibody or binding fragment thereof, and typically in the context of the present disclosure this refers to oxidized IL-33, reduced IL-33, and reduced IL-33 / sST2 complexes.

[0034] "ST2-mediated signaling / effects," as used herein, refers to the IL-33 / ST2 system in which reduced IL-33 recognition by ST2 promotes dimerization with IL-1RAcP on the cell surface and intracellularly and recruitment of receptor complex components MyD88, TRAF6, and IRAK1-4 to the intracellular TIR domain. Thus, ST2-dependent signaling / effects can be disrupted and attenuated by disrupting the interaction of IL-33 with ST2 or, alternatively, by preventing its interaction with IL-1RAcP.

[0035] "RAGE-EGFR-mediated signaling / effect," as used herein, refers to the oxidized IL-33 / RAGE-EGFR system in which recognition of oxidized IL-33 by RAGE promotes its complexation with EGFR in the cell membrane. Thus, RAGE-EGFR-mediated signaling / effect can be disrupted or attenuated by disrupting the interaction between oxidized IL-33 and RAGE or by preventing the conversion of reduced IL-33 to oxidized IL-33.

[0036] "Attenuating the activity of," as used herein, refers to reducing or inhibiting the relevant activity or to stopping the relevant activity. Generally, attenuating and inhibiting are used interchangeably herein.

[0037] It should be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "an anti-IL-33 antibody" is understood to refer to one or more anti-IL-33 antibodies. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0038] As used herein, the term "treat" or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, where the goal is to prevent or slow (alleviate) an undesirable physiological change or disorder. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in extent of disease, stabilized (i.e., not worsening) disease, delay or slowing of disease progression, amelioration or palliation, and remission (partial or total), whether detectable or undetectable, of the disease state. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those susceptible to the condition or disorder or those in whom the condition or disorder is to be prevented.

[0039] "Subject" or "individual" or "animal" or "patient" or "mammal" means any subject for which diagnosis, prognosis, or treatment is desired, particularly a mammalian subject, except where the subject is defined as a "healthy subject." Mammalian subjects include humans; livestock; agricultural animals; for example, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, cows, etc.

[0040] IL-33 antagonist The present disclosure relates to the medical use of IL-33 antagonists, particularly for preventing or treating disease by inhibiting IL-33-mediated EGFR signaling. In certain cases, the disclosure relates to the use of IL-33 antagonists for the prevention or treatment of abnormal epithelial physiology that may be found in EGFR-mediated diseases.

[0041] "IL-33 antagonist," as used herein, refers to any agent that attenuates IL-33 activity, e.g., reduced IL-33 activity, oxidized IL-33 activity, or both. Preferably, the IL-33 antagonist is specific for reduced and / or oxidized IL-33. Preferably, attenuation is by binding IL-33 in its reduced or oxidized form. Preferably, if the antagonist attenuates reduced IL-33 activity and oxidized IL-33 activity, attenuation is by binding to IL-33 in its reduced form (i.e., by binding to reduced IL-33).

[0042] Preferably, the IL-33 antagonist is a binding molecule or a fragment thereof.

[0043] As used herein, the term "binding molecule" or "antigen-binding molecule" refers in its broadest sense to a molecule that specifically binds to an antigenic determinant. Preferably, the binding molecule specifically binds to IL-33, particularly red IL-33 or oxidized IL-33.

[0044] Suitably, the binding molecule may be selected from: an antibody, an antigen-binding fragment thereof, an aptamer, 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.

[0045] Preferably, the IL-33 antagonist is an antibody or a binding fragment thereof. Preferably, the IL-33 antagonist is an anti-IL-33 antibody or a binding fragment thereof. Preferably, the anti-IL-33 antibody or a binding fragment thereof specifically binds to IL-33, particularly reduced IL-33 or oxidized IL-33.

[0046] "Antibody," as used herein, refers to an immunoglobulin molecule, as discussed in more detail below, particularly a full-length antibody or a molecule comprising a full-length antibody, such as a DVD-Ig molecule.

[0047] "Binding fragment thereof" is interchangeable with "antigen-binding fragment thereof" and refers to an epitope / antigen-binding fragment of an antibody fragment, e.g., comprising a binding region, in particular comprising six CDRs, such as three CDRs of the heavy chain variable region and three CDRs of the light chain variable region.

[0048] Preferably, the antibody or binding fragment thereof is selected from: naturally occurring, polyclonal, monoclonal, multispecific, murine, human, humanized, primatized, or chimeric. Preferably, the antibody or binding fragment thereof may be an epitope-binding fragment, such as Fab', F(ab')2, Fd, Fvs, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv), fragments containing either the VL domain or the VH domain, or fragments produced by a Fab expression library. Preferably, the antibody or binding fragment thereof may be a minibody, diabody, triabody, tetrabody, or single-chain antibody. Preferably, the antibody or binding fragment thereof is a monoclonal antibody. ScFv molecules are known in the art and are described, for example, in U.S. Pat. No. 5,892,019.

[0049] Immunoglobulin or antibody molecules of the present disclosure can be of 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 molecule.

[0050] Preferably, the IL-33 antagonist inhibits the activity of oxidized IL-33, preferably by inhibiting the formation of oxidized IL-33. Preferably, the IL-33 antagonist inhibits the conversion of reduced IL-33 to oxidized IL-33.

[0051] Preferably, the IL-33 antagonist is a reduced IL-33 antagonist. In other words, the IL-33 antagonist attenuates the activity of reduced IL-33. Preferably, the attenuation is due to binding to reduced IL-33. Preferably, by binding to reduced IL-33, the antagonist also inhibits / attenuates the activity of oxidized IL-33 by preventing its conversion to the oxidized IL-33 form.

[0052] Preferably, inhibiting the activity of oxidized IL-33 downregulates or turns off RAGE-dependent signaling and / or RAGE-mediated effects. Preferably, inhibition downregulates or turns off RAGE-EGFR-dependent signaling and / or RAGE-EGFR-mediated effects. Preferably, inhibition downregulates or turns off EGFR-dependent signaling. Preferably, inhibition downregulates or turns off EGFR-mediated effects. In particular, it has been shown that IL33 antagonists that bind to reduced IL-33 can prevent oxidized IL-33 from binding to RAGE, thereby inhibiting RAGE-EGFR signaling.

[0053] Preferably, inhibition of oxidized IL-33 activity downregulates or prevents RAGE-EGFR complexation. Preferably, inhibition downregulates or prevents EGFR activation, preferably RAGE-mediated EGFR activation.

[0054] Preferably, the IL-33 antagonist has all of the above inhibitory effects. Preferably, the reduced IL-33 antagonist has all of the above inhibitory effects.

[0055] Preferably, the IL-33 antagonist is a reduced IL-33 binding molecule or a fragment thereof. Preferably, the IL-33 antagonist is a reduced IL-33 antibody or a binding fragment thereof, preferably an anti-reduced IL-33 antibody or a binding fragment thereof.

[0056] Preferably, the binding molecule or fragment thereof is 5×10 -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, 5 x 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 Specifically binds to redIL-33 with a binding affinity (Kd) of less than 5×10 M. Preferably, the binding affinity to redIL-33 is less than 5×10 -14The binding affinity is less than M (i.e., 0.05 pM). Preferably, the binding affinity is as measured using Kinetic Exclusion Assays (KinExA) or BIACORE™, preferably using KinExA, such as the protocol described in WO 2016 / 156440, which is incorporated herein by reference in its entirety (see, e.g., Example 11). Binding molecules that bind redIL-33 with this binding affinity appear to bind redIL-33 tightly enough to prevent dissociation of the binding molecule / redIL-33 complex within a biologically relevant timescale. Without being bound by theory, it is believed that this binding strength prevents release of the antigen prior to degradation of the antibody / antigen complex in vivo, such that redIL-33 is not released and conversion of redIL-33 to oxIL-33 is not possible. Thus, when binding to redIL-33 with this binding affinity, the binding molecule can inhibit or attenuate the activity of oxIL-33 by preventing the formation of oxIL-33 and thereby inhibiting RAGE signaling.

[0057] Preferably, the binding molecule or fragment thereof is 10 3 M -1 seconds -1 , 5×10 3 M -1 seconds -1 , 10 4 M -1 seconds -1 , or 5 x 10 4 M -1 seconds -1 For example, the binding molecules of the present disclosure may specifically bind to redIL-33 with an on-rate (k(on)) of 10 or greater. 5 M -1 seconds -1 , 5×10 5 M -1 seconds -1 , 10 6 M -1 seconds -1 , or 5 x 10 6 M -1 seconds -1 or 10 7 M -1 seconds-1 The antibody may bind to redIL-33 or a fragment or variant thereof with an on-rate (k(on)) of 10 or greater. Preferably, the k(on) rate is 10 7 M -1 seconds -1 That's all.

[0058] Preferably, the binding molecule or fragment thereof is 5×10 -1 seconds -1 , 10 -1 seconds -1 , 5X10 -2 seconds -1 , 10 -2 seconds -1 , 5X10 -3 seconds -1 , or 10 -3 seconds -1 For example, a binding molecule of the present disclosure may specifically bind to redIL-33 with an off rate (k(off)) of 5×10 -4 seconds -1 , 10 -4 seconds -1 , 5×10 -5 seconds -1 , 10 -5 seconds -1 , 5×10 -6 seconds -1 , 10 -6 seconds -1 , 5×10 -7 seconds -1 , or 10 -7 seconds -1 It can be said to bind to redIL-33 or a fragment or variant thereof with an off rate (k(off)) of less than or equal to 10. Suitably, the k(off) rate is less than or equal to 10. -3 seconds -1IL-33 is an alarmin cytokine that is released rapidly and at high concentrations in response to inflammatory stimuli. Red IL-33 is converted to its oxidized state approximately 5 to 45 minutes after release into the extracellular environment. Thus, to prevent the conversion of red IL-33 to ox IL-33, the binding molecules described herein may bind to red IL-33 with these k(on) and / or k(off) rates. Without being bound by theory, these k(on) / k(off) rates are believed to ensure that the binding molecules can rapidly bind to red IL-33 before it is converted to ox IL-33, thereby reducing the formation of ox IL-33, thereby attenuating RAGE signaling, preferably RAGE / EGFR signaling, and thereby attenuating RAGE / EGFR-mediated effects.

[0059] Suitably, the IL-33 binding molecule may competitively inhibit the binding of IL-33 to the binding molecule 33_640087-7B (as described in WO 2016 / 156440). Suitably, WO 2016 / 156440 describes that 33_640087-7B binds to redIL-33 with particularly high affinity and attenuates both ST-2- and RAGE-dependent IL-33 signaling. Thus, binding molecules that competitively inhibit the binding of IL-33 to the binding molecule 33_640087-7B are likely to inhibit both redIL-33 and oxIL-33 signaling and are therefore particularly suitable for use in the methods described herein.

[0060] A binding molecule or fragment thereof is said to competitively inhibit the binding of a reference antibody to a given epitope if it specifically binds to that epitope to the extent that it blocks the binding of the reference antibody to that epitope to some extent. Competitive inhibition can be determined by any method known in the art, such as solid-phase assays such as competitive ELISA assays, dissociation-promoted lanthanide fluorescence immunoassays (DELFIA®, Perkin Elmer), and radioligand binding assays. For example, one skilled in the art can determine whether a binding molecule or fragment thereof competes for binding to redIL-33 by using an in vitro competitive binding assay, such as the derivation of the HTRF assay described in Example 1 of WO 2016 / 156440. For example, one skilled in the art can label a recombinant antibody in Table 1 with a donor fluorophore and mix multiple concentrations with a fixed concentration sample of acceptor fluorophore-labeled redIL-33. The binding characteristics can then be confirmed by measuring fluorescence resonance energy transfer between the donor and acceptor fluorophores in each sample. To identify competitive binding molecules, one skilled in the art can first mix various concentrations of a test binding molecule with a fixed concentration of labeled antibody from Table 1. A decrease in FRET signal when the mixture is incubated with labeled IL-33, compared to a positive control of labeled antibody only, indicates competitive binding to IL-33. A binding molecule or fragment thereof can be said to competitively inhibit binding of a reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0061] In some embodiments, the binding molecule is selected from the following anti-IL-33 antibodies: 33_640087-7B (as described in WO 2016 / 156440), ANB020, also known as Etokimab (as described in WO 2015 / 106080), 9675P (as described in U.S. Patent Application Publication No. 2014 / 0271658), A25-3H04 (as described in U.S. Patent Application Publication No. 2017 / 0283494), Ab43 (as described in WO 2018 / 081075), IL33-158 (as described in U.S. Patent Application Publication No. 2018 / 0037644), 10C12.38.H6.87Y.581 IgG4 (as described in WO 2016 / 077381), or a binding fragment thereof, each of which is incorporated herein by reference. All of these antibodies are referenced in Table 1.

[0062] Preferably, the IL-33 antagonist is an antibody or antigen-binding fragment comprising the complementarity-determining regions (CDRs) of a pair of variable heavy (VH) and variable light (VL) domains selected from Table 1. Pair 1 corresponds to the VH and VL domain sequences of 33_640087-7B described in WO 2016 / 156440. Pairs 2 to 7 correspond to the VH and VL domain sequences of antibodies described in U.S. Patent Application Publication No. 2014 / 0271658. Pairs 8 to 12 correspond to the VH and VL domain sequences of antibodies described in U.S. Patent Application Publication No. 2017 / 0283494. Pair 13 corresponds to the VH and VL domain sequences of ANB020 described in WO 2015 / 106080. Pairs 14 to 16 correspond to the VH and VL domain sequences of antibodies described in WO 2018 / 081075, Pair 17 corresponds to the VH and VL domain sequences of IL33-158 described in U.S. Patent Application Publication No. 2018 / 0037644, and Pair 18 corresponds to the VH and VL domain sequences of 10C12.38.H6.87Y.581 IgG4 described in WO 2016 / 077381.

[0063] [Table 1]

[0064] [Table 2]

[0065] [Table 3]

[0066] [Table 4]

[0067] [Table 5]

[0068] Suitably, the IL-33 antagonist is an antibody or antigen-binding fragment comprising a heavy chain variable region (HCVR) complementarity determining region (CDR) comprising the sequence of SEQ ID NO: 1 and a light chain variable region (LCVR) complementarity determining region (CDR) comprising the sequence of SEQ ID NO: 19. These CDRs correspond to those derived from 33_640087-7B (as described in WO 2016 / 156440), which binds reduced IL-33 and inhibits its conversion to oxidized IL-33. 33_640087-7B is fully described in WO 2016 / 156440, which is incorporated herein by reference.

[0069] Preferably, the IL-33 antagonist is an antibody or antigen-binding fragment comprising a heavy chain variable region (HCVR) complementarity determining region (CDR) comprising the sequence of SEQ ID NO: 7 and a light chain variable region (LCVR) complementarity determining region (CDR) comprising the sequence of SEQ ID NO: 25. These CDRs correspond to those derived from antibody 9675P, which is fully described in U.S. Patent Application Publication No. 2014 / 0271658, which is incorporated herein by reference.

[0070] Preferably, the IL-33 antagonist is an antibody or antigen-binding fragment comprising a heavy chain variable region (HCVR) complementarity determining region (CDR) comprising the sequence of SEQ ID NO: 11 and a light chain variable region (LCVR) complementarity determining region (CDR) comprising the sequence of SEQ ID NO: 29. These CDRs correspond to those derived from antibody A25-3H04. A25-3H04 is fully described in U.S. Patent Application Publication No. 2017 / 0283494, which is incorporated herein by reference.

[0071] Preferably, the IL-33 antagonist is an antibody or antigen-binding fragment comprising a heavy chain variable region (HCVR) complementarity determining region (CDR) comprising the sequence of SEQ ID NO: 13 and a light chain variable region (LCVR) complementarity determining region (CDR) comprising the sequence of SEQ ID NO: 31. These CDRs correspond to those derived from antibody ANB020, which is fully described in WO 2015 / 106080, which is incorporated herein by reference.

[0072] Preferably, the IL-33 antagonist is an antibody or antigen-binding fragment comprising a heavy chain variable region (HCVR) complementarity determining region (CDR) comprising the sequence of SEQ ID NO: 16 and a light chain variable region (LCVR) complementarity determining region (CDR) comprising the sequence of SEQ ID NO: 34. These CDRs correspond to those derived from antibody Ab43. Ab43 is fully described in WO 2018 / 081075, which is incorporated herein by reference.

[0073] Preferably, the IL-33 antagonist is an antibody or antigen-binding fragment comprising a heavy chain variable region (HCVR) complementarity determining region (CDR) comprising the sequence of SEQ ID NO: 17 and a light chain variable region (LCVR) complementarity determining region (CDR) comprising the sequence of SEQ ID NO: 35. These CDRs correspond to those derived from the antibody IL33-158. IL33-158 is fully described in U.S. Patent Application Publication No. 2018 / 0037644, which is incorporated herein by reference.

[0074] Preferably, the IL-33 binding molecule is an antibody or antigen-binding fragment comprising a heavy chain variable region (HCVR) complementarity determining region (CDR) comprising the sequence of SEQ ID NO: 18 and a light chain variable region (LCVR) complementarity determining region (CDR) comprising the sequence of SEQ ID NO: 36. These CDRs correspond to those derived from antibody 10C12.38.H6.87Y.581 IgG4. 10C12.38.H6.87Y.581 IgG4 is fully described in WO 2016 / 077381, which is incorporated herein by reference.

