How to use the IL-33 Antagonist

Abstract

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.

Description

[Technical Field]

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

[0002] This disclosure relates to IL-33 antagonists for use in the prevention or treatment of abnormal epithelial physiological function or EGFR-mediated diseases, and to corresponding preventive or therapeutic methods, including administering the IL-33 antagonist to patients in need. [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: reduced (redIL-33) and oxidized (oxIL-33). Previous studies have shown that the reduced form is rapidly oxidized under physiological conditions, forming at least one disulfide bond in the oxidized form, and that the two forms may have different binding patterns and effects.

[0004] Reduced IL-33 binds to ST2 and has been previously discovered to be the only known ligand for the ST2 receptor expressed by Th2 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, which promote inflammation. Soluble ST2 (sST2) is thought to be a decoy receptor that interferes with reduced IL-33 signaling.

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

[0006] There is considerable interest in IL-33 as a therapeutic target, primarily due to its ability to stimulate ST2 and produce potent inflammatory effects, particularly in its reduced form. However, there is little research or interest in the oxidized IL-33 pathway as a therapeutic target. This is partly due to the fact, as later discoveries have shown, that RAGE has many ligands and its downstream interactions are not well understood.

[0007] At least one of these downstream RAGE interactions arising 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's involvement is significant because EGFR is a key therapeutic target for many diseases, including aspects of abnormal epithelial physiological function.

[0008] As a result of this discovery, it is believed that antagonists capable of binding to any form of IL-33 can effectively inhibit the signaling of oxidized IL-33. This can be done 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 offers therapeutic benefits in any EGFR-mediated disease, particularly in conditions where EGFR is excessively stimulated.

[0009] EGFR is known to exert various homeostatic effects on epithelial physiological function. Stimulation of EGFR increases epithelial cell differentiation, epithelial cell migration, and epithelial mucosa production. Inhibition of EGFR-mediated signaling is thought to treat or prevent disorders involving abnormal epithelial physiological function, such as abnormal remodeling of airway epithelial tissue or excessive mucus production.

[0010] IL-33 has long been associated with airway tissue remodeling (Non-Patent Literature 1; Non-Patent Literature 2; Non-Patent Literature 3). However, this is thought to occur indirectly via a self-persistent amplification loop, thereby upregulating the expression of both IL-33 and its homologous 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 lymphoid cells, it has not been established or suggested that IL-33 itself directly influences airway epithelial biological function.

[0011] As stated above, this disclosure is based on the discovery that IL-33 also acts directly through a different mechanism; via the RAGE-EGFR pathway; directly affecting epithelial physiological function. This new understanding is important because it will broaden the therapeutic applications of IL-33 antagonists so that they can be used to treat more diseases, more disease symptoms, and more patients. The therapeutic opportunity to directly control and inhibit IL-33-mediated EGFR-mediated signaling by targeting IL-33 has not been realized until now.

[0012] The disclosures of this application are for the first time to show that the use of IL-33 antagonists, by directly inhibiting RAGE / EGFR-mediated oxIL-33 activity, can directly affect impaired epithelial repair responses in patients with abnormal epithelial physiological function, such as those with COPD or bronchitis, reducing the differentiation and proliferation of epithelial goblet cells, decreasing mucus production, and improving mucociliary motility. Accordingly, the studies presented herein support the therapeutic use of IL-33 antagonists in the direct prevention or treatment of abnormal epithelial physiological function, which is typically due to EGFR-mediated effects and is therefore 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 [Overview of the Initiative] [Means for solving the problem]

[0014] According to the first embodiment, an IL-33 antagonist is provided for use in the prevention or treatment of abnormal epithelial physiological function by modulating or inhibiting the RAGE-EGFR-mediated effect.

[0015] An alternative first embodiment provides a method for preventing or treating abnormal epithelial physiological function in a patient, comprising: administering an effective amount of an IL-33 antagonist to a patient in need to modulate or inhibit the RAGE-EGFR-mediated effect.

[0016] According to an alternative first embodiment, the use of an IL-33 antagonist in the manufacture of a drug for the prevention or treatment of abnormal epithelial physiological function is provided.