[0075] Preferably, those skilled in the art are aware of methods available in the art for identifying CDRs within the heavy and light chain variable regions of an antibody or antigen-binding fragment thereof. Preferably, those skilled in the art can perform, for example, sequence-based annotation. Because the regions between CDRs are generally highly conserved, logical rules can be used to determine the location of CDRs. Those skilled in the art can use a set of sequence-based rules for conventional antibodies (Pantazes and Maranas, Protein Engineering, Design and Selection, 2010). Alternatively, or additionally, those skilled in the art can refine the rules based on multiple sequence alignments. Alternatively, those skilled in the art can compare antibody sequences to publicly available databases operating on the Kabat, Chothia, or IMGT methods using the BLASTP command in BLAST+ to identify the most similar annotated sequences. Each of these methods devise a unique residue numbering scheme for numbering residues in the hypervariable regions, and then the start and end of each of the six CDRs are determined according to specific key positions. For example, by aligning with the most similar annotated sequence, CDRs can be extrapolated from the annotated sequence to the unannotated sequence, thereby identifying the CDRs. Suitable tools / databases include, for example, the Kabat database, Kabatman, Scalinger, IMGT, and Abnum.

[0076] Preferably, the IL-33 antagonist is an antibody or antigen-binding fragment comprising a pair of variable heavy domain (VH) and variable light domain (VL) selected from Table 1.

[0077] Suitably, the IL33 antibody or antigen-binding fragment thereof comprises a VH domain of sequence SEQ ID NO: 1 and a VL domain of sequence SEQ ID NO: 19.

[0078] Suitably, the IL33 antibody or antigen-binding fragment thereof comprises a VH domain of sequence SEQ ID NO: 7 and a VL domain of sequence SEQ ID NO: 25.

[0079] Suitably, the IL33 antibody or antigen-binding fragment thereof comprises a VH domain of sequence SEQ ID NO: 11 and a VL domain of sequence SEQ ID NO: 29.

[0080] Suitably, the IL33 antibody or antigen-binding fragment thereof comprises a VH domain of sequence SEQ ID NO: 13 and a VL domain of sequence SEQ ID NO: 31.

[0081] Suitably, the IL33 antibody or antigen-binding fragment thereof comprises a VH domain of sequence SEQ ID NO: 16 and a VL domain of sequence SEQ ID NO: 34.

[0082] Suitably, the IL33 antibody or antigen-binding fragment thereof comprises a VH domain of sequence SEQ ID NO: 17 and a VL domain of sequence SEQ ID NO: 35.

[0083] Thus, suitably, the IL-33 antagonist is a binding molecule that may comprise, for example, three CDRs in a heavy chain variable region independently selected from SEQ ID NOs: 1, 7, 11, 13, 16, 17, and 18.

[0084] Suitably, the IL-33 antagonist is a binding molecule comprising three CDRs in the heavy chain variable region according to SEQ ID NO:1.

[0085] Suitably, the IL-33 antagonist is a binding molecule that may comprise three CDRs in a light chain variable region independently selected from SEQ ID NOs: 19, 25, 29, 31, 34, 35, and 36.

[0086] Suitably, the IL-33 antagonist is a binding molecule comprising three CDRs in the light chain variable region according to SEQ ID NO:19.

[0087] Thus, suitably, the IL-33 antagonist is a binding molecule that may comprise, for example, three CDRs in a heavy chain variable region independently selected from SEQ ID NOs: 1, 7, 11, 13, 16, 17, and 18, and three CDRs in a light chain variable region independently selected from SEQ ID NOs: 19, 25, 29, 31, 34, 35, and 36.

[0088] Thus, preferably, the IL-33 antagonist is a binding molecule comprising three CDRs in a heavy chain variable region according to SEQ ID NO:1 and three CDRs in a light chain variable region according to SEQ ID NO:19.

[0089] Thus, suitably, the IL-33 antagonist is a binding molecule that may comprise a variable heavy domain (VH) and a variable light domain (VL) having VH CDRs 1-3 with the sequences of SEQ ID NOs: 37, 38, and 39, respectively, wherein one or more VH CDRs have no more than three single amino acid substitutions, insertions, and / or deletions.

[0090] Thus, preferably, the IL-33 antagonist is a binding molecule comprising a VH domain comprising VHCDRs 1 to 3 of SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39, respectively.

[0091] Therefore, preferably, the IL-33 antagonist is a binding molecule comprising a VH domain comprising VHCDR1 to 3 consisting of SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39, respectively.

[0092] Thus, suitably, the IL-33 antagonist is a binding molecule that may comprise a variable heavy domain (VH) and a variable light domain (VL) having VLCDRs 1-3 with the sequences of SEQ ID NOs: 40, 41, and 42, respectively, wherein one or more VLCDRs have no more than three single amino acid substitutions, insertions, and / or deletions.

[0093] Thus, preferably, the IL-33 antagonist is a binding molecule comprising a VL domain comprising VLCDR1-3 of SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42, respectively.

[0094] Therefore, preferably, the IL-33 antagonist is a binding molecule comprising a VL domain comprising VLCDR1 to 3 consisting of SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42, respectively.

[0095] Thus, suitably, the IL-33 antagonist is a binding molecule that may comprise a VHCDR1 having the sequence of SEQ ID NO: 37, a VHCDR2 having the sequence of SEQ ID NO: 38, a VHCDR3 having the sequence of SEQ ID NO: 39, a VLCDR1 having the sequence of SEQ ID NO: 40, a VLCDR2 having the sequence of SEQ ID NO: 41, and a VLCDR3 having the sequence of SEQ ID NO: 42.

[0096] Thus, preferably, the IL-33 antagonist is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VH has an amino acid sequence at least 90%, such as 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, identical to a VH according to SEQ ID NOs: 1, 7, 11, 13, 16, 17, and 18.

[0097] Thus, preferably, the IL-33 antagonist is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VH has an amino acid sequence at least 90%, such as 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, identical to the VH according to SEQ ID NO:1.

[0098] Thus, preferably, the IL-33 antagonist is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VH disclosed above has a sequence in which 1, 2, 3, or 4 framework amino acids have been deleted, inserted, and / or independently substituted with different amino acids.

[0099] Thus, preferably, the IL-33 antagonist is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VL has an amino acid sequence at least 90%, e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, identical to a VL according to SEQ ID NOs: 19, 25, 29, 31, 34, 35, and 36.

[0100] Thus, preferably, the IL-33 antagonist is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VL has an amino acid sequence at least 90%, such as 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, identical to the VL according to SEQ ID NO: 19.

[0101] Thus, preferably, the IL-33 antagonist is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VL has a sequence as disclosed above in which 1, 2, 3, or 4 framework amino acids have been independently deleted, inserted, and / or substituted with different amino acids.

[0102] Thus, preferably, the IL-33 antagonist is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VH has an amino acid sequence at least 90%, such as 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, identical to a VH according to SEQ ID NOs: 1, 7, 11, 13, 16, 17, and 18, and the VL has an amino acid sequence at least 90%, such as 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to a VL according to SEQ ID NOs: 19, 25, 29, 31, 34, 35, and 36.

[0103] Thus, preferably, the IL-33 antagonist is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VH has the amino acid sequence consisting of SEQ ID NOs: 1, 7, 11, 13, 16, 17, and 18, and the VL has the amino acid sequence consisting of SEQ ID NOs: 19, 25, 29, 31, 34, 35, and 36.

[0104] Thus, preferably, the IL-33 antagonist is an antibody or binding fragment thereof comprising a VH and a VL, wherein the VH has the amino acid sequence consisting of SEQ ID NO: 1 and the VL has the amino acid sequence consisting of SEQ ID NO: 19.

[0105] Composition and Administration The IL-33 antagonists in the medical uses and methods described herein may be administered to a patient in the form of a pharmaceutical composition.

[0106] Suitably, reference herein to an "IL-33 antagonist" may also refer to a pharmaceutical composition comprising an IL-33 antagonist. Suitably, a pharmaceutical composition may comprise one or more IL-33 antagonists.

[0107] Suitably, the IL-33 antagonist may be administered in a pharmaceutically effective amount for the in vivo treatment of abnormal epithelial physiology, or an EGFR-mediated disease, or a respiratory disease as defined in the methods of medical use and treatment aspects herein.

[0108] Suitably, a "pharmaceutically effective amount" or "therapeutically effective amount" of an IL-33 antagonist should be held to mean an amount sufficient to achieve effective binding with IL-33 and achieve a benefit, e.g., ameliorate the symptoms of a disease or condition as described in the medical uses / methods herein.

[0109] Suitably, the IL-33 antagonist or pharmaceutical composition thereof may be administered to humans or other animals in accordance with the above-mentioned methods of treatment / medical uses in an amount sufficient to produce a therapeutic effect.

[0110] Suitably, the IL-33 antagonist or pharmaceutical composition thereof may be administered to such humans or other animals in conventional dosage forms prepared by combining the IL-33 antagonist with conventional pharmaceutically acceptable carriers or diluents in accordance with known techniques.

[0111] Those skilled in the art will recognize that the form and nature of the pharmaceutically acceptable carrier or diluent will be dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables. Those skilled in the art will further appreciate that cocktails comprising more than one species of IL-33 antagonist may prove particularly effective.

[0112] The amount of IL-33 antagonist that can be combined with carrier materials to produce a single dosage form will vary depending on the subject being treated and the particular method of administration. Suitably, the pharmaceutical composition can be administered as a single dose, multiple doses, or over an established period of time via infusion. Suitably, the dosage regimen can also be adjusted to achieve the optimum desired response (e.g., a therapeutic or prophylactic response).

[0113] Preferably, the IL-33 antagonist is formulated to facilitate ease of administration and to promote stability of the IL-33 antagonist.

[0114] Preferably, the pharmaceutical compositions are formulated to include a pharmaceutically acceptable, non-toxic, sterile carrier, such as physiological saline, non-toxic buffers, preservatives, and the like.

[0115] Suitably, the pharmaceutical composition may contain a pharmaceutically acceptable carrier, including, for example, water, an ion exchanger, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicic acid, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol, and wool fat.

[0116] Preferably, the pharmaceutical composition may include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include 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, such as saline and buffered media.

[0117] Suitable pharmaceutically acceptable carriers include, but are not limited to, 0.01-0.1M, preferably 0.05M, phosphate buffer or 0.8% saline. Other common parenteral carriers include sodium phosphate solutions, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as antimicrobials, antioxidants, chelating agents, and inert gases and the like.

[0118] Preferably, pharmaceutical compositions for injectable use may include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In such cases, the composition must be sterile and fluid to the extent that easy syringability exists. The composition must be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. Preferably, the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0119] Suitable formulations for use in the therapeutic methods disclosed herein are described in Remington's Pharmaceutical Sciences (Mack Publishing Co.), 16th ed. (1980).

[0120] Preferably, the prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride, in the pharmaceutical composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0121] Preferably, sterile injectable solutions can be prepared by incorporating the active compound (e.g., an IL-33 antagonist, alone or in combination with other active agents) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as appropriate, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound in a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, preparation methods include vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution thereof.

[0122] Methods of administering an IL-33 antagonist or a pharmaceutical composition thereof to a subject in need thereof are well known to, or readily determined by, those of skill in the art.

[0123] Suitably, the route of administration of the IL-33 antagonist or pharmaceutical composition thereof may be, for example, oral, parenteral, by inhalation or topical. Suitably, the term parenteral as used herein includes, for example, intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or intravaginal administration.

[0124] Suitably, the IL-33 antagonist or pharmaceutical composition thereof may be administered orally in an acceptable dosage form including, for example, a capsule, a tablet, an aqueous suspension, or a solution.

[0125] Suitably, the IL-33 antagonist or pharmaceutical composition thereof may be administered by nasal aerosol or inhalation. Such compositions may be prepared as a solution in saline using benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, and / or other conventional solubilizing or dispersing agents.

[0126] Suitably, parenteral formulations can be a single bolus dose, an infusion, or a loading bolus dose followed by a maintenance dose. These compositions may be administered at specific fixed or variable intervals, for example, once daily, or "as needed."

[0127] Preferably, the IL-33 antagonist or pharmaceutical composition thereof is delivered directly to the site of the disease or condition, e.g., abnormal epithelial physiology, thereby increasing the exposure of the affected tissue to the therapeutic agent. Preferably, the IL-33 antagonist or pharmaceutical composition thereof is administered directly to the site of the disease or condition. Thus, preferably, the IL-33 antagonist or pharmaceutical composition thereof is administered to the site of abnormal epithelial physiology, EGFR-mediated disease, or respiratory disease.

[0128] In one embodiment, the administration of the IL-33 antagonist or pharmaceutical composition thereof is to the respiratory tract. Preferably, it is by intranasal administration. Preferably, it is by intranasal inhalation. Preferably, the IL-33 antagonist or pharmaceutical composition thereof may be provided in an inhalation device. Suitable inhalation devices are well known in the art.

[0129] In one embodiment, there is provided an inhaler comprising an IL-33 antagonist or a pharmaceutical composition thereof for use in the prevention or treatment of a condition or disease as defined herein.

[0130] Thus, preferably, the IL-33 antagonist or pharmaceutical composition thereof is formulated as a liquid composition, preferably as a liquid composition that can be aerosolized.

[0131] In one embodiment, the IL-33 antagonist or pharmaceutical composition thereof is provided as an aerosol.

[0132] Preferably, the above-described components for preparing the pharmaceutical compositions described herein may be packaged and sold in the form of a kit, which may preferably bear a label or package insert indicating that the associated pharmaceutical composition is useful for treating a subject suffering from or susceptible to a disease or disorder.

[0133] Preferably, the ingredients for the liquid formulation are processed according to methods known in the art, filled into containers such as ampoules, bags, bottles, syringes, or vials, and sealed under sterile conditions. Preferably, the containers are pressurizable, and preferably, they may be aerosol containers. These containers may be included in the kits as described above. Preferably, the kits may further include an inhalation device. Preferably, the inhalation device contains an IL-33 antagonist or pharmaceutical composition described herein, or is operable to contain such a container that may contain an IL-33 antagonist or pharmaceutical composition described herein.

[0134] Abnormal epithelial physiology The present disclosure relates to the medical use of IL-33 antagonists to prevent or treat abnormal epithelial physiology.

[0135] "Abnormal epithelial physiology," as used herein, refers to any abnormality in the function of epithelia in the human body. The functions of epithelia in the human body include: acting as a barrier to protect underlying tissues; regulating and exchanging chemicals between tissues and cavities; secreting chemicals into cavities; and sensation. Abnormalities in any of these functions can have devastating physiological effects. Epithelia are present in a wide range of tissues in the body, including the skin, respiratory tract, gastrointestinal tract, reproductive tract, urinary tract, exocrine glands, and endocrine glands, and as such, abnormalities in epithelia can be involved in a wide range of diseases or pathologies. Preferably, the epithelium is respiratory tract epithelium, and the abnormal epithelial physiology is abnormal respiratory tract epithelial physiology.

[0136] "Abnormal," as used herein, refers to a difference in function compared to the function in a healthy subject, typically an increase or decrease in function compared to the function in a healthy subject.

[0137] Preferably, the epithelium is selected from: squamous, cuboidal, columnar, and pseudostratified. Preferably, the epithelium is columnar.

[0138] Preferably, the epithelium is ciliated. Preferably, the epithelium is ciliated columnar. Preferably, the abnormal epithelial physiology is abnormal ciliated columnar epithelial physiology.

[0139] Preferably, the abnormal epithelial physiology includes abnormal epithelial cell migration. Preferably, the abnormal epithelial physiology may include decreased epithelial cell migration. Preferably, the abnormal epithelial physiology may include abnormal epithelial cell proliferation. Preferably, the abnormal epithelial physiology may include decreased epithelial cell proliferation.

[0140] Preferably, reduced epithelial cell migration leads to an impaired ability of the epithelium to repair wounds. Preferably, the abnormal epithelial physiology includes impaired wound repair. Impaired wound repair may include impaired wound closure and reduced wound cell density.

[0141] Preferably, treating abnormal epithelial physiology may include increasing or improving epithelial cell migration. Preferably, treating abnormal epithelial physiology may include increasing or improving epithelial wound repair. Preferably, treating abnormal epithelial physiology may include increasing or improving wound closure. Preferably, treating abnormal epithelial physiology may include increasing or improving wound cell density.

[0142] Preferably, the abnormal epithelial physiology is abnormal mucociliary physiology.

[0143] "Abnormal mucociliary physiology," as used herein, refers specifically to any abnormality in the function of the mucociliary role of the epithelium. Abnormal function of the mucociliary role of the epithelium may be due to abnormal function of ciliated columnar cells and / or goblet cells, which are key to mucociliary function. Preferably, the abnormal mucociliary physiology is due to abnormal function of goblet cells.

[0144] "Mucociliary" as used herein refers to the function of ciliated columnar cells and goblet columnar cells in the epithelium to secrete and transport mucus. The roles of the mucociliary body of the epithelium can include: goblet cell proliferation; goblet cell differentiation; mucus secretion; regulation of mucus composition; and / or mucus transport or clearance.

[0145] In one embodiment, there is provided an IL-33 antagonist for use in the prevention or treatment of abnormal mucociliary physiology, such as abnormal mucociliary physiology of the epithelium.

[0146] In one embodiment, a method of preventing or treating abnormal mucociliary physiology, such as abnormal mucociliary physiology of the epithelium, in a patient is provided, comprising: administering to a patient in need thereof an effective amount of an IL-33 antagonist.

[0147] Abnormal mucociliary physiology can include any abnormal function of ciliated columnar cells or goblet cells of the epithelium. Preferably, the abnormal mucociliary physiology includes: abnormal mucus production; abnormal goblet cell differentiation; abnormal goblet cell proliferation; abnormal epithelial thickness; abnormal mucus clearance; and / or abnormal mucus composition.

[0148] Preferably, the abnormal mucus production comprises abnormal MUC5AC production. Preferably, the abnormal goblet cell differentiation comprises abnormal MUC5AC+ goblet cell differentiation. Preferably, the abnormal goblet cell proliferation comprises abnormal MUC5AC+ goblet cell proliferation. Preferably, the abnormal thickness of the epithelium comprises an abnormal amount of MUC5AC+ in the total tissue area of ​​the epithelium. + Contains goblet cells.

[0149] Preferably, the abnormal mucociliary physiology includes: an increased number of goblet cells; increased mucus production; increased goblet cell differentiation; increased epithelial thickness; and / or decreased mucus clearance.

[0150] Preferably, the increased mucus production comprises increased MUC5AC production. Preferably, the increased goblet cell differentiation comprises increased MUC5AC+ goblet cell differentiation. Preferably, the increased goblet cell proliferation comprises increased MUC5AC+ goblet cell proliferation. Preferably, the increased epithelial thickness comprises increased amounts of MUC5AC in the total tissue area of ​​the epithelium. + Contains goblet cells.

[0151] Preferably, the increased production of MUC5AC is caused by increased MUC5AC gene expression. Preferably, the abnormal mucociliary physiology comprises increased MUC5AC gene expression in epithelial cells. Preferably, the abnormal mucociliary physiology comprises increased expression of MUC5AC in epithelial goblet cells.

[0152] Preferably, the abnormal mucociliary physiology includes an alteration in mucus composition, which may include an increase or decrease in the ratio of various mucus compounds contained 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.

[0153] The change in mucus composition can include an increase or decrease in the ratio of different mucins, such as an increase or decrease in the ratio of mucins MUC5AC to MUC5B.

[0154] The alteration in mucus composition may include an increase or decrease in the concentration of mucins. Preferably, the alteration in mucus composition includes a decrease in the concentration of mucin 5AC. Preferably, the alteration in mucus composition includes a decrease in the number of goblet cells with upregulated MUC5AC expression.

[0155] Such changes in mucins contained in mucus can be measured and calculated as described in WO 2018 / 204598, which is incorporated herein by reference.

[0156] Preferably, the abnormal mucus composition comprises an increased ratio of MUC5AC:MUC5B. Preferably, the abnormal mucus composition comprises an increase in MUC5AC contained in the mucus. Preferably, the abnormal mucus composition comprises an increase in mucus thickness.

[0157] Abnormal mucociliary physiology may include any one or more of the above symptoms in combination.

[0158] Preferably, the abnormal epithelial physiology includes abnormal tissue remodeling, such as abnormal epithelial remodeling. Preferably, the abnormal epithelial physiology includes increased tissue remodeling. Preferably, the abnormal epithelial physiology includes increased epithelial remodeling.

[0159] Abnormal epithelial physiology may include any one or more of the above symptoms in combination.

[0160] Treatment or prevention of abnormal epithelial physiology or treatment or prevention of abnormal mucociliary physiology: Improved or increased mucociliary clearance; Reducing or inhibiting mucus production; Inhibition of abnormal mucus composition; reducing or inhibiting epithelial remodeling; and / or This may include reducing or inhibiting goblet cell differentiation and / or proliferation.

[0161] Suitably, reducing or inhibiting mucus production comprises reducing or inhibiting MUC5AC production. Thus, suitably, the treatment or prevention reduces or inhibits MUC5AC production.

[0162] Preferably, inhibiting abnormal mucus composition may include restoring normal mucus composition. Preferably, this includes reducing the ratio of MUC5AC:MUC5B. Thus, preferably, treatment or prevention reduces the ratio of MUC5AC:MUC5B. Preferably, prevention or treatment inhibits or reduces MUC5AC in mucus. Preferably, prevention or treatment reduces mucus thickness.

[0163] Suitably, the reduction or inhibition of goblet cell differentiation and / or proliferation is due to the induction of MUC5AC + The treatment or prevention includes reducing or inhibiting goblet cell differentiation or proliferation. + Reduces or inhibits goblet cell differentiation or proliferation.

[0164] Preferably, reducing or inhibiting epithelial remodelling comprises reducing the thickness of the respiratory epithelium. Thus, preferably, the treatment or prevention reduces the thickness of the respiratory epithelium.

[0165] Preferably, the reduction or inhibition of epithelial remodeling is achieved by increasing MUC5AC in all tissue regions of the epithelium. + Thus, preferably, the treatment or prevention involves reducing the amount of goblet cells in the entire tissue area of ​​the epithelium. + Reduce or inhibit goblet cell mass.

[0166] Improving or increasing mucociliary clearance includes improving or increasing mucociliary motility. Thus, preferably, the treatment or prevention improves or increases mucociliary motility.

[0167] Preferably, the epithelium is a respiratory epithelium. Preferably, the abnormal epithelial physiology is abnormal epithelial physiology in a respiratory epithelium.

[0168] In one embodiment, there is provided an IL-33 antagonist for use in treating abnormal epithelial physiology in respiratory disease.

[0169] In one embodiment, a method of preventing or treating abnormal epithelial physiology in a patient with a respiratory disease is provided, comprising: administering to a patient in need thereof an effective amount of an IL-33 antagonist.

[0170] Suitable respiratory disorders are defined elsewhere herein.

[0171] Preferably, the abnormal epithelial physiology is abnormal mucociliary physiology in the respiratory epithelium.

[0172] In one embodiment, there is provided an IL-33 antagonist for use in the prevention or treatment of abnormal mucociliary physiology in respiratory epithelia.

[0173] In one embodiment, a method of preventing or treating abnormal mucociliary physiology of the respiratory epithelium in a patient is provided, comprising: administering to a patient in need thereof an effective amount of an IL-33 antagonist.

[0174] Preferably, the abnormal epithelial physiology is abnormal mucociliary physiology in respiratory disease.

[0175] In one embodiment, there is provided an IL-33 antagonist for use in the prevention or treatment of abnormal mucociliary physiology in respiratory disease.

[0176] In one embodiment, a method of preventing or treating abnormal mucociliary physiology in a patient with a respiratory disease is provided, comprising: administering to a patient in need thereof an effective amount of an IL-33 antagonist.

[0177] Preferably, the abnormal epithelial physiology is present in the respiratory tract. Preferably, the abnormal epithelial physiology is abnormal epithelial physiology of the respiratory tract. Preferably, the abnormal epithelial physiology is abnormal mucociliary physiology of the respiratory tract.

[0178] The respiratory tract includes the upper and lower respiratory tract. Typically, the upper respiratory tract includes the nasal cavity, paranasal sinuses, pharynx, and larynx. Typically, the lower respiratory tract includes the trachea, bronchi, bronchioles, alveolar ducts, and alveoli.

[0179] Preferably, the abnormal epithelial physiology is abnormal epithelial physiology of the lower respiratory tract, such as the bronchi.

[0180] Preferably, the abnormal epithelial physiology is abnormal epithelial physiology of the lower respiratory tract. Preferably, the abnormal epithelial physiology is abnormal epithelial physiology of the bronchi. Preferably, the abnormal lower respiratory tract epithelial physiology is abnormal mucociliary physiology of the lower respiratory tract. Preferably, the abnormal mucociliary physiology of the lower respiratory tract is abnormal mucociliary physiology of the bronchi.

[0181] In one embodiment there is provided an IL-33 antagonist for use in the prevention or treatment of abnormal mucociliary physiology of the lower respiratory tract.

[0182] In one embodiment, a method of preventing or treating abnormal mucociliary physiology of the lower respiratory tract in a patient is provided, comprising: administering to a patient in need thereof an effective amount of an IL-33 antagonist.

[0183] In one embodiment there is provided an IL-33 antagonist for use in the prevention or treatment of abnormal mucociliary physiology of the bronchi.

[0184] In one embodiment, a method of preventing or treating abnormal bronchial mucociliary physiology in a patient is provided, comprising: administering to a patient in need thereof an effective amount of an IL-33 antagonist.

[0185] EGFR signaling This disclosure is based on the discovery that oxidized IL-33 binds to RAGE, which then complexes with EGFR and acts to disrupt epithelial homeostasis. The use of an IL-33 antagonist can inhibit oxidized IL-33 signaling, thereby inhibiting RAGE activation and RAGE-EGFR complexation. The data disclosed herein demonstrate that preventing the formation of the RAGE-EGFR complex disrupts IL-33-mediated EGFR signaling and restores normal epithelial physiology.

[0186] Preferably, the IL-33 antagonist inhibits oxidized IL-33 signaling.

[0187] Preferably, the IL-33 antagonist inhibits the binding of oxidized IL-33 to RAGE.

[0188] Preferably, the IL-33 antagonist inhibits the formation of the RAGE-EGFR complex. Preferably, the IL-33 antagonist inhibits the formation of the oxidized IL33-RAGE-EGFR complex.

[0189] Preferably, the IL-33 antagonist inhibits EGFR clustering. Preferably, the IL-33 antagonist inhibits EGFR clustering in the cell membrane. Preferably, the IL-33 antagonist inhibits EGFR internalization. Preferably, the IL-33 antagonist inhibits co-localization of RAGE and EGFR in the cell membrane. Preferably, the IL-33 antagonist inhibits internalization of the RAGE-EGFR complex.

[0190] Preferably, the IL-33 antagonist inhibits activation of EGFR. Preferably, the IL-33 antagonist inhibits phosphorylation of EGFR.

[0191] Preferably, the IL-33 antagonist inhibits a RAGE-EGFR mediated effect. Preferably, the IL-33 antagonist inhibits an effect mediated by the RAGE-EGFR complex. Preferably, the IL-33 antagonist inhibits an effect mediated by the oxidized IL33-RAGE-EGFR complex.

[0192] Preferably, the IL-33 antagonist inhibits EGFR signaling. Preferably, the IL-33 antagonist inhibits RAGE-EGFR signaling. Preferably, the IL-33 antagonist inhibits oxidized IL33-RAGE-EGFR signaling.

[0193] Suitably, the IL-33 antagonist inhibits the binding of oxidized IL-33 to RAGE, thereby inhibiting RAGE-EGFR complexation and thereby inhibiting RAGE-EGFR mediated effects such as downstream signaling.

[0194] Preferably, the IL-33 antagonist inhibits IL-33-mediated EGFR effects. Preferably, the IL-33 antagonist inhibits IL-33-mediated EGFR signaling. Preferably, the IL-33 antagonist inhibits oxidized IL-33-mediated EGFR effects. Preferably, the IL-33 antagonist inhibits oxidized IL-33-mediated EGFR signaling. Preferably, the IL-33 antagonist inhibits oxidized IL-33-mediated RAGE-EGFR effects. Preferably, the IL-33 antagonist inhibits oxidized IL-33-mediated RAGE-EGFR signaling.

[0195] Preferably, the RAGE-EGFR mediated effect is caused by RAGE-EGFR complexation, preferably by oxidized IL-33-RAGE-EGFR complexation.

[0196] Preferably, such effects may include downstream signaling, which may typically be referred to herein as EGFR signaling or RAGE-EGFR signaling. Preferably, such EGFR signaling may include phosphorylation and / or chemokine release.

[0197] Preferably, such EGFR signaling includes phosphorylation of EGFR and subsequent phosphorylation of components of the EGFR pathway, such as EGFR, PLC, JNK, MAPK / ERK1 / 2, p38, and STAT5. Preferably, EGFR signaling includes phosphorylation of tyrosine kinases, such as JNK, MAPK / ERK, and p38.

[0198] Suitably, EGFR signalling includes increased release of chemokines such as IL-8.

[0199] Thus, preferably, the IL-33 antagonist inhibits EGFR-mediated phosphorylation and / or chemokine release.

[0200] Thus, preferably, the IL-33 antagonist inhibits phosphorylation of a component of the EGFR pathway. Preferably, the IL-33 antagonist inhibits phosphorylation of any one of: EGFR, PLC, JNK, MAPK / ERK1 / 2, p38, and STAT5. Preferably, the IL-33 antagonist inhibits EGFR-mediated phosphorylation of any one of: EGFR, PLC, JNK, MAPK / ERK1 / 2, p38, and STAT5. Preferably, the IL-33 antagonist inhibits phosphorylation of a tyrosine kinase. Preferably, the IL-33 antagonist inhibits phosphorylation of a tyrosine kinase selected from: JNK, MAPK / ERK, and p38. Preferably, the IL-33 antagonist inhibits EGFR-mediated phosphorylation of a tyrosine kinase selected from: JNK, MAPK / ERK, and p38.

[0201] Thus, preferably, the IL-33 antagonist inhibits the release of chemokines. Preferably, the IL-33 antagonist inhibits the release of IL-8. Preferably, the IL-33 antagonist inhibits EGFR-mediated release of chemokines. Preferably, the IL-33 antagonist inhibits EGFR-mediated release of IL-8.

[0202] In one embodiment, there is provided an IL-33 antagonist for use in the prevention or treatment of an EGFR-mediated disease.

[0203] In another embodiment, the IL-33 antagonist may be for use in the prevention or treatment of respiratory disease by inhibiting EGFR-mediated effects.

[0204] Additionally, IL-33 antagonists may be for use in the prevention or treatment of abnormal epithelial physiology in EGFR-mediated diseases.

[0205] In one embodiment, there is provided an IL-33 antagonist for use in the prevention or treatment of EGFR-mediated diseases by ameliorating abnormal epithelial physiology.

[0206] Preferably, the EGFR-mediated disease is a RAGE-EGFR-mediated disease.

[0207] Preferably, the EGFR-mediated effect is a RAGE-EGFR-mediated effect.

[0208] Preferably, the EGFR-mediated effect is RAGE-EGFR-mediated signaling.

[0209] Suitably, the IL-33 antagonist inhibits an EGFR-mediated effect. Suitably, the IL-33 antagonist treats or prevents a disease or condition by inhibiting an EGFR-mediated effect.

[0210] Suitably, the IL-33 antagonist inhibits a RAGE-EGFR mediated effect. Suitably, the IL-33 antagonist treats or prevents a disease or condition by inhibiting a RAGE-EGFR-mediated effect.

[0211] As used herein, a "RAGE-EGFR mediated effect" refers to any physiological effect caused by the complexation of RAGE with EGFR at the cell membrane and the resulting abnormal EGFR activity. A RAGE-EGFR mediated effect 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 found in epithelia and manifest as abnormal epithelial physiology. Abnormal epithelial physiology is defined above, but may include adverse effects on: barrier integrity; regulation and exchange of chemicals between tissues and cavities; secretion of chemicals into cavities; and sensation.

[0212] Preferably, the RAGE-EGFR mediated disease and / or effect is characterised by aberrant EGFR activity. Preferably, the RAGE-EGFR mediated disease and / or effect is characterised by aberrant RAGE-EGFR signalling. Preferably, the RAGE-EGFR mediated effect and / or RAGE-EGFR signalling characterises the RAGE-EGFR mediated disease.

[0213] Suitably, the RAGE-EGFR mediated disease may be a disease characterized by abnormal epithelial physiology.

[0214] Suitably, the RAGE-EGFR mediated disease may be a disease characterized by abnormal epithelial physiology in the respiratory epithelium.

[0215] Suitably, the RAGE-EGFR mediated disease may be a disease characterized by abnormal mucociliary physiology.

[0216] Suitably, the RAGE-EGFR mediated disease may be a disease characterized by abnormal mucociliary physiology in the respiratory epithelium.

[0217] Suitable RAGE-EGFR mediated diseases may be selected from any of the respiratory diseases defined herein below.

[0218] respiratory disease The present disclosure relates to the medical use of IL-33 antagonists for the prevention or treatment of respiratory diseases by improving epithelial physiology or by modulating EGFR-mediated effects, preferably by inhibiting EGFR-mediated, preferably by inhibiting RAGE / EGFR-mediated effects.

[0219] Suitably, abnormal epithelial physiology may be a symptom of a respiratory disease, and therefore suitably, the IL-33 antagonist may be for use in the treatment or prevention of a respiratory disease characterized by abnormal epithelial physiology.

[0220] As defined in a further embodiment, there is provided an IL-33 antagonist for use in the prevention or treatment of respiratory disease by improving epithelial physiology.

[0221] Abnormal epithelial physiology is defined elsewhere herein.

[0222] Suitable improvement of epithelial physiology may include improvement of abnormal epithelial physiology.

[0223] Suitable means of ameliorating abnormal epithelial physiology are described herein above.

[0224] Preferably, the treatment of respiratory disorders by improving abnormal epithelial physiology comprises: Improved or increased mucociliary clearance; Reducing or inhibiting mucus production; Inhibition of abnormal mucus composition; Reducing or inhibiting aberrant epithelial remodeling; and / or This may include reducing or inhibiting goblet cell differentiation or proliferation.

[0225] Further details regarding each of these effects are provided above in relation to the treatment or prevention of abnormal epithelial physiology, and may be combined herein with the treatment of respiratory disease.

[0226] Suitably, aberrant EGFR activity may be a symptom of a respiratory disease, and therefore suitably, the IL-33 antagonist may be for use in the treatment or prevention of a respiratory disease characterized by aberrant EGFR activity.

[0227] As defined in a further embodiment, there is provided an IL-33 antagonist for use in the prevention or treatment of respiratory disease by inhibiting EGFR-mediated effects.

[0228] EGFR-mediated effects are defined elsewhere herein.

[0229] Preferably, the respiratory disorder is a lower respiratory disorder, and preferably, the respiratory disorder is a disorder affecting the trachea, bronchi, bronchioles, alveolar ducts, and / or alveoli. Preferably, the respiratory disorder is a bronchial disorder.

[0230] Suitably, the respiratory disease may be selected from: COPD, bronchitis, emphysema, bronchiectasis such as CF-bronchiectasis or non-CF-bronchiectasis, asthma, overlap of asthma and COPD (ACO).

[0231] Preferably, the respiratory disease is COPD. Preferably, the respiratory disease is bronchitis-associated COPD. Bronchitis-associated COPD is a specific form of COPD in which patients with COPD experience chronic bronchitis. Bronchitis-associated COPD patients experience a faster decline in lung function, increased symptoms, and a higher risk of secondary infections, resulting in a higher mortality rate than COPD patients. In particular, patients with bronchitis-associated COPD have higher total mucin levels and mucus hypersecretion. Therefore, due to the discovery that IL-33 antagonists inhibit EGFR activity and improve mucociliary physiology, patients with bronchitis-associated COPD may particularly benefit from treatment with IL-33 antagonists as described herein.

[0232] Preferably, for the same reason, the respiratory disease may be bronchial asthma.

[0233] In one embodiment, an IL-33 antagonist is provided for preventing or treating bronchitis COPD.