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

[0018] An alternative second embodiment provides a method for preventing or treating an EGFR-mediated disease in a patient, comprising administering an effective amount of an IL-33 antagonist to a patient in need thereof.

[0019] According to an alternative second embodiment, the use of an IL-33 antagonist in the manufacture of a drug for the prevention or treatment of EGFR-mediated diseases is provided.

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

[0021] An alternative third embodiment provides a method for preventing or treating a respiratory disease by improving the epithelial physiological function of a patient, comprising administering an effective amount of an IL-33 antagonist to a patient in need thereof.

[0022] According to an alternative third embodiment, the use of an IL-33 antagonist in the manufacture of a drug for the prevention or treatment of respiratory diseases by improving epithelial physiological function is provided.

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

[0024] An alternative fourth embodiment provides a method for preventing or treating a respiratory disease by inhibiting the EGFR-mediated effect in a patient, comprising administering an effective amount of an IL-33 antagonist to a patient in need thereof.

[0025] According to an alternative fourth embodiment, the use of an IL-33 antagonist in the manufacture of a drug for the prevention or treatment of respiratory disease by inhibiting EGFR-mediated effects is provided.

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

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

[0028] In an alternative further embodiment, the use of an IL-33 antagonist in the manufacture of a drug for the prevention or treatment of disease by inhibiting IL-33-mediated EGFR signaling is provided.

[0029] Further features and embodiments of the embodiments defined above are described in the following sections. Each section can be combined with any of the embodiments above in any compatible combination. [Modes for carrying out the invention]

[0030] definition As used herein, the "IL-33" protein refers to interleukin-33, particularly the mammalian interleukin-33 protein, e.g., the human protein deposited under UniProt number 095760. However, it is clear that this entity is not a single species, but rather exists in both reduced and oxidized forms. Given that the reduced form is rapidly oxidized in vivo (e.g., within 5 to 40 minutes) and in vitro, references to IL-33 in the prior art 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 other embodiments, IL-33 is mature truncated IL-33 (amino acids 112 to 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 IL-33 fragment 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 the form of IL-33 that binds to RAGE and induces RAGE-EGFR-mediated signaling. Oxidized IL-33 refers to a protein that appears as a characteristic band, for example, by Western blotting under non-reducing conditions, and in particular has a mass 4 Da less than the corresponding reduced form. More specifically, it refers to a protein having one or two disulfide bonds between cysteines independently selected from cysteine ​​208, 227, 232, and 259. In one embodiment, oxidized IL-33 does not show 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 induces ST2-mediated signaling. In particular, the reduced cysteine ​​208, 227, 232, and 259 are not disulfide-bonded. In one embodiment, reduced IL-33 does not bind to RAGE. References to "WT IL-33" or "IL-33" should be understood to refer to either the reduced or oxidized form, or both, unless it is clear from the context in which they are used that one of the forms is intended.

[0033] As used herein, “antigenically distinct types of IL-33” refers to any type of IL-33 that acts as an antigen and can be conjugated by an antibody or its binding fragment, and typically in the context of this disclosure, this means oxidized IL-33, reduced IL-33, and reduced IL-33 / sST2 complexes.

[0034] As used herein, "ST2-mediated signaling / effect" refers to the IL-33 / ST2 system in which ST2-mediated recognition of reduced IL-33 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. Therefore, ST2-dependent signaling / effect can be disrupted and attenuated by disrupting the interaction between IL-33 and ST2, or by interfering with the interaction with IL-1RAcP.

[0035] As used herein, "RAGE-EGFR-mediated signaling / effect" refers to the oxidized IL-33 / RAGE-EGFR system, in which RAGE recognizes oxidized IL-33 and promotes its complexation with EGFR within the cell membrane. Therefore, RAGE-EGFR-mediated signaling / effect can be disrupted and 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] When used herein, "to reduce the activity of ~" means to decrease or inhibit the relevant activity, or to suspend the relevant activity. Generally, "reduction" and "inhibition" are used interchangeably herein.

[0037] It should be noted that the term “a” or “an” refers to one or more entities; for example, “an anti-IL-33 antibody” is understood to represent one or more anti-IL-33 antibodies. Thus, the terms “a” (or “an”), “one or more” and “at least one” may be used interchangeably in this specification.