[0234] In one embodiment, there is provided a method of preventing or treating bronchitic COPD in a patient, comprising: administering to a patient in need thereof an effective amount of an IL-33 antagonist.

[0235] ST2 signaling Although the present disclosure relates to the medical use of IL-33 antagonists to inhibit RAGE-EGFR mediated signaling and effects, it is already known that IL-33 antagonists can inhibit ST2 mediated signaling and effects, and therefore the medical uses described herein contemplate modulation of both EGFR- and ST2-mediated effects.

[0236] Preferably, the IL-33 antagonist is for use in the prevention and treatment of abnormal epithelial physiology by modulating EGFR- and ST2-mediated effects. Preferably, the IL-33 antagonist is for use in the prevention and treatment of abnormal epithelial physiology by inhibiting EGFR- and ST2-mediated effects. Preferably, the IL-33 antagonist is for use in the prevention and treatment of abnormal epithelial physiology by inhibiting RAGE / EGFR- and ST2-mediated effects. Abnormal epithelial physiology is as defined elsewhere herein.

[0237] Preferably, the IL-33 antagonist is for use in the prevention and treatment of EGFR-mediated diseases by modulating EGFR- and ST2-mediated effects. Preferably, the IL-33 antagonist is for use in the prevention and treatment of EGFR-mediated diseases by inhibiting EGFR- and ST2-mediated effects. Preferably, the IL-33 antagonist is for use in the prevention and treatment of EGFR-mediated diseases by inhibiting RAGE / EGFR- and ST2-mediated effects. EGFR-mediated diseases are as defined elsewhere herein.

[0238] Preferably, the ST2-mediated effect includes inflammation. Accordingly, preferably, the IL-33 antagonist is for use in the prevention and treatment of ST2-mediated diseases by modulating EGFR- and ST2-mediated effects. Preferably, the IL-33 antagonist is for use in the prevention and treatment of ST2-mediated diseases by inhibiting EGFR- and ST2-mediated effects. Preferably, the IL-33 antagonist is for use in the prevention and treatment of ST2-mediated diseases by inhibiting RAGE / EGFR- and ST2-mediated effects. Suitable ST2-mediated diseases may include diseases characterized by inflammation. Suitable ST2-mediated diseases may include inflammatory diseases.

[0239] ST2-mediated or inflammatory diseases may include: COPD; allergic diseases such as asthma, chronic sinusitis, food allergies, eczema, and dermatitis; fibroproliferative diseases such as pulmonary fibrosis; pulmonary eosinophilia; pleural malignancies; rheumatoid arthritis; collagen vascular disease; atherosclerotic vascular disease; uticaria; inflammatory bowel disease; Crohn's disease; coeliac disease; systemic lupus erythematosus; progressive systemic sclerosis; Wegener's granulomatosis; septic shock; and Bechet's disease.

[0240] Preferably, the ST2-mediated or inflammatory disease is respiratory. Preferably, the ST2-mediated or inflammatory disease is in the respiratory tract as defined above.

[0241] Preferably, the IL-33 antagonist is for additional use in the prevention and treatment of inflammation or inflammatory diseases. Preferably, the IL-33 antagonist may be for additional use in the prevention and treatment of ST2-mediated inflammation and ST2-mediated inflammatory diseases.

[0242] Preferably, the ST2-mediated disease and the EGFR-mediated disease overlap. In other words, ST2-mediated effects and EGFR-mediated effects, preferably RAGE-EGFR-mediated effects, contribute to the disease pathology. Advantageously, it is believed that the medical use of a single IL-33 antagonist may inhibit the activation of both RAGE and ST2 by IL-33. Thus, a single IL-33 antagonist may simultaneously treat both RAGE-EGFR-mediated disease and ST2-mediated disease.

[0243] In one embodiment, an IL-33 antagonist is provided for use in preventing or treating abnormal epithelial physiology and inflammation. In one embodiment, a method is provided for preventing or treating abnormal epithelial physiology and inflammation in a patient, comprising: administering an effective amount of an IL-33 antagonist.

[0244] Preferably, abnormal epithelial physiology and inflammation may be symptoms of respiratory disease, and therefore references to the treatment and prevention of these symptoms may be in the context of respiratory disease and may preferably include the treatment or prevention of abnormal epithelial physiology and inflammation in respiratory disease.

[0245] In one embodiment, there is provided an IL-33 antagonist for use in the prevention or treatment of EGFR-mediated diseases and ST2-mediated diseases.

[0246] In one embodiment, a method of preventing or treating an EGFR-mediated disease and an ST2-mediated disease in a patient is provided, comprising: administering an effective amount of an IL-33 antagonist.

[0247] Preferably, the IL-33 antagonist is a reduced IL-33 antagonist. Preferably, the reduced IL-33 antagonist is as defined above.

[0248] Preferably, the IL-33 antagonist is as defined above. Preferably, the IL-33 antagonist is 33_640087-7B.

[0249] Alternatively, various IL-33 antagonists can be used as a combination therapy to inhibit both RAGE and ST2 activation by IL-33. Thus, combinations of IL-33 antagonists are envisioned to simultaneously treat both RAGE-EGFR- and ST2-mediated diseases.

[0250] Preferably, the respiratory disease is as defined above. Preferably, the respiratory disease is characterised by abnormal EGFR activity and abnormal ST2 activity.

[0251] Thus, suitably, in one embodiment there is provided a combination of a first IL-33 antagonist for use in the prevention or treatment of abnormal epithelial physiology and a second IL-33 antagonist for use in the prevention or treatment of inflammation.

[0252] Thus, suitably, in one embodiment, there is provided a method of preventing or treating abnormal epithelial physiology and inflammation in a patient, comprising: administering an effective amount of a first IL-33 antagonist in combination with an effective amount of a second IL-33 antagonist.

[0253] Thus, suitably, in one embodiment there is provided a combination of a first IL-33 antagonist for use in the prevention or treatment of an EGFR-mediated disease and a second IL-33 antagonist for use in the prevention or treatment of an ST2-mediated disease.

[0254] Thus, suitably, in one embodiment, there is provided a method of preventing or treating an EGFR-mediated disease and an ST2-mediated disease in a patient, the method comprising: administering an effective amount of a first IL-33 antagonist in combination with an effective amount of a second IL-33 antagonist.

[0255] Suitably, the first IL-33 antagonist is for preventing or treating abnormal epithelial physiology and / or an EGFR-mediated disease.

[0256] Preferably, the second IL-33 antagonist is for preventing and treating inflammation and / or ST2-mediated diseases.

[0257] Preferably, the first and second IL-33 antagonists are different.

[0258] Preferably, the first IL-33 antagonist is as defined above. Preferably, the second IL-33 antagonist may be any other IL-33 antagonist known to inhibit ST2-mediated effects. Preferably, the second IL-33 antagonist is also as defined above.

[0259] 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.

[0260] Preferably, at least one of the IL-33 antagonists is 33_640087-7B. Preferably, the first antagonist is 33_640087-7B.

[0261] Preferably, the first and second IL-33 antagonists may be administered in combination. Preferably, the first and second IL-33 antagonists may be administered in combination simultaneously or at different times. Suitable dosing regimens can be determined by a medical professional.

[0262] These statements apply equally to the above medical uses / methods of treatment in which ST-2 mediated diseases may also be prevented or treated.

[0263] Alternatively, in a further embodiment, the 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 inhibit the ST2 receptor by other means. Suitably, the ST2 inhibitor may function to treat or prevent an ST2-mediated disease, as described above.

[0264] Thus, in one embodiment there is provided a combination of an IL-33 antagonist for use in the treatment or prevention of abnormal epithelial physiology and an ST2 inhibitor for use in the treatment or prevention of inflammation.

[0265] In one embodiment, a method of preventing or treating abnormal epithelial physiology and inflammation in a patient is provided, comprising: administering an effective amount of an IL-33 antagonist in combination with an effective amount of an ST2 inhibitor.

[0266] Preferably, abnormal epithelial physiology and inflammation may be symptoms of respiratory disease, and therefore references to the treatment and prevention of these symptoms may be in the context of respiratory disease and may preferably include the treatment or prevention of abnormal epithelial physiology and inflammation in respiratory disease.

[0267] Thus, in one embodiment there is provided a combination of an IL-33 antagonist for use in the prevention or treatment of an EGFR mediated disease and an ST2 inhibitor for use in the treatment or prevention of an ST2 mediated disease.

[0268] In one embodiment, a method of preventing or treating an EGFR-mediated disease in combination with an ST2-mediated disease in a patient is provided, comprising: administering an effective amount of an IL-33 antagonist in combination with an effective amount of an ST2 inhibitor.

[0269] Suitably, the IL-33 antagonist is as defined elsewhere herein. Suitably, the EGFR-mediated disease and the ST2-mediated disease are as defined elsewhere herein.

[0270] Suitably, the ST2 inhibitor may be any such inhibitor known in the art, for example GSK3772847 (described in WO 2013 / 165894) and RG6149 (WO 2013 / 173761), both of which are incorporated herein by reference.

[0271] Preferably, the IL-33 antagonist and the ST2 inhibitor may be administered in combination. Preferably, the IL-33 antagonist and the ST2 inhibitor may be administered in combination at the same time or at different times. The appropriate dosing regimen can be determined by a medical professional.

[0272] patient The methods and medical uses are practiced in relation to a patient or subject, which may be one in need of identification, diagnosis, or treatment of a physiological condition or disease, such as abnormal epithelial physiology, an EGFR-mediated disease, or a respiratory disease.

[0273] Preferably, the patient may be a human. The patient may be receiving medical care or may be an individual seeking medical care. Preferably, the patient is male or female. Preferably, the patient is an adult or a child.

[0274] Preferably, in the methods described herein, a suitable patient may be one believed to have abnormal epithelial physiology, or an EGFR-mediated disease, or a respiratory disease, for example, a suitable patient may have symptoms consistent with such a condition.

[0275] Alternatively, suitable patients in the context of the methods described herein may be considered to be at risk for developing abnormal epithelial physiology, or an EGFR-mediated disease, or a respiratory disease, for example, such patients may have been in contact with an individual suffering from such a condition, may be suffering from a related condition, or may meet risk factors associated with such conditions, such as smoking, old age, allergies, etc.

[0276] Embodiment Portions of the present disclosure can be characterized by the following embodiments: Embodiment 1 describes an IL-33 antagonist for use in the prevention or treatment of disease by inhibiting EGFR-mediated effects. Embodiment 2 describes an IL-33 antagonist for use according to embodiment 1, wherein the EGFR-mediated effect is a RAGE-EGFR-mediated effect. Embodiment 3 describes an IL-33 antagonist for use according to embodiment 1 or 2, wherein the EGFR-mediated effect is RAGE-EGFR-mediated signaling. Embodiment 4 describes an IL-33 antagonist for use according to any one of embodiments 1 to 3, wherein the disease is a respiratory disease. Embodiment 5 describes an IL-33 antagonist for use according to any one of embodiments 1 to 4, wherein the disease is characterized by abnormal epithelial physiology and / or abnormal EGFR activity. Embodiment 6 describes an IL-33 antagonist for use according to any one of embodiments 1 to 5, wherein the disease is selected from: COPD, bronchitis, emphysema, bronchiectasis such as CF-bronchiectasis or -CF-bronchiectasis, asthma, or asthma and COPD overlap (ACO). Embodiment 7 describes an IL-33 antagonist for use according to any one of embodiments 4 to 6, wherein the respiratory disease is bronchitis COPD. Embodiment 8 describes an IL-33 antagonist for use according to any one of embodiments 1 to 7, wherein the treatment improves mucus clearance; inhibits abnormal mucus production; inhibits abnormal epithelial remodeling; and / or inhibits abnormal goblet cell differentiation. Embodiment 9 describes an IL-33 antagonist for use according to any one of embodiments 1 to 8, wherein the IL-33 antagonist inhibits the activity of oxidized IL-33. Embodiment 10 describes an IL-33 antagonist for use according to any one of embodiments 1 to 9, wherein the IL-33 antagonist prevents oxidized IL-33 from binding to RAGE, thereby inhibiting RAGE-EGFR signaling. Embodiment 11 describes an IL-33 antagonist for use according to any one of Embodiments 1 to 10, wherein the IL-33 antagonist is an anti-IL-33 antibody or antigen-binding fragment thereof, preferably an anti-reduced IL-33 antibody or antigen-binding fragment thereof. Embodiment 12 describes an IL-33 antagonist for use according to embodiment 11, wherein the anti-IL-33 antibody or antigen-binding fragment thereof comprises the complementarity-determining regions (CDRs) of a pair of variable heavy domain (VH) and variable light domain (VL) selected from Table 1. Example 13 describes an IL-33 antagonist for use according to Example 12, wherein the anti-IL-33 antibody or antigen-binding fragment thereof comprises a variable heavy domain (VH) and a variable light domain (VL) pair selected from Table 1. Embodiment 14 describes an IL-33 antagonist for use according to any one of Embodiments 11 to 13, wherein the anti-IL-33 antibody or antigen-binding fragment thereof comprises a VHCDR1 having the sequence of SEQ ID NO: 37, a VHCDR2 having the sequence of SEQ ID NO: 38, a VHCDR3 having the sequence of SEQ ID NO: 39, a VLCDR1 having the sequence of SEQ ID NO: 40, a VLCDR2 having the sequence of SEQ ID NO: 41, and a VLCDR3 having the sequence of SEQ ID NO: 42. Embodiment 15 describes an IL-33 antagonist for use according to any one of embodiments 11 to 14, wherein the IL-33 antagonist is an IL33 antibody or antigen-binding fragment thereof, comprising a VH domain of sequence SEQ ID NO: 1 and a VL domain of sequence SEQ ID NO: 19.

[0277] Embodiments will now be described, by way of example only, with reference to the following drawings in which: [Brief explanation of the drawings]

[0278] [Figure 1] Figure 1 shows a grayscale heat map of the fold increase in kinase phosphorylation compared to untreated controls for each of the MAP kinase phosphorylation antibody array detection assays. Reduced IL-33 (IL-33-01 and IL-33-16, respectively) did not generate a signal above baseline. oxIL-33 (oxidized IL-33-01) caused increased phosphorylation of multiple kinases. [Figure 2]Figure 2 shows the signal patterns for each stimulation condition in a receptor tyrosine kinase (RTK) activity array. oxIL-33 elicited a positive signal in the RTK array corresponding to the epidermal growth factor receptor (EGFR), whereas reduced IL33-01 and IL33-16 did not. Dot intensity correlates with receptor tyrosine kinase phosphorylation. [Figure 3] Figure 3A shows pEGFR(Tyr1068) activity in normal human bronchial epithelial (NHBE) cells stimulated with increasing concentrations of IL-33 or EGFR ligands. oxIL-33, but not reduced IL-33 (IL33-01), promoted EGFR phosphorylation, similar to EGF, HB-EGF, and TGFα. Figure 3B shows pEGFR(Tyr1068) activity in A549 cells stimulated with increasing concentrations of IL-33 or EGFR ligands. oxIL-33 (oxidized IL-33-01) promoted EGFR phosphorylation, similar to EGF, HB-EGF, and TGFα, in a pattern similar to that seen in NHBE cells, but not reduced IL-33 (IL-33-01). [Figure 3C] Figure 3C shows pEGFR(Tyr1068) activity in A549 cells stimulated with increasing concentrations of IL-33, EGFR ligand, or RAGE ligand. oxIL-33, but not wild-type (WT) IL-33 (IL-33-01), C->S mutant (mut) IL-33 (IL-33-16), or RAGE ligand, promoted EGFR phosphorylation similarly to EGF. [Figure 4] Figure 4 shows that oxidized IL-33 induces phosphorylation of multiple molecules involved in the EGFR pathway (EGFR, PLC, AKT, JNK, ERK 1 / 2, p38) when analyzed by Western blot. [Figure 5] Figure 5 shows that increasing doses of anti-EGFR antibody reduce oxIL-33-01-induced STAT5 phosphorylation compared to isotype control. [Figure 6]Figure 6 shows the immunoprecipitation with anti-EGFR followed by Western blotting to detect EGFR, RAGE, or IL-33. IL-33 and RAGE co-precipitated with EGFR after NHBE stimulation with oxIL-33, suggesting that they formed a complex. RAGE appears to be unique to the oxIL-33 signaling complex compared with EGF. [Figure 7] [Figure 7A] Figure 7A shows that oxIL-33 binds directly to RAGE. HMGB1 is a known RAGE ligand and serves as a positive control in this study. [Figure 7B] Figure 7B shows that oxIL-33 does not bind directly to EGFR (although the known EGFR ligand EGF does). However, when RAGE is added to this assay in combination with oxIL-33, EGFR binding is observed. [Figure 8] Figure 8 shows immunoprecipitation with anti-EGFR or anti-RAGE followed by Western blot of EGFR, RAGE, and IL-33 in wild-type and RAGE-deficient A549 cells after activation with oxIL-33 at the indicated time points. [Figure 9] FIG. 9 shows that STAT5 phosphorylation induced by oxIL-33-01 is reduced by anti-RAGE antibody but not by anti-ST2 antibody. [Figure 10] Figure 10 shows that EGF and oxidized IL33 (oxIL33) induce EGFR clustering and internalization in EGFR-GFP A549 cells. Representative images are shown after 5 minutes of stimulation. The histograms show depletion of EGFR in the non-clustered regions of cells treated with EGF and oxIL33 (a left shift in the bell-shaped peak of the histogram) and an increase in the number of saturated pixels in these cells caused by clustering (intensity 255). [Figure 11]Figure 11 shows the fold increase in IL-8 secretion by NHBES and DHBEs after 24 hours of stimulation with medium alone (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 from four NHBE and three DHBE donors. [Figure 12] [Figure 12A] Figure 12A shows the relative wound-healing density of A549 cells after treatment with reduced IL-33, oxIL-33, or EGF. Bar graphs show the mean and SEM from six technical replicates per condition. [Figure 12B] Figure 12B shows the relative wound-healing density of NHBE cells after treatment with reduced IL-33, oxIL-33, or EGF. Bar graphs show the mean and SEM from six technical replicates per condition. [Figure 13] Figure 13 shows the scratch wound closure rate of NHBE cells treated with medium alone (unstimulated control), reduced IL-33, oxidized IL-33, or oxidized IL-33 in the presence of anti-ST2, anti-RAGE, or anti-EGFR. Bar graphs show the mean and SEM from six technical replicates per condition. [Figure 14] FIG. 14 shows the relative wound healing density in human bronchial epithelial cells from healthy subjects, smokers, and COPD with and without stimulation with oxidized IL-33. [Figure 15] Figure 15 shows % wound closure at 24 hours for 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. Bar graphs show the mean and SEM from n=5 individual donors. [Figure 16][Figure 16A] Figure 16A shows representative immunohistochemical staining of basal cells (p63+; blue), ciliated cells (α-tubulin; purple), and goblet cells (mucin 5ac+mucin B; yellow) from ALI cultures derived from a healthy donor. [Figure 16B] Figure 16B shows immunohistochemical quantification of various epithelial cell types using HALO software after 7 days of treatment with anti-IL-33 (33_640087-7B) or an isotype control antibody. Data shown are the mean and SEM from n = 2-3 individual donors. [Figure 16C] Figure 16C shows goblet cell quantification using HALO software after 7 days of treatment with anti-IL-33 (33_640087-7B) or an isotype control antibody. Data shown are the mean and SEM from n = 2-3 individual donors. [Figure 17] Figure 17 shows example staining of individual mucins (mucin 5AC and mucin 5B) in ALI cultures from healthy (1 donor) or COPD (1 donor), and the reduction of mucin staining in COPD cultures after 7 days of treatment with anti-IL-33 (33_640087-7B). [Figure 18] FIG. 18 shows tSNE plots demonstrating the different proportions of cell subtypes found in COPD ALI cultures from individual donors treated with anti-IL-33 compared to untreated. [Figure 19][Figure 19A] Figure 19A shows a representative flow cytometry contour plot detecting goblet cells in ALI cultures from a normal donor. Muc5B is on the x-axis, and Muc5AC is on the y-axis. ALI cultures were treated with proteins for 7 days. Treatment with 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, similar to IL-13. IL-13 is known to increase goblet cells in ALI cultures and is used as a positive control in this study. Numbers in the quadrants indicate the percentage of the total population: Muc5AC single-positive goblet cells in the upper left quadrant, Muc5B single-positive goblet cells in the lower right quadrant, and Muc5AC and Muc5B double-positive goblet cells in the upper right quadrant. [Figure 19B] Figure 19B shows combined flow cytometry data from ALI cultures from normal donors (n = 6) showing the percentage of goblet cells (combined Muc5AC single-positive, Muc5B single-positive, and Muc5AC and Muc5B double-positive goblet cells) relative to the total epithelial population. 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. Violin plots show all data points and medians. [Figure 19C] Figure 19C shows combined flow cytometry data from ALI cultures from 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, similar to IL-13. Violin plots show all data points and medians. [Figure 19D] Figure 19D shows combined RT-qPCR data from ALI cultures from normal donors (n=4) showing fold changes in MUC5AC mRNA. Reduced IL-33 (IL-33[C->S]) did not increase MUC5AC mRNA. OxIL-33 (oxidized IL-33-01) increased MUC5AC mRNA, similar to IL-13.The violin plot shows all data points and the median. [Figure 20] [Figure 20A] Figure 20A shows representative immunohistochemical staining of basal cells (p63+; purple), ciliated cells (α-tubulin; dark grayish blue), and goblet cells (Muc5ac+Muc5B; yellow) from ALI cultures derived from healthy donors. Reduced IL-33 (IL-33[C->S]) did not visually increase goblet cells. oxIL-33 (oxidized IL-33-01) caused a visual increase in goblet cells. [Figure 20B] Figure 20B shows quantification of the area of ​​mucin 5ac+mucin 5b (% total epithelial tissue area) from immunohistochemical images (minimum n = 3 donors per condition) using HALO software. Compared to untreated and reduced IL-33-treated controls, oxIL-33 and IL-13 increase the area of ​​mucin staining. [Figure 21][Figure 21A] Figure 21A shows representative flow cytometry contour plots detecting goblet cells in ALI cultures from COPD donors. Muc5B is on the x-axis, and Muc5AC is depicted on the y-axis. ALI cultures were treated with antibodies for 7 days. Anti-IL-33 (33_640087-7B) treatment reduced total goblet cell numbers. Numbers in the quadrants indicate the percentage of the total population: Muc5AC single-positive goblet cells in the upper left quadrant, Muc5B single-positive goblet cells in the lower right quadrant, and Muc5AC and Muc5B double-positive goblet cells in the upper right quadrant. [Figure 21B] Figure 21B shows combined flow cytometry data from ALI cultures from COPD donors (n=6) showing total goblet cells (combined Muc5AC single-positive, Muc5B single-positive, and Muc5AC and Muc5B double-positive goblet cells). ALI cultures were treated with antibodies for 7 days. Anti-IL-33 (33_640087-7B) treatment reduced total goblet cell numbers. Violin plots represent all data points and medians. [Figure 21C] Figure 21C shows combined flow cytometry data from ALI cultures from COPD donors (n=6) showing 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. Violin plots represent all data points and medians. [Figure 21D] Figure 21D shows combined RT-qPCR data from ALI cultures from COPD donors (n=5) showing fold change in MUC5AC mRNA. Anti-IL-33 (33_640087-7B) treatment reduced MUC5AC expression. Violin plots represent all data points and medians. [FIG. 21E] FIG. 21E shows combined flow cytometry data from ALI cultures from COPD donors (n=6) showing overall viability across treatment conditions, as judged by LD-negative cell staining. [Figure 22][Figure 22A] Figure 22A shows representative immunohistochemical staining of basal cells (p63+; purple), ciliated cells (α-tubulin; dark grayish blue), and goblet cells (Muc5ac+MucB; yellow) from ALI cultures derived from COPD donors. Treatment with anti-IL-33 (33_640087-7B) for 7 days caused a visible reduction in goblet cells. [Figure 22B] Figure 22B shows quantification of the area of ​​Muc5ac+Muc5b (% total epithelial tissue area) from immunohistochemical images (n = 4 donors) using HALO software. Compared to untreated and human IgG1-treated controls, anti-IL-33 (33-640087_7B) reduces the area of ​​mucin staining. [Figure 23] [Figure 23A] Figure 23A shows quantification of Muc5AC in apical lavage fluid obtained from COPD and healthy ALI cultures. COPD cultures have elevated Muc5AC levels as judged by Muc5AC ELISA. [Figure 23B] Figure 23B shows quantification of Muc5AC in apical lavage fluid obtained from healthy ALI cultures. ALI cultures were treated with reduced IL-33mut16 (IL-33[C->S]), oxIL-33, and wild-type IL-33, as measured by Muc5AC ELISA. [Figure 23C] Figure 23C shows quantification of Muc5AC in apical lavage fluid obtained from COPD ALI cultures. Cells were treated with human and mouse IgG1 controls (hIgG1 and mIgG1), 33-640087_7B, or anti-ST2 antibody. Treatment with anti-IL-33 (33-640087_7B) reduced Muc5AC levels as measured by Muc5AC ELISA. [Example]

[0279] Example 1 - Oxidized IL-33 promotes the formation of a signaling complex between RAGE and EGFR In Cohen, ES et al. Nat. Commun. 6:8327 (2015), Applicants described the discovery of an oxidized disulfide-bonded form of IL-33 (DSB IL-33) and showed that this form does not bind to ST2 and cannot activate ST2-dependent signaling. Subsequently (see WO 2016156440 A1), 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.

[0280] To further investigate the function of oxIL-33, epithelial cells were stimulated with reduced or oxidized IL-33 and the signaling pathway was examined. Herein, we show that oxIL-33 is a novel ligand for the receptor for advanced glycation end products (RAGE)-epidermal growth factor receptor (EGFR) complex and has a significant impact on epithelial function.

[0281] 1. Cloning and Expression of Human Mature and Cysteine-Mutated Variants of IL33 cDNA molecules encoding the mature component of human IL-33 (112-270; accession number (UniProt) 095760 (also referred to as IL33-01 or IL-33) and a variant in which four cysteine ​​residues were mutated to serine (IL33-16 or IL-33[C->S]) were synthesized by primer extension PCR and cloned into pJexpress411 (DNA2.0). The wild-type (WT) and mutant IL-33 coding sequences were modified to contain 10xHis, Avitag, and a factor Xa protease cleavage site (MHHHHHHHHHHAAGLNDIFEAQKIEWHEAAIEGR, SEQ ID NO: 43) at the N-terminus of the protein. N-terminally tagged His10 / Avitag IL33-01 (WT, SEQ ID NO: 44) and N-terminally tagged His10 / Avitag IL33-16 (WT, SEQ ID NO: 45) were generated by transformation of E. coli BL21(DE3) cells. Transformed cells were cultured in autoinduction medium (Overnight Express™ Autoinduction System 1, Merck Millipore, 71300-4) at 37°C for 18 hours, after which the cells were harvested by centrifugation and stored at -20°C. Cells were resuspended in 2x DPBS containing cOmplete EDTA-free protease inhibitor cocktail tablets (Roche, 11697498001) and 50 U / mL benzonase nuclease (Merck Millipore, 70746-3) and lysed by sonication. Cell lysates were clarified by centrifugation at 50,000 x g for 30 minutes at 4°C. IL-33 protein was purified from the supernatant by immobilized metal affinity chromatography and loaded onto a HisTrap Excel column (GE Healthcare, 17371205) equilibrated with 2x DPBS, 1 mM DTT at 5 mL / min. The column was washed with 2x DPBS, 1 mM DTT, 20 mM imidazole, pH 7.4 to remove impurities, and then with 2x DPBS, 0.1% Triton X-114 to remove endotoxin and immobilized protein.After further washing with 2x DPBS, 1 mM DTT, 20 mM imidazole, pH 7.4, the sample was eluted with 2x DPBS, 1 mM DTT, 400 mM imidazole, pH 7.4. IL-33 was further purified by size-exclusion chromatography using a HiLoad Superdex75 26 / 600 pg column (GE Healthcare, 28989334) at 2.5 mL / min in 2x DPBS. Peak fractions were analyzed by SDS-PAGE. Fractions containing pure IL-33 were pooled, and the concentration was determined by absorbance at 280 nm. The final sample was analyzed by SDS-PAGE.

[0282] To generate untagged IL-33, N-terminally tagged His10 / Avitag IL-33 was incubated with 10 units of factor Xa (GE Healthcare, 27084901) per mg of protein in 2x DPBS buffer at room temperature for 1 hour. Untagged IL-33 was purified using SEC chromatography on a HiLoad Superdex 75pg column (GE Healthcare, 28989333) in 2x DPBS at a flow rate of 1 mL / min.

[0283] 2. Preparation and purification of oxidized IL-33 (oxIL-33) Reduced IL33-01 was diluted to a final concentration of 0.5 mg / mL in 60% IMDM medium (phenol red-free), 40% DPBS, and oxidized by incubation at 37°C for 18 hours. Aggregates formed during the oxidation process were removed from the sample by loading onto a HiTrap Capto Q ImpRes anion-exchange column (GE Healthcare, 17547055). Prior to loading, the sample was adjusted by adding 1 M Tris, pH 9.0, until the pH reached 8.3, and 5 M NaCl to a final concentration of 125 mM. Under these loading conditions, aggregates bound to the column and monomeric oxIL-33 flowed through and were collected. The tag was cleaved from oxIL-33 by incubation with factor Xa (NEB, P8010L) at a final concentration of 1 μg of factor Xa per 50 μg of oxIL-33 for 120 minutes at 22°C. To remove residual reduced IL-33 from the sample, soluble human ST2S extracellular domain fused to human IgG1 Fc-His6 was incubated with the sample at 22°C for 30 min to bind to reduced IL-33. The sample was concentrated using a 3,000 Da cutoff centrifugal concentrator and loaded onto a HiLoad Superdex 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 pooled and concentrated, and the final concentration of the sample was measured by UV absorbance spectroscopy at 280 nm. The quality of the final product was assessed by SDS-PAGE, HP-SEC, and RP-HPLC.

[0284] 3. Cloning, Expression, and Purification of Human ST2ECD A cDNA encoding the naturally occurring ST2S soluble isoform of ST2 (UniProt accession number Q01638-2), lacking the endogenous signal peptide (amino acid residues 19-328), was amplified by PCR using primers encoding Gibson assembly-compatible extensions and a CD33 signal peptide fused to the N-terminus of the ST2S coding sequence. The coding sequence for human IgG1 Fc with a C-terminal His6 tag was similarly amplified. The ST2S cDNA and IgG1 Fc-His6 cDNA were assembled using Gibson assembly in pDEST12.2OriP, a mammalian CMV promoter-driven expression vector containing the OriP origin of replication from EBV, allowing episomal maintenance in cell lines expressing EBNA-1 protein. For protein expression, the plasmids were primarily transformed into suspension cultures of CHO cells overexpressing EBNA-1 using polyethyleneimine as the transfection reagent. Conditioned medium containing the secreted ST2S-Fc-His6 fusion protein was collected 7 days after transfection and loaded onto a HiTrap MabSelect SuRe (Protein A, GE Healthcare, 11-0034-95) affinity chromatography column at 2 mL / min. The column was washed with 2x DPBS, and the protein was eluted with 25 mM sodium acetate, pH 3.6. Fractions containing ST2S-Fc-His6 were pooled and loaded onto a HiLoad Superdex 200 26 / 600 pg column (GE Healthcare, 28989336) equilibrated with 2x DPBS at 2 mL / min. Fractions containing pure ST2S-Fc-His6 protein were pooled, and the concentration was determined by absorbance at 280 nm. The final sample was analyzed by SDS-PAGE.

[0285] 4. Cloning, Expression, and Purification of Human Asialoglycoprotein Receptor (ASGPR) ECD A cDNA encoding the extracellular domain (ECD) of the asialoglycoprotein receptor (UniProt accession number P07306), lacking the cytoplasmic and transmembrane domains (amino acid residues 62–291), was chemically synthesized at Geneart with a CD33 signal peptide followed by a His10_Avi tag sequence fused to the N-terminus of the ECD domain. The construct was directly cloned into pDEST12.2OriP, a mammalian CMV promoter-driven expression vector containing the OriP origin of replication from EBV, allowing episomal maintenance in cell lines expressing the EBNA-1 protein. For protein expression, the plasmid was primarily transformed into suspension cultures of HEK Freestyle 293F cells using 293fectin as the transfection reagent. Conditioned medium containing the secreted HisAVi_hASGPR ECD fusion protein was collected 7 days after transfection by immobilized metal affinity chromatography and loaded onto a HisTrap Excel column (GE Healthcare, 17371205) equilibrated with 2x DPBS at 4 mL / min. The column was washed with 2x DPBS, 40 mM imidazole, pH 7.4 to remove impurities, and the sample was eluted with 2x DPBS, 400 mM imidazole, pH 7.4. The human ASGPR ECD was further purified by size exclusion chromatography using a HiLoad Superdex75 16 / 600 pg column (GE Healthcare, 28-9893-33) at 1 mL / min in 2x DPBS. Peak fractions were analyzed by SDS-PAGE. Fractions containing pure monomeric ASGPR were pooled, and the concentration was determined by absorbance at 280 nm. The final sample was analyzed by SDS-PAGE.

[0286] 5. Oxidized 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. NHBE cells were harvested with Accutase (PAA, #L1 1-007) and plated at 1 × 10 cells per well in a 6-well dish (Corning Costar, 3516) in culture medium [BEGM (Lonza CC-3171) and supplement kit (Lonza CC-4175)]. 6 Plates were seeded at 2 mL / well. Cells were incubated at 37°C, 5% CO2 for 18-24 hours. After this time, the medium was aspirated, and cells were washed twice with 1 mL PBS before adding starvation medium (BEGM (Lonza CC-3171) supplemented with 1% penicillin / streptomycin). Plates were then incubated at 37°C, 5% CO2 for an additional 18-24 hours before stimulation.

[0287] The MAP kinase phosphorylation antibody array kit (ab211061) was purchased from Abcam, and experiments were performed according to the manufacturer's instructions. Six-well dishes of NHBE cells starved for 18–24 h were either left untreated or treated with 30 ng / mL of either reduced IL-33, IL-33-16, or oxidized IL-33, then returned to the incubator at 37°C and 5% CO2 for 10 min (see Table 2 for the activators used in this assay). The plates were removed from the incubator, and the cells were washed with ice-cold PBS. 100 μL of 1x lysis buffer provided with the kit was then added per well. Protein extracts were transferred to 1.5 mL tubes and clarified at 14,000 rpm at 4°C. Protein concentration was determined using the BCA method (Thermo, 23225), using 250 μg of total protein per array membrane. All subsequent steps were performed according to the manufacturer's instructions. Membranes were visualized with LiCor C-digit and quantified using Image Lite studio.

[0288] [Table 6]

[0289] In contrast to wild-type (IL-33) and C→S (IL-33[C→S]) reduced forms of IL-33 (IL33-01 and IL33-16, respectively), oxidized IL33-01 (oxIL-33) activated multiple key signaling molecules consistent with pathways involving receptor tyrosine kinases (RTKs) (Fig. 1).