[0038] As used herein, the terms “to treat” or “treatment” refer to both therapeutic measures and prophylactic or preventative measures, the aim of which is to prevent or slow (mitigate) undesirable physiological changes or impairments. Favorable or desired clinical outcomes include, but are not limited to, symptom reduction, reduction of disease severity, stabilization (i.e., non-exacerbating) of the disease, delay or slowing of disease progression, recovery or mitigation of the condition, and remission (partial or complete), detectable or undetectable. “Treatment” may also mean extending survival compared to the expected survival without treatment. Those in need of treatment include those who already have a condition or impairment, as well as those who are susceptible to or should be prevented from developing a condition or impairment.

[0039] "Subject," "individual," "animal," "patient," or "mammal" means any subject for which diagnosis, prognosis, or treatment is desirable, especially a mammalian subject, unless 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, cattle, cows, etc.

[0040] IL-33 Antagonist This disclosure relates to the medical use of IL-33 antagonists, particularly for the medical use of preventing or treating diseases by inhibiting IL-33-mediated EGFR signaling. In specific cases, this disclosure relates to the use of IL-33 antagonists for the prevention or treatment of abnormal epithelial physiological functions that may be found in EGFR-mediated diseases.

[0041] As used herein, "IL-33 antagonist" refers to any agent that attenuates IL-33 activity, such as reduced IL-33 activity, oxidized IL-33 activity, or both. Preferably, the IL-33 antagonist is specific to reduced and / or oxidized IL-33. Preferably, the attenuation is due to binding of IL-33 in its reduced or oxidized form. Preferably, when the antagonist attenuates reduced IL-33 activity and oxidized IL-33 activity, the attenuation is due to binding of 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] In its broadest sense, the term "binding molecule" or "antigen-binding molecule" in this disclosure refers 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] Preferably, the binding molecule may be selected from: an antibody, its antigen-binding fragment, an aptamer, at least one heavy or light chain CDR from a reference antibody molecule, and at least six CDRs derived from one or more reference antibody molecules.

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

[0046] As used herein, "antibody" refers to an immunoglobulin molecule, more specifically a full-length antibody or a molecule containing a full-length antibody, as will be discussed in more detail below, such as the DVD-Ig molecule.

[0047] "The binding fragment" refers to an epitope / antigen-binding fragment of an antibody fragment that is interchangeable with "the antigen-binding fragment" and contains six CDRs, including, for example, the binding region, particularly three CDRs in the heavy chain variable region and three CDRs in the light chain variable region.

[0048] Preferably, the antibody or its conjugated fragment is selected from: naturally occurring, polyclonal, monoclonal, multispecific, mouse, human, humanized, primated, or chimeric. Preferably, the antibody or its conjugated fragment may be an epitope-binding fragment, e.g., a fragment containing Fab' and any of F(ab')2, Fd, Fvs, single-chain Fvs(scFv), disulfide-bonded Fvs(sdFv), VL domain, or VH domain, or a fragment produced by a Fab expression library. Preferably, the antibody or its conjugated fragment may be a minibody, diabody, triabody, tetrabody, or single-chain antibody. Preferably, the antibody or its conjugated fragment is a monoclonal antibody. The ScFv molecule is known in the art and is described, for example, in U.S. Patent No. 5,892,019.

[0049] The immunoglobulin or antibody molecules of this disclosure may be immunoglobulin molecules of any kind (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), of any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, etc.), or of any subclass.

[0050] Preferably, the IL-33 antagonist inhibits the activity of oxidized IL-33 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 reducing IL-33 antagonist. In other words, the IL-33 antagonist weakens the activity of reducing IL-33. Preferably, the weakening occurs by binding to reducing IL-33. Preferably, by binding to reducing IL-33, the antagonist also inhibits / weakens the activity of oxidized IL-33 by preventing its conversion to the oxidized IL-33 form.

[0052] Preferably, inhibition of 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 leads to downregulation or inhibition of RAGE-EGFR complexation. Preferably, inhibition leads to downregulation or inhibition of EGFR activation, preferably RAGE-mediated EGFR activation.