[0290] 6. Oxidized IL-33 activates the epidermal growth factor receptor (EGFR) To identify receptor tyrosine kinases (RTKs) activated by oxIL-33, a 71RTK array was used for screening. The RTK phosphorylation antibody array kit (ab193662) was purchased from Abcam, and experiments were performed according to the manufacturer's instructions. NHBEs were cultured at 1 × 10 cells / well in a 6-well plate (Corning Costar, 3516) in culture medium [BEGM (Lonza CC-3171) and supplement kit (Lonza CC-4175)]. 6 Cells were incubated at 37°C in 5% CO2 for 18-24 hours. After this time, the medium was aspirated, and the cells were washed twice with 1 mL PBS before adding starvation medium (BEGM (Lonza CC-3171) without supplementation kit). Plates were then incubated at 37 °C and 5% CO for an additional 18–24 h before stimulation. Cells were activated (activators in Table 2) and lysed, using 250 μg of total protein per array membrane, following the same procedure as described above for the MAP kinase array. All subsequent steps were performed according to the manufacturer's instructions. Membranes were visualized with LiCor C-digit and quantified using Image Lite Studio. No response was detected to either 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) elicited a positive signal on the RTK array corresponding to the epidermal growth factor receptor (EGFR) (Figure 2).

[0291] The ability of oxIL-33 (oxidized IL-33-01) to stimulate EGFR signaling was confirmed by further methods. After activation, EGFR is phosphorylated at Tyr1068, and this phospho-EGFR can be detected using a homogeneous FRET (Fluorescence Resonance Energy Transfer) HTRF® (Homogeneous Time-Resolved Fluorescence, Cisbio International) assay (Cisbio kit #64EG1PEH). Briefly, NHBEs were cultured at 5 × 10 in a 96-well plate (Corning Costar, 3598) in culture medium [BEGM (Lonza CC-3171) and supplement kit (Lonza CC-4175)]. 5 Plates were plated at 100 μL per well. Plates were incubated at 37°C and 5% CO2 for 18–24 hours. After this time, the medium was aspirated, and cells were washed twice with 0.2 mL PBS before adding starvation medium (BEGM (Lonza CC-3171) without supplementation kit). Plates were then incubated at 37°C and 5% CO2 for an additional 18–24 hours before being stimulated with increasing concentrations of IL-33-01, IL-33-16, and oxIL-33 (oxidized IL-33-01), and EGFR ligands (Tables 2 and 3), and then returned to the incubator at 37°C and 5% CO2 for 10 minutes. The medium was aspirated and replaced with 50 μL of lysis buffer (Cisbio, 64EG1PEH) per well. The assay was then performed according to the manufacturer's instructions (Cisbio, 64EG1PEH). An EnVision plate reader (Perkin Time-resolved fluorescence was read at 620 nm and 665 nm emission wavelengths using a Fluorescence Intensity Analyzer (Elmer). Data were analyzed by calculating the 665 / 620 nm ratio, and EC values ​​were determined by curve fitting with a four-parameter logistic equation using GraphPad Prism software.

[0292] [Table 7]

[0293] Similarly, EGFR phosphorylation was assessed in the epithelial cell line A549 using the HTRF assay, as previously described 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 with Accutase (PAA, #L1 1-007) and 5 × 10 5 Cells were seeded into 96-well plates at 100 μL per well and incubated at 37°C and 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 incubation at 37°C and 5% CO2 for 18–24 hours. Cells were stimulated with increasing concentrations of IL-33-01, IL-33-16, and oxIL-33-01 (another name for oxidized IL-33-01), EGFR ligands, and RAGE ligands (Tables 2 and 3), and then returned to the incubator for 10 minutes at 37°C and 5% CO2. The medium was aspirated and replaced with 50 μL of lysis buffer (Cisbio, 64EG1PEH) per well. The assay was then performed according to the manufacturer's instructions (Cisbio, 64EG1PEH). Time-resolved fluorescence was read at 620 nm and 665 nm emission wavelengths using an EnVision plate reader (Perkin Elmer). Data were analyzed by calculating the 665 / 620 nm ratio, and EC values ​​were determined by curve fitting with a four-parameter logistic equation using GraphPad Prism software.

[0294] In both NHBE and A549 cells, oxIL-33 promoted EGFR phosphorylation similar to the bona fide agonist EGF (Fig. 3), a finding that was not replicated by other RAGE ligands tested.

[0295] 7. Western Blot of Signaling Components Western blot experiments were performed to further investigate which components of the EGFR signaling complex are activated in response to oxIL-33 (oxIL33-01). NHBEs were cultured and plated in 6-well dishes as described in section 5 above. After serum starvation, cells were stimulated with oxIL-33 (30 ng / mL) for 5–240 min. The medium was then aspirated, and the cells were washed with ice-cold PBS before adding 150 μL of lysis buffer [1x LDS sample buffer (Thermo, NP0008), 10 mM MgCl2 (VWR, 7786-30-3), 2.5% β-mercaptoethanol (Sigma, M6250), and 0.4 μg / mL benzonase (Millipore, 70746)]. After placing the cells on ice for 10 min, the lysate was transferred to a 1.5 mL tube and heated to 90°C for 5 min. The solution was transferred to a new 1.5 mL tube, and 10 μL of sample and 5 μL of protein ladder (BioRad, 1610374) were loaded onto a 4-12% SDS-PAGE gel (Thermo, NW04127BOX) in MES running buffer (B0002). Using a Transblot Turbo (BioRad), the gel was transferred to a PVDF membrane (BioRad, 1704156). The PVDF membrane was blocked with PBS-Tween containing 5% skim milk powder (Marvel) for 10 minutes. The membrane was then incubated overnight at 4°C with the primary antibody in PBS-Tween containing 5% BSA. The membrane was then washed five times with PBS-Tween and incubated with the secondary HRP-tagged antibody in PBS-Tween containing 5% skim milk powder for 1 hour at room temperature. The membrane was then washed five times with PBS-tween, and ECL (BioRad, 1705062) was added to visualize Licor C-digit.

[0296] The results show that oxIL-33-01 activated several EGFR signaling components (Fig. 4).

[0297] 8.ox-IL-33 induces STAT-5 phosphorylation, which is blocked by EGFR-neutralizing Abs Next, we sought to determine whether we could inhibit oxIL33-mediated STAT5 activation by preventing its binding to EGFR. Briefly, A549 cells were cultured in RPMI GlutaMax medium supplemented with 1% penicillin / streptomycin and 10% FBS. Cells were harvested with Accutase and 5 × 10 cells were cultured. 5 The cells were seeded into a 96-well plate at 100 μL / well and incubated at 37°C, 5% CO for 18-24 hours. The wells were then washed twice with 100 μL of PBS, after which 100 μL of starvation medium (RPMI GlutaMax medium supplemented with 1% penicillin / streptomycin) was added and the wells were incubated at 37°C, 5% CO for 18-24 hours. Anti-EGFR antibody (clone LA1 (05-101, Millipore) or isotype control (MAB002, R&D Systems) was added to the wells in a dose-dependent manner, and the plate was returned to the incubator for 30 min. The plate was then stimulated with oxIL-33 (30 ng / mL) for 30 min, after which it was lysed using phosho-STAT5 ELISA kit lysis buffer (85-86112-11, ThermoFischer Scientific), developed according to the manufacturer's instructions, and absorbance was read at 450 nM. As shown in Figure 5, cells activated with oxIL-33-01 exhibited STAT5 phosphorylation, which was reduced in the presence of anti-EGFR antibody (Figure 5).

[0298] Example 2 - Oxidized IL-33 induces complex formation between EGFR and RAGE 9. oxIL-33 induces complex formation between EGFR and RAGE To understand how RAGE and EGFR are involved in promoting oxIL-33 signaling, we performed immunoprecipitation experiments to examine the signaling complex. First, anti-EGFR antibody was covalently coupled to Dynabeads. Two 100 μg vials of anti-EGFR antibody (R&D Systems, AF231) were incubated with 40 mg of Dynabeads (Thermo, 14311D) and covalently coupled according to the manufacturer's instructions. After successful coupling, the beads were resuspended in PBS at 30 mg / mL and kept at 4°C.

[0299] NHBE was obtained from Lonza (CC-2540), and frozen vials were placed in 15-cm dishes (Thermo, 157150) at 1 × 10 per dish. 6NHBEs were directly seeded with 1000kJ / well of cells. NHBEs were maintained in complete BEGM medium (Lonza) according to the manufacturer's protocol for 1 month, with medium changes every 3 days until cells reached confluence. During this time, plates were incubated at 37°C and 5% CO2. The day before stimulation, plates were washed twice with 20 mL of PBS, followed by the addition of 15 mL of starvation medium (BEGM (Lonza CC-3171) without supplement kit). Plates were then incubated at 37°C, 5% CO for an additional 18–24 hours before being stimulated with medium alone (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% CO for 10 minutes. The medium was aspirated, and plates were washed twice with ice-cold PBS. Then, 1 mL of lysis buffer (Abcam, ab152163) containing phosphatase and protease inhibitors (Thermo, 78440) was added per 15 cm dish. Cells were scraped into the lysis buffer, followed by the addition of 2 mL of Proteinase inhibitors. The extract was transferred to a LoBind tube (Eppendorf, Z666513) and clarified by spinning at 14,000 rpm at 4°C. Protein concentration was determined using a BCA kit (Thermo, 23225), and all protein extracts were normalized to 3 mg / mL in lysis buffer. Six mg of total protein extract was incubated with 100 μL of anti-EGFR Dynabeads (described above) in a clear 2 mL LoBind tube. The tube was then placed on an end-over-end mixer at 4°C for 5 hours. A magnet (BioRad, 1614916) was used to immobilize the Dynabeads, and the protein extract was aspirated and eluted with 2 mL of Wash Buffer 1 (50 mM Tris-HCl pH 7.5 (Thermo, 15567027), 0.5% Triton X). The solution was replaced with 100 ml (Sigma, X100) and 0.3 M NaCl. This was repeated four more times. The beads were then washed an additional 10 times with wash buffer 2 (50 mM Tris-HCl pH 7.5) in the same manner. After the final wash step, 50 μL of 1% Rapigest (w / v) (Waters, 186001861) in 50 mM Tris-HCl pH 8.0 was added to the beads and heated at 60°C for 10 minutes.The supernatant was then transferred to a new LoBind 2 mL tube. An additional 100 μL of 50 mM Tris-HCl pH 8.0 was added to the resin, mixed, and combined with the first eluate. TCEP (Sigma, 646547) was then added to a final concentration of 5 mM, and the sample was heated at 60°C for 10 minutes. The eluate was then alkylated by adding iodoacetamide (Sigma, 16125) to 10 mM for 20 minutes at room temperature in the dark. Alkylation was quenched by adding DTT (Sigma, D5545) to 10 mM. Tris-HCl buffer 50 mM pH 8.0 was then added to bring the final sample volume to 500 μL. 0.5 μg of trypsin (Promega, V5111) was added per tube, and the sample was digested overnight at 30°C on a shaking platform at 400 rpm. The samples were then acidified with trifluoroacetic acid (Sigma, 302031) to a final concentration of 2.0% (v / v) and incubated at 37°C for 1 hour. The samples were then centrifuged at 14,000 rpm for 30 minutes, and the supernatant was transferred to a new 2 mL LoBind tube. The samples were then processed on a C18 column (Thermo, 87784) according to the manufacturer's instructions. The samples were then dried using a speed-vac before being stored at -20°C. The samples were then analyzed by peptide mass fingerprinting-mass spectrometry (PMF-LC-MS). Results were analyzed using Scaffold software.

[0300] EGFR was similarly detected in all four conditions, suggesting that immunoprecipitation worked well in all samples. RAGE and IL-33 were detected in samples treated with oxIL-33, in contrast to samples treated with IL33-01 (IL-33) or EGF, suggesting that oxIL-33 and RAGE associated with EGFR during signaling. Consistent with previous observations of EGFR activation in these cells by oxIL-33 and EGF, proteins previously reported to be involved in EGFR signaling and endocytosis were detected after stimulation with these ligands but not with reduced IL33-01 (Table 4).

[0301] Table 4 shows LCMS analysis of NHBE stimulated with reduced IL-33-01 (IL-33), oxIL-33 (oxidized IL-33-01), or EGF. IL-33 and RAGE are detected in complex with EGFR after stimulation with oxIL-33, but not after stimulation with reduced IL-33-01 (IL-33) or EGF. Brackets indicate the number of unique peptides identified for each protein.

[0302] [Table 8]

[0303] To confirm these observations, immunoprecipitation and Western blot analysis were also performed on cell lysates prepared according to the above protocol. After determining the concentration of NHBE protein extracts, 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 hours. Next, 1.5 mg of Protein A / G magnetic beads (Thermo, 88802) were added to each tube, and the tubes were then returned to 4°C for an additional hour with mixing. The beads were then collected with a magnet (BioRad, 1614916) and washed three times with 500 μL of 50 mM Tris (pH 7.5), 1% TritonX, and 0.25 M NaCl, and once with 500 μL of 10 mM Tris (pH 7.5). Proteins were then released from the magnetic beads using 35 μL of LDS sample buffer (Thermo, NP0008) containing a reducing agent (Thermo, NP0004) and heated at 95°C for 5 minutes. The solution was transferred to a new 1.5 mL tube, and 10 μL of sample and 5 μL of protein ladder (BioRad, 1610374) were loaded onto a 4-12% SDS-PAGE gel (Thermo, NW04127BOX) in MES running buffer (B0002). The gel was transferred to a PVDF membrane (BioRad, 1704156) using a Transblot Turbo (BioRad). The PVDF membrane was blocked for 10 minutes with PBS-Tween solution containing 5% skim milk powder (Marvel). The membranes were then incubated with primary antibodies (anti-EGFR (Cell Signaling Technology, 2232), anti-RAGE (Cell Signaling Technology, 6996), or anti-IL-33 (R&D Systems, AF3625) in PBS-Tween containing 5% BSA overnight at 4°C. The membranes were then washed five times with PBS-Tween and then incubated with anti-rabbit secondary HRP-tagged antibody (Cell Signaling Technology, 7074) or anti-goat secondary HRP-tagged antibody (R&D Systems, HAF109) in PBS-Tween containing 5% skim milk powder for 1 hour at room temperature.The membrane was then washed five times with PBS-tween, followed by the addition of ECL (BioRad, 1705062) and visualization of Licor C-digits. Western blot analysis confirmed that RAGE co-precipitated with EGFR in the presence of oxIL-33, but was not detected upon EGF stimulation (Figure 6). These findings demonstrate that RAGE and EGFR are functional parts of the oxidized IL-33 signaling complex.

[0304] 10. RAGE is required for oxIL-33 to form a complex with EGFR The experiments described above demonstrate that oxIL-33 is a ligand for the EGF receptor (EGFR) complex, resulting in downstream signaling. The experiments in this section are designed to determine whether oxIL-33 is a direct-binding ligand of either RAGE or EGFR. To further understand the formation of the signaling complex and assess whether oxIL-33 interacts directly with EGFR, we examined the binding of oxIL-33 to RAGE, ST2-Fc, and EGFR using an ELISA format.

[0305] Proteins and Modifications: Proteins containing the Avitag sequence motif (GLNDIFEAQKIEWHE, SEQ ID NO: 46) were biotinylated using biotin ligase (BirA) enzyme (Avidty, Bulk BirA) according to the manufacturer's protocol. All modified proteins without Avitag used herein were biotinylated via free amines using EZ link sulfo-NHS-LC-biotin (Thermo / Pierce, 21335) according to the manufacturer's protocol. Table 5 lists the biotinylated proteins used.

[0306] [Table 9]

[0307] Streptavidin plates (Thermo Scientific, AB-1226) were coated with 100 μL / well of biotinylated antigen (10 μg / mL in PBS) for 1 hour at room temperature. The plates were washed three times with 200 μL of PBS-T (PBS + 1% (v / v) Tween-20) and blocked for 1 hour with 300 μL / well of blocking buffer (PBS containing 1% BSA (Sigma, A9576)). The plates were washed three times with PBS-T. RAGE-Fc (R&D Systems #1145-RG) or ST2-Fc (R&D Systems #523-ST) was diluted to 10 μg / mL in PBS in blocking buffer, added to the relevant wells, and incubated for 1 hour at room temperature. Alternatively, 100 μL of 10 μg / mL EGFR-Fc (R&D Systems #344-ER-050) in PBS was added for 1 hour in the presence or absence of 10 μg / mL untagged RAGE (Sino Biological, 11629-HCCH). The plate was washed three times with 200 μL of PBS-T. RAGE-Fc, ST2-Fc, and EGFR-Fc were then detected with 100 μL / well of anti-human IgG HRP (Sigma AO170, 5.1 mg / mL) diluted 1:10,000 in blocking buffer for 1 hour at room temperature. 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. The absorbance was read at 450 nm on a CytationGen5 or similar instrument. The results showed that oxIL-33 exhibited a clear interaction with RAGE (Figure 7A), whereas direct binding of oxIL-33 to EGFR was negligible (Figure 7B). EGFR binding to oxIL-33 was only observed upon addition of sRAGE to the assay (Figure 7B). This could not be reproduced when oxIL-33 was used in place of HMGB1, a bona fide RAGE agonist (Figure 7B).

[0308] The requirement for RAGE in oxIL-33-induced EGFR signaling was further confirmed using a RAGE-deficient cell line, A549, which was generated as follows.

[0309] 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 the Cas9 endonuclease was generated. A549 conditioned medium was generated by growing A549 cells in F12K nut mix (Gibco, supplemented with 10% FBS and 1% penicillin / streptomycin) for 2 days in a T-175 flask. Spent 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 placed in three T-75 flasks at 2 × 10 5The cells were seeded at 1500 cells / mL in a total volume of 15 mL and placed in a 37°C, 5% CO2 incubator overnight. The transfection mixture was prepared using 1.6 mL of F12K nut mix (supplemented with 1% penicillin / streptomycin) containing 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 left at room temperature for 15 minutes. 0.75 mL of the transfection mixture was then added to each T-75 flask. The flasks were returned to the incubator for 2 days. A549 cells were then detached using Accutase, transferred to PBS containing 1% FBS, and single cells were sorted into 96-well dishes using an Aria cell sorter (BD) based on RFP expression. Cells were fed with conditioned medium every 3–5 days. Once the cells reached >50% confluence, they were transferred to 24-well plates for expansion. This upscaling process continued until each successful clone was split into a T15 flask. Cells were then split into 12-well plates and grown to greater than 50% confluence before being analyzed by genomic PCR for knockout success. Cells were lysed in 100 μL of DNA lysis buffer (Viagen Biotech, 301-C, supplemented with 0.3 μg / mL proteinase K) per well. These samples were incubated at 55°C for 4 hours, followed by incubation at 85°C for 15 minutes. PCR for RAGE 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 cycle setup was as follows: 50 μL reaction volume (25 μL Q5 polymerase mix, 2.5 μL forward primer (10 μM stock), 2.5 μL reverse primer (10 μM stock), 2 μL template DNA lysate, 18 μL nuclease-free water). PCR reactions were performed with an initial denaturation at 98°C for 30 seconds, followed by 35 cycles of 98°C for 5 seconds, 57°C for 10 seconds, and 72°C for 20 seconds, followed by a final step at 72°C for 2 minutes.Four microliters 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 (1:10,000 SYBR Safe) at 90 V for 1 hour and then visualized on a Versadoc Imager. The remaining PCR product was then cleaned up using a QIAquick PCR Purification Kit (Qiagen, 28104) according to the manufacturer's protocol. DNA-50 concentration was measured using a nanodrop. Several clones (selected from the results) were sent for in-house sequencing. The results showed that the insertion of a stop codon into clones RAGE09 and RAGE10 was successful.

[0310] To confirm the essentiality of RAGE for oxIL-33-mediated EGFR signaling, we then performed immunoprecipitation and Western blot analysis in A549 cells and RAGE-deficient A549 cells. Briefly, the cell lines were activated with oxIL-33-01 for various time points (0–15 min). Subsequent immunoprecipitation of EGFR or RAGE was followed by Western blot analysis using anti-RAGE, anti-EGFR, and anti-IL-33 antibodies, according to the relevant experimental protocol detailed in Section 9. The results demonstrate the essential role of RAGE in the formation of a complex with oxIL-33 and EGFR (Figure 8).

[0311] 11. Oxidized IL-33 induces STAT5 phosphorylation, which is blocked by RAGE but not by ST2-neutralizing antibodies. To confirm the importance of RAGE over ST2 in oxIL-33 signaling, we tested blocking antibodies. Briefly, A549 cells were cultured in RPMI GlutaMax medium supplemented with 1% penicillin / streptomycin and 10% FBS. Cells were harvested with Accutase and 5 × 10 cells were cultured. 5The cells were seeded into a 96-well plate at 100 μL / well 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 incubation at 37°C, 5% CO2 for 18–24 hours. Anti-RAGE (M4F4; WO 2008137552), anti-ST2 (AF532; RnD Systems), or isotype control (MAB002, R&D Systems) were added to the wells in a dose-dependent manner, and the plate was returned to the incubator for 30 minutes. Plates were then stimulated with oxidized IL-33 (30 ng / mL) for 30 minutes, then lysed using phosho-STAT5 ELISA kit lysis buffer (85-86112-11, ThermoFischer Scientific), developed according to the manufacturer's instructions, and absorbance was read at 450 nM. As shown in Figure 9, cells activated with oxIL-33-01 exhibited phosphorylation of STAT5, which was reduced in the presence of anti-RAGE antibody but not in the presence of anti-ST2 antibody (Figure 9).

[0312] Example 3 - oxIL-33 induces EGFR internalization in epithelial cells We next investigated whether oxIL-33 induces changes in EGFR dynamics compared with EGF.

[0313] 12. Confocal Experiments of EGF Internalization This experiment aimed to investigate the dynamics of EGFR in epithelial cells after stimulation with EGF, reduced, or oxidized IL-33 using confocal imaging. The EGFR-GFP A549 epithelial cell line (Sigma, CLL1141-1VL) was plated at a concentration of 20,000 cells / mL (RPMI medium + 10% FCS + penicillin / streptomycin) in 1 mL of 24-well glass-bottom plates (Greiner, 662892). EGF receptor linked to green fluorescent protein (GFP) allows for tracking of EGFR membrane dynamics and internalization. Cells were washed once with PBS and incubated in RPMI medium (without FCS). After 24 hours of starvation, cells were washed with RPMI and incubated with 0.5 mL of RPMI medium containing CellMask (Invitrogen C10046) Deep Red at a 1:5,000 dilution. Cells were briefly stained with CellMask before treatment for membrane marking and then live-imaged at 1 frame / min on a confocal microscope immediately after treatment to record EGFR-GFP dynamics. Cells were stained for 5 min at 37°C, washed once with PBS, and stimulated with oxIL-33 (oxidized IL-33-01) or IL-33-16 at a concentration of 200 ng / mL in 0.5 mL of serum-free RPMI per well. Confocal images were taken immediately with a 40x oil objective, 1 min / frame, and 5 stacks spaced 2 μm apart for 25 min (approximately 30 min after protein addition). The disorganized (dotted) pattern of GFP signal indicates receptor clustering on the membrane and internalization. Pixel intensity histograms of membrane regions (masked with CellMask) and intracellular regions (masked with inverse CellMask, not shown) generated at different time points from live imaging showed EGFR depletion in non-clustered regions (a left shift of the bell-shaped peak in the histogram) and an increased number of saturated pixels (intensity 255), which is caused by clustering. Although oxIL-33 induced EGF receptor clustering and internalization, EGF stimulation resulted in the most obvious EGFR clustering. In contrast, reduced IL-33 (IL-33-16) did not significantly alter EGFR cellular distribution (Figure 10).

[0314] Example 4 - oxIL-33 induces IL-8 secretion by epithelial cells similar to EGF 13.Selective secretion of IL-8 by oxIL-33 Human bronchial epithelial cells from healthy subjects (NHBE; Lonza CC-2540) and human bronchial epithelial cells from chronic obstructive pulmonary disease (COPD) patients (DHBE; Lonza 00195275) were maintained in complete BEGM medium (Lonza) for 1 month according to the manufacturer's protocol, with medium changes every 3 days until cells reached confluence. Cells were harvested with Accutase and plated at 5 × 10 cells in 96-well plates (Corning 3596) in culture medium. 5 Plates were seeded at 100 μL / well. Plates were incubated at 37°C, 5% CO2 for 18–24 hours. After this time, the medium was aspirated, and cells were washed twice with 100 μL of PBS before adding starvation medium (BEGM (Lonza CC-3171) supplemented with 1% penicillin / streptomycin without supplementation kit). Plates were then incubated at 37°C, 5% CO2 for an additional 18–24 hours before stimulation with medium alone (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. After 24 hours of stimulation, supernatants were collected and analyzed in a multiplex assay (Mesoscale Discovery Chemokine production was assessed using a 20-kDa oxIL-33-activated cell line (K15047D-2). As shown in Figure 11, NHBE and DHBE showed a 4-fold increase in IL-8 secretion upon stimulation with oxIL-33 compared to unstimulated cells (medium alone). No significant differences were observed with other chemokines (TARC, MIP-1a, MIP-1b, MCP4, MCP1, IP10, eotaxin, eotaxin-3, MDC - data not shown).

[0315] Example 5 - oxIL-33 impairs the repair response of scratch wounds in deep monolayer epithelial cultures 14.oxIL-33, in contrast to EGF, impairs scratch wound 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 Accutase (PAA, #L1 1-007) and collected at 5 × 10 5 Cells were seeded into 96-well plates at 100 μL per well 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 incubation at 37°C, 5% CO2 for 18–24 hours. Cells were scratched using a WoundMaker™ (Essen Bioscience). The wells were then washed twice with 200 μL of PBS, followed by the addition of RPMI GlutaMax medium supplemented with 0.1% FBS (v / v) and 1% (v / v) penicillin / streptomycin containing the indicated stimuli: medium alone (unstimulated control), 30 ng / mL reduced IL-33-01, 30 ng / mL oxidized IL-33-01, or 30 ng / mL EGF. The plates were then returned to 37°C, 5% CO2. Plates were placed in IncucyteZoom for 48 hours for imaging and analysis of wound healing. Relative wound density was calculated via a wound healing algorithm within the IncucyteZoom software.

[0316] NHBE (CC-2540) were 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 Accutase and plated at 5 × 10 in 96-well ImageLock plates (Sartorius, 4379) in culture medium. 5Plates were incubated at 37°C and 5% CO2 for 18-24 hours. After this time, the medium was aspirated, and cells were washed twice with 100 μL of PBS before adding starvation medium (BEGM (Lonza CC-3171) supplemented with 1% penicillin / streptomycin without supplementation kit). Plates were then incubated at 37°C and 5% CO2 for an additional 18-24 hours before scratch wounding. Cells were scratched using a WoundMaker™ (Essen Bioscience), and wells were then washed twice with 200 μL of PBS before inoculation with the indicated stimuli: medium alone (unstimulated control), 30 ng / mL reduced IL-33-01, 30 ng / mL oxidized IL-33-01, or 30 ng / mL oxidized IL-33-01. BEBM medium (Lonza) containing EGF, supplemented with 0.1% FBS (v / v) and 1% (v / v) penicillin / streptomycin, was added and the cells were returned to 37°C and 5% CO2. The plates were placed in an IncucyteZoom for 48 hours for imaging and analysis of wound healing. Relative wound density was calculated via the wound healing algorithm within the IncucyteZoom software. As shown in Figure 12, oxIL-33 inhibited wound healing in submerged cultures of A549 cells (Figure 12A) and NHBE cells (Figure 12B), with the opposite effect to EGF, in which an increase in wound cell density was observed.

[0317] 15. Impairment of scratch wound closure by oxidized IL-33 can be prevented by antibodies neutralizing RAGE or EGFR, but not ST2. To understand whether these functional effects of oxIL-33 are mediated through RAGE / EGFR, we performed scratch assays with NHBE cells as described in Section 14, but in the presence of antibodies neutralizing different receptor components. NHBE cells were treated with medium alone (unstimulated control), reduced IL-33, or oxidized IL-33 in the presence of 10 μg / mL of 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, again indicating that RAGE and EGFR are essential receptors involved in the oxidized IL-33 signaling pathway (Figure 13).

[0318] Example 6 - Anti-IL-33 improves the phenotype of COPD cells in submerged culture 16.oxIL-33 can induce a COPD-like response in healthy NHBEs in a scratch wound closure assay Next, we examined the effects of oxidized IL-33 in healthy, smoker, and COPD bronchial epithelial cells. NHBE (CC-2540), NHBE from a smoker (CC-2540), 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 medium alone (unstimulated control) or 30 ng / mL oxidized IL-33. Bronchial epithelial cells from smokers or COPD exhibited impaired scratch closure compared with cells from healthy subjects, similar to the impairment observed after treatment of healthy cells with oxIL-33 (Figure 14). In contrast to healthy cells, oxIL-33 did not further impair the scratch wound closure response in smoker and COPD HBE cells (Figure 14).

[0319] 17. Blockade of endogenous IL-33 via the RAGE / EGFR pathway may improve the impaired scratch wound repair phenotype of COPD basal cells. Epithelial cells are known to produce IL-33, so autocrine IL-33 secretion may be responsible for the impaired scratch wound repair phenotype observed in COPD cells. To investigate this, we performed a scratch closure assay with COPD-derived bronchial epithelial cells in the presence of IL-33 neutralization. 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 Accutase and plated at 5 × 10 cells per well in a 96-well ImageLock plate (Sartorius, 4379) in culture medium. 5Plates were incubated at 37°C and 5% CO2 for 18-24 hours. After this time, the medium was aspirated, and cells were washed twice with 100 μL of PBS before adding starvation medium (BEGM (Lonza CC-3171) supplemented with 1% penicillin / streptomycin without supplement kit). Plates were then incubated at 37°C and 5% CO2 for an additional 18-24 hours before scratch wounding. Cells were scratched using a WoundMaker™ (Essen Bioscience). Wells were then washed twice with 200 μL of PBS before adding 10 μg / mL of anti-IL-33 (33_640087-7B, described in WO 2016 / 156440), anti-ST2 (AF532, R&D BEBM medium (Lonza) supplemented with 0.1% FBS (v / v) and 1% (v / v) penicillin / streptomycin containing IgG1 (Biotin Systems), anti-RAGE (M4F4, WO 2008137552), or NIP228 (IgG1 isotype control) was added and returned to 37°C, 5% CO2. The plates were placed in an IncucyteZoom for 48 hours for wound healing imaging and analysis. Relative wound density was calculated via a wound healing algorithm within the IncucyteZoom software. As previously observed, COPD cells exhibited impaired scratch closure responses compared with cells derived from healthy subjects. Anti-IL-33 and anti-RAGE, but not anti-ST2, were able to improve the scratch closure response of COPD cells to levels similar to those of healthy cells (Figure 15), indicating that epithelial cells produce autocrine IL-33 that signals through the RAGE / EGFR pathway (Figure 15).

[0320] Example 7 - Anti-IL-33 reduces goblet cells in 3D epithelial cultures 18. Air-liquid Interface (ALI) Culture of Airway Basal Cells Next, we sought to determine the relevance of IL-33 signaling in air-liquid interface cell culture ("ALI culture"). ALI culture is a method in which basal cells are grown with the basal surface in contact with medium and the upper (apical) cell layer exposed to air. ALI culture allows the development of a three-dimensional cellular structure in vitro with a mucociliary phenotype of pseudostratified epithelium, similar to tracheal epithelium. Therefore, ALI culture can be used to study fundamental aspects of the respiratory epithelium, such as cell-cell signaling, disease modeling, and respiratory regeneration.

[0321] Cryovials of 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 onto 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 (276-201, Propagenix, Rockville, MD). After reaching confluence, these cells were split once into the appropriate number of T-75 flasks and then harvested for ALI culture. Transwells containing 12 mm, 0.4 μM polyester membrane inserts (Costar, Corning, NY) for ALI culture were prepared by coating the inserts with 1:70 Purecol solution and incubating at 37°C for 1–16 h. The Purecol solution was removed, and the transwells were placed under UV light for 30 min and then washed with PBS. Basement cells from a T-75 flask were detached using 4 mL of trypsin solution (ThermoFisher, 15400054). The cell suspension was added to a 50 mL tube containing 5 mL of FBS, then counted using a ViCell counter (Beckman Coulter, Brea, CA) and spun down at 1,000 RPM for 5 minutes. The cells were then resuspended in Pneumacult ALI medium (Stemcell Tech, Vancouver, BC) at a concentration of 3.57 × 10 5The cells were resuspended at a density of 1 / mL and 700 μL was dispensed into each transwell. 1 mL of ALI medium was added to the space below the insert. Cells were left immersed in ALI medium until confluent and tight junctions formed (typically 7 days), at which point the medium was removed from the apical side and the cells were allowed to differentiate for 2 weeks, with medium changes on the basal side every other day. Fully differentiated cultures were treated for 7 days with no antibody, 1 μg / mL anti-IL-33 (33_640087-7B), or 1 μg / mL NIP228 (IgG1 isotype control) by using the treatment in the medium supplied to the basal side of the culture. Medium changes were performed every other day (including the relevant treatment).

[0322] 19. IHC Triple Staining (Basal, Caulic, and Ciliary) and Quantification ALI cultures from COPD donors were prepared 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 with a sequential 3-plex chromogenic assay using Ventana Discovery Ultra. Antigen retrieval was performed with Cell Conditioner 1 (CC1) (Cat. No. 5424569001, Roche), and endogenous peroxidase was blocked with Discovery Inhibitor (Cat. No. 7017944001, Roche) for 12 minutes. Anti-p63 (clone 4A4) (catalog no. 790-4509, Roche, Basel, Switzerland) was applied for 24 min at 36°C, visualized with mouse anti-HQ (12 min) (catalog no. 7017782001, Roche) and anti-HQ HRP (12 min) (catalog no. 7017936001, Roche), and incubated with Teal substrate (catalog no. 8254338001, Roche) for 12 min. Slides were treated with an antibody denaturing step (100°C for 24 min) using Cell Conditioner 2 (CC2) (Cat. No. 5424542001, Roche), followed by incubation with anti-tubulin (Cat. No. ab24610, Abcam, Cambridge, UK) diluted to 0.01 μg / mL in Dako antibody diluent (Cat. No. S3022) for 16 min, detected with mouse OmniMap-HRP (8 min) (Cat. No. 5269652001, Roche), and visualized with Discovery Purple substrate (Cat. No. 7053983001, Roche) for 16 min. Slides were subjected to additional antibody denaturation with CC2, followed by a cocktail of rabbit anti-mucin 5AC 1.1 μg / mL and rabbit anti-mucin 5B 7 μg / mL (catalog numbers ab198294 and ab87376, respectively, Abcam) for 20 minutes, followed by visualization with anti-rabbit NP (4 minutes) (catalog number 7425317001, Roche), anti-NP-AP (8 minutes) (catalog number 7425325001, Roche), and then Discovery Yellow (catalog number 7698445001, Roche) for 20 minutes.Stained slides were rinsed in Dawn detergent, counterstained with hematoxylin (catalog no. 5277965001, Roche), rinsed, dehydrated through a graded series of ethanol and xylene, and mounted with permanent mounting medium. Quantification using HALO software demonstrated a reduction in goblet cells in ALI cultures derived from healthy donors treated with anti-IL-33 (Figure 16).