[0054] Preferably, an IL-33 antagonist has all of the above inhibitory effects. Preferably, a 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 its fragments are 5 × 10 -2 M, 10-2 M, 5×10 -3 M, 10 -3 M, 5×10 -4 M, 10 -4 M, 5×10 -5 M, 10 -5 M, 5×10 -6 M, 10 -6 M, 5×10 -7 M, 10 -7 M, 5×10 -8 M, 10 -8 M, 5×10 -9 M, 10 -9 M, 5×10 -10 M, 10 -10 M, 5×10 -11 M, 10 -11 M, 5×10 -12 M, 10 -12 M, 5×10 -13 M, 10 -13 M, 5×10 -14 M, 10 -14 M, 5×10 -15 M, or 10 -15 Bind specifically to redIL-33 with a binding affinity (Kd) less than M. Preferably, the binding affinity with redIL-33 is 5×10 -14The binding affinity is less than M (i.e., 0.05 pM). Preferably, the binding affinity is measured using Kinetic Exclusion Assays (KinExA) or BIACORE®, preferably using KinExA, as described in the protocol in International Publication No. 2016 / 156440 (which is incorporated herein by reference in its entirety) (see, for example, Example 11). A binding molecule that binds to redIL-33 with this binding affinity appears to bind sufficiently tightly to redIL-33 to prevent dissociation of the binding molecule / redIL-33 complex within a biologically relevant timescale. While not bound by theory, this binding strength is thought to prevent the release of the antigen before degradation of the antibody / antigen complex in vivo, resulting in the redIL-33 not being released and thus unable to be converted to oxIL-33. Therefore, when binding to redIL-33 with this binding affinity, the binding molecule can inhibit or attenuate oxIL-33 activity 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 5×10 4 M -1 seconds -1 It can specifically bind to redIL-33 at an on-rate (k(on)) of 10 5 M -1 seconds -1 , 5×10 5 M -1 seconds -1 , 10 6 M -1 seconds -1 , or 5×10 6 M -1 seconds -1 Or 10 7 M -1 seconds-1 It can bind to redIL-33 or its fragments or variants at the above on-rate (k(on)). Preferably, the k(on) rate is 10 7 M -1 seconds -1 That's all.

[0058] Preferably, the binding molecule or fragment thereof is 5X10 -1 seconds -1 , 10 -1 seconds -1 , 5X10 -2 seconds -1 , 10 -2 seconds -1 , 5X10 -3 seconds -1 , or 10 -3 seconds -1 The following off-rates (k(off)) can specifically bind to redIL-33. For example, the binding molecule of this disclosure can bind to 5 × 10⁻¹⁶. -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 that redIL-33 or its fragments or variants bind at the following off-rates (k(off)). Preferably, the k(off) rate is 10 -3 seconds -1The following applies: IL-33 is an alarmin cytokine that is rapidly and rapidly released at high concentrations in response to inflammatory stimuli. Red IL-33 is converted to an oxidized state approximately 5–45 minutes after being released into the extracellular environment. Therefore, in order to prevent the conversion of red IL-33 to ox IL-33, the binding molecules described herein can bind to red IL-33 at these k(on) and / or k(off) rates. While not theoretically bound, these k(on) / k(off) rates are thought to ensure that the binding molecules can rapidly bind to red IL-33 before being converted to ox IL-33, thereby reducing ox IL-33 formation, which in turn attenuates RAGE signaling, preferably RAGE / EGFR signaling, and thereby attenuates the RAGE / EGFR-mediated effect.

[0059] Preferably, the IL-33 binding molecule can competitively inhibit the binding of IL-33 to the binding molecule 33_640087-7B (as described in International Publication No. 2016 / 156440). Preferably, International Publication No. 2016 / 156440 describes that 33_640087-7B binds to redIL-33 with particularly high affinity, attenuating both ST-2 and RAGE-dependent IL-33 signaling. Therefore, a binding molecule that competitively inhibits the binding of IL-33 to the binding molecule 33_640087-7B is likely to inhibit both redIL-33 and oxIL-33 signaling and is 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 degree. Competitive inhibition can be determined by any method known in the art, such as solid-phase assays including competitive ELISA assays, dissociation-promoting lantanide fluorescence immunoassays (DELFIA®, Perkin Elmer), and radioligand binding assays. For example, a person 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 induction of the HTRF assay described in Example 1 of International Publication No. 2016 / 