[0323] Example 8 - Anti-IL-33 modulates mucins and improves mucus movement in 3D epithelial cultures from COPD 20. IHC double IF staining (mucin 5B + mucin 5AC) ALI cultures from COPD donors were prepared 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 in a sequential double immunofluorescence assay on a Ventana Discovery Ultra. Antigen retrieval was performed with Cell Conditioner 1 (Ultra CC1), endogenous peroxidase was blocked with Discovery Inhibitor for 12 minutes, followed by 8 minutes of blocking with S Block (RUO) Roche Diagnostics (Cat. No. 760-4212). Cells were incubated for 24 minutes at 36°C with anti-mucin 5B at 7 μg / mL diluted in Dako Ab Diluent, S3022. Detection was performed with anti-rabbit HQ (Roche Diagnostics Cat. No. 760-4815) for 4 minutes and anti-HQ-HRP (Roche Diagnostics Cat. No. 760-4820) for 8 minutes. Samples were then incubated for 8 minutes with Discovery FITC tyramide conjugate (Roche Diagnostics Cat. No. 760-232). Dual sequencing was selected in the Discovery Ultra program, and samples were treated with Cell Conditioner 2 (CC2) for an antibody denaturation step (100°C for 24 minutes), followed by neutralization with Discovery Inhibitor (40°C for 24 minutes). Anti-mucin 5AC (1.1 μg / mL) was then added for 20 minutes at 36°C. Mucin 5AC was detected with anti-rabbit HQ (Roche Diagnostics catalog no. 760-4815) for 4 minutes, anti-HQ-HRP (Roche Diagnostics catalog no. 760-4820) for 8 minutes, and visualized with tyramide conjugate Discovery Red 610 for 8 minutes. After completion of this step, the stained slides were removed from the Discovery Ultra Autostainer and rinsed with Dawn detergent followed by deionized water.Samples were incubated with 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI nucleic acid stain), Thermo Fisher catalog number D1306, diluted to 1 μg / mL in deionized water for 2 minutes. Samples were rinsed with deionized water, coverslipped with ProLong Gold Antifade mounting medium (Thermo Fisher, catalog number P36930), and stored in a light-tight slide box. Stained slides were imaged with a Zeiss LSM 880 confocal microscope (Carl Zeiss Microscopy, LLC, White Plains, NY). Figure 17 shows that anti-IL-33 treatment of ALI cultures can lead to downregulation of various mucins in COPD cultures.

[0324] 21. Anti-IL-33 reverses the impaired mucociliary clearance observed in COPD ALI cultures ALI cultures from COPD donors were generated and processed as described in section 18. Then, 30 μL of 0.2 μM FluoSpheres (ThermoFisher, F8811) diluted 1:33 in PBS was added to the apical surface, and a short video of FluoSphere movement was captured using a Zeiss LSM800 microscope, demonstrating increased mucus ciliary movement after treatment with anti-IL-33 (33_640087-7B), but not the control antibody.

[0325] 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 prepared and processed as described in Section 18. To obtain single-cell suspensions, filter inserts were incubated with 0.25% trypsin at 37°C for 5 minutes. Epithelial cells were gently detached from the filters by pipetting up and down with PBS and then transferred to a 15 mL Falcon tube. The cells were centrifuged at 1000 RPM for 5 minutes at 4°C. After removing the supernatant, the cells were resuspended in 0.4% BSA in PBS, and the cell concentration was adjusted to 1000 cells / µL for sequencing. The cell suspension was loaded according to the standard protocol of the Chromium Single Cell 3' Kit, capturing 5000–10,000 cells / channel. Version 2 chemistry was used. Single-cell 3' libraries for Illumina sequencing were obtained according to the manufacturer's protocol (Chromium™ Single Cell 3' Reagent Kit, v2 Chemistry). Library quality was assessed (TapeStation 4200, Agilent) and then sequenced on a NextSeq500 or NovaSeq6000 instrument (Illumina). Initial data processing was performed using the Cell Ranger version 2.0 pipeline (10x Genomics). Postprocessing was performed using the Seurat package to eliminate low-quality cells and normalize them. Each sample was analyzed as an independent dataset to capture intra-sample heterogeneity (cell subtyping). Clustering and visualization were achieved using t-distributed stochastic neighbor embedding (tSNE). Identification of COPD cell clusters was guided by marker genes. For other samples, initial clusters were manually inspected, and then Seurat's label transfer algorithm was applied to identify subtypes. MUC5AC and MUC5B gene expression analysis was performed for each cluster among cells from COPD ALI cultures with or without anti-IL-33 treatment, as described in Section 18. Heatmaps and tSNE plots were generated using Seurat. FIG. 18 shows a tSNE plot showing the different proportions of cell subtypes found in ALI cultures treated with anti-IL-33 (33_640087-7B) compared to untreated.As shown in Figure 18, a decrease in MUC5B-high cells was observed after anti-IL-33 treatment.

[0326] Example 10 - Anti-IL-33 reduces goblet cells in COPD 3D epithelial cultures 22. Air-liquid interface (ALI) culture of airway basal cells To quantify and investigate the effects of oxIL-33 in a physiologically relevant air-liquid interface (ALI) culture system, we deployed a flow cytometry assay aimed at distinguishing between goblet cell types (MUC5ac vs. MUC5b) and the remaining epithelial population (mucin-negative).

[0327] Cryovials of frozen lung basal cells from healthy controls (CC-2540) or COPD patients (195275) were received from Lonza. One vial per donor was thawed and plated into four T-175 flasks in Epix medium (276-201, Propagenix, Rockville, MD). After reaching confluence, these cells were frozen at 1e6 cells per vial in P2. Cells from P2 were placed in two T-75 flasks in Epix medium and grown to 80% confluence. Transwells for ALI culture containing 12 mm or 6.5 mm, 0.4 μM polyester membrane inserts (Costar, Corning, NY) were prepared by coating the inserts with 1x collagen I solution (Celladhere™ Collagen I-Stemcell #07001, prepared in dH2O) and incubating at 37°C for 1–16 h. The collagen I solution was removed, and the transwells were washed with PBS. The basement cells in the T-75 flask were washed with PBS and detached using 6 mL of trypsin solution (Lonza Trypsin Subculture Pack - #CC-5034). The trypsin was neutralized with 6 mL of trypsin neutralizing solution (Lonza Trypsin Subculture Pack - #CC-5034), the cell suspension was added to a 15 mL tube, counted, and the tube was spun down at 1,200 RPM for 5 minutes. The cells were then soaked in Pneumacult ALI medium (Stemcell Tech, Vancouver, BC) at 8 × 105 The cells were resuspended at a density of 1000 μg / mL and 0.5 mL was dispensed into each 12 mm transwell and 0.25 mL into each 6.5 mm transwell. 1 mL of ALI medium was added to the space below the 12 mL insert and to the 0.5 mL below the 6.5 mm insert. Cells were left immersed in ALI medium until confluent and tight junctions formed (typically 7 days), at which point the medium was removed from the apical side and the cells were differentiated for 3 weeks, with medium changes on the basal side every Monday, Wednesday, and Friday. Fully differentiated normal cultures were either left untreated or treated for 7 days with reduced or oxidized untagged IL33-01 (30 ng / mL), untagged IL33-16 (30 ng / mL), IL-13 (10 ng / mL), EGF (30 ng / mL), or HMGB1 (30 ng / mL) by using treatment agents in the medium supplied to the basal side of the culture (7-day treatment). Fully differentiated COPD cultures were either left untreated or treated for 7 days with no antibody, 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 by using the treatment in the medium supplied to the basal side of the culture. Medium changes (including the relevant treatments) were performed every Monday, Wednesday, and Friday.

[0328] [Table 10]

[0329] 23. FACS analysis of goblet cells in ALI cultures Following 7 days of treatment (Table 6), 4-week-old normal control or COPD ALI cultures in 6.5 mm inserts were analyzed by flow cytometry. 200 μL of 37°C PBS was added to the apical surface of each transwell (transwell surface) and placed in an incubator for 30 minutes. Apical washes were stored at -80°C for mucin analysis. 150 μL of trypsin (Lonza Trypsin Subculture Pack - #CC-5034) was added to both the apical and basolateral compartments (below the transwell). The transwells were returned to the incubator for 30 minutes. ALI were dissociated by gently pipetting the trypsin up and down. 150 μL of trypsin neutralizing solution (Lonza Trypsin Subculture Pack - #CC-5034) was added to each apical chamber and mixed. The cell suspension was transferred to a U-shaped 90-well plate, the cells were counted, and centrifuged at 1200 RPM for 5 minutes at 4°C. The trypsin / TNS was removed, and 200 μL of live / dead dye (eBioscience™ Fixable Viability Dye eFluor™ 780 Thermo 65-0865-14, diluted 1:2000 in PBS) was added to each well. The cells were resuspended and incubated on ice in the dark for 10 minutes. The plate was centrifuged at 1200 RPM for 5 minutes at 4°C, the live / dead dye was removed, and 200 μL of PBS was added to each well. The plate was centrifuged at 1200 RPM for 5 minutes at 4°C, the PBS was removed, and replaced with 200 μL of Fixation / Permeabilization Solution (Thermo 00-5123 and 00-5223). The plate was incubated on ice in the dark for 40 minutes. The plate was centrifuged at 1200 RPM for 5 minutes at 4°C, and the solution was removed. The cells were resuspended in 300 μL of 1x permeabilization solution (Thermo 00-8333). 5e4 cells from each well were added to a new 96-well U-bottom plate, centrifuged at 1200 RPM for 5 minutes at 4°C, and the cells were resuspended in 50 μL of 1x permeabilization solution. 50 μL of antibody staining cocktail (anti-Muc5AC at 1:400 and anti-Muc5B at 1:800) or isotype staining cocktail at the same dilution was added. The plate was incubated on ice in the dark for 30 minutes. The plate was centrifuged at 1200 RPM for 5 minutes at 4°C, and the solution was removed.Cells were washed with PBS, centrifuged, and then resuspended in 150 μL of PBS. Data were then acquired on a BD FACSymphony™ and analyzed using FlowJo software.

[0330] [Table 11]

[0331] 24. qPCR / Bulk RNA-seq Analysis of ALI Cultures Following 7 days of treatment (Table 6), 4-week-old normal control or COPD ALI cultures in 6.5 mm inserts were lysed for RNA analysis. First, 200 μL of 37°C PBS was added to the apical surface of each ALI, and the plate was returned to the incubator for 30 minutes. For mucin analysis, the apical washes were stored at -80°C. ALI cultures were lysed and RNA was extracted using a MagMAX™-96 Total RNA Isolation Kit (Thermo, AM1830). The RNA was then used to synthesize cDNA using a High-Capacity RNA-to-cDNA™ Kit (Thermo, 4388950). To this end, 9 μL of each RNA sample was incubated with 10 μL of 2X RT buffer mix and 1 μL of 20X RT enzyme mix in a PCR tube (Thermo, AM12230), placed in a thermocycler, and incubated at 37°C for 60 minutes. The reaction was terminated by heating to 95°C for 5 minutes and holding at 4°C. 60 μL of nuclease-free water (Thermo, 750024) was added to each tube containing 20 μL of cDNA. For RT-qPCR, 4 μL of cDNA was added to a barcoded MicroAmp™ EnduraPlate™ optical 384-well clear reaction plate (Thermo, 4483273) along with 5 μL of TaqMan Fast Advanced Master Mix (Thermo, 4444557), 0.5 μL of Muc5AC FAM probe (Thermo, Hs01365616_m1), and 0.5 μL of GAPDH VIC probe (Thermo, Hs02786624_g1). The plate was sealed, briefly centrifuged, and then analyzed using a QuantStudio™ 7Flex Real-Time PCR System (Thermo). Delta-delta-ct was then calculated by normalizing the data to untreated normal controls.

[0332] oxIL-33, but not reduced IL-33, led to an increase in goblet cell numbers, particularly the MUC5AC+ goblet cell subset (Figures 19A-C). Accordingly, MUC5AC mRNA copies increased upon treatment with oxIL-33, as determined by qPCR (Figure 19D).

[0333] 25. IHC Triple Staining (Basal, Caulic, and Ciliary) and Quantification Next, quantitative image analysis from ALI immunohistochemistry was evaluated. ALI cultures from COPD donors were prepared 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 with Ventana Discovery Ultra in a sequential triple chromogenic assay. Antigen retrieval was performed with Cell Conditioner 1 (Ultra CC1) (Cat. No. 5424569001, Roche), and endogenous peroxidase was blocked with Discovery Inhibitor (Cat. No. 7017944001, Roche) for 12 minutes. Anti-p63 (clone 4A4) (catalog no. 790-4509, Roche, Basel, Switzerland) was applied for 24 min, visualized with anti-mouse HQ (12 min) (catalog no. 7017782001, Roche) and anti-HQ HRP (12 min) (catalog no. 7017936001, Roche), and incubated with Discovery Purple kit (catalog no. 07053983001, Roche) for 12 min. Slides were treated with an antibody denaturing step (92°C for 24 min) using Cell Conditioner 2 (Ultra CC2) (Cat. No. 5424542001, Roche), followed by denaturing with anti-tubulin (Cat. No. ab24610, Abcam, Cambridge, UK) diluted in Dako antibody diluent (Cat. No. S3022) for 16 min (on-slide concentration 0.003 μg / mL), detected with mouse OmniMap-HRP (8 min) (Cat. No. 5269652001, Roche), and visualized with the Discovery Teal HRP kit (Cat. No. 82544338001, Roche).Slides were subjected to additional antibody denaturation with CC2, followed by a cocktail of rabbit anti-mucin 5AC at 1.1 μg / mL (dispenser concentration) and rabbit anti-mucin 5B at 7 μg / mL (dispenser concentration) (catalog numbers ab198294 and ab87376, respectively, Abcam) for 20 minutes, followed by visualization with anti-rabbit NP (4 minutes) (catalog number 7425317001, Roche), anti-NP-AP (8 minutes) (catalog number 7425325001, Roche), and then Discovery Yellow (catalog number 7698445001, Roche) for 20 minutes. Stained slides were counterstained with hematoxylin II (8 min) (catalog no. 5277965001, Roche) and Bluing reagent (4 min) (catalog no. 5266769001, Roche), rinsed with dish soap, dehydrated through a graded series of ethanol and xylene, and mounted with permanent mounting medium.

[0334] IHC images were analyzed using HALO v3.1 (Indica Labs) and first manually annotated to exclude out-of-focus and tissue-damaged areas. A random forest classifier was trained to recognize epithelium and separate it from transmembrane and slide background. For cilia area quantification, another random forest classifier was trained for coarse detection of tubulin staining, followed by fine detection using the Area Quantification v2.1.7 algorithm. For mucin area quantification, Area Quantification v2.1.7 was used directly to detect staining. For basal (p63+) cell counting, the algorithm CytoNuclear 2.0.9 was used to segment cells based on nuclear staining, and basal cells were further detected by counting p63-positive nuclei. All quantification methods were validated against human recognition and had an accuracy of over 90%.

[0335] Consistent with previous findings, oxIL-33 significantly affected the number of goblet cells (MUC5ac+b) (Figures 20A and 20B).

[0336] Together, these studies demonstrated a role for oxIL-33 in promoting goblet cell differentiation within the lung epithelium, suggesting that epithelium chronically exposed to ox-IL33 evolves toward a goblet cell hyperplastic phenotype that adversely affects lung function.

[0337] 26. Reversal of COPD goblet cell phenotype by blocking agents A key feature of COPD is excess mucus due to increased goblet cells and mucus secretion (Gohy et al., 2019 Sci Rep 9:17963). To investigate whether oxidized IL-33 could play a direct role in the goblet cell COPD phenotype, ALI cultures from COPD donors were established with readout as described in sections 22–25.

[0338] COPD ALI cells were 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-D). None of the treatments affected the viability of ALI cultures (Figure 21E), confirming that the treatment phenomenon was not an artifact or the result of antibody toxicity. Consistent with previous results, anti-ST2 treatment did not result in a reduction in goblet cell numbers, providing further evidence that this is a disease phenotype directly mediated by IL-33, primarily ox-IL-33, via the ox-IL-33-RAGE-EGFR pathway. The effect of anti-IL-33 antibody (33-640087_7B) on COPD ALI cultures was further confirmed by immunohistochemical analysis, where IL-33 blockade resulted in a reduction in goblet cell numbers in pairwise analyses (Figures 22A and 22B). After treatment with anti-IL-33 antibody (33-640087_7B), the epithelium of COPD ALI cultures resembled healthy epithelium, as shown in Figure 20A.

[0339] Finally, MUC5AC and MUC5B released into apical mucus from both healthy and COPD ALI cultures were measured using ELISA. To quantify mucin released from ALI cultures, apical supernatants were analyzed for MUC5AC levels by immunoassay (Novus NBP2-76703) according to the manufacturer's protocol. Samples were diluted 1:2000 in sample diluent, and concentrations were estimated from a recombinant MUC5AC protein standard curve. As shown in Figure 23, ALI cultures from COPD patients shed increased levels of MUC5AC compared to ALI from healthy donors (Figure 23A). Treatment with exogenous oxIL-33 resulted in increased mucin secretion from healthy ALI cultures (Figure 23B). In COPD ALI donors, elevated mucin levels were reduced by blockade with anti-IL-33 (33_640087_7B), which inhibits MUC5AC protein levels released from ALI cultures, but not by anti-ST2 or isotype mAb control (Figure 23C).

[0340] Overall, this example highlights the role of oxidized IL-33 in the dysregulation of lung epithelial cell differentiation. The results imply that, if uncontrolled, oxidized IL-33 may contribute to the goblet cell hyperplasia and excessive mucus production seen in some phenotypes of COPD. Therefore, treatment with oxIL-33 signaling axis antagonists, such as anti-IL-33, anti-RAGE, or anti-EGFR binding molecules, may have a significant therapeutic effect on COPD patients by restoring normal epithelial physiology, for example, by reducing goblet cell numbers and excessive mucus production.

[0341] Additional arrays In addition to the sequences listed in Table 1, the following additional CDR sequences are provided: 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: [ka] IL-33-16 SEQ ID NO:45: [ka] Avitag sequence motif SEQ ID NO: 46: GLNDIFEAQKIEWHE gRNA vector targeting RAGE exon 3 SEQ ID NO: 47: TGAGGGGATTTTCCGGTGC RAGE forward primer SEQ ID NO: 48: gttgcagcctcccaacttc RAGE reverse primer SEQ ID NO: 49: aatgaggccagtggaagtca Human ST2S (signal peptide is underlined) SEQ ID NO:50: [ka] Human ST2S-huIgG1 Fc-His6 (signal peptide is underlined) SEQ ID NO:51: [ka] SEQ ID NO:52: His10 / Avitag human ASGPR ECD (signal peptide underlined, tag double underlined) [ka]

Claims

[Claim 1] The invention described in the present specification and drawings.

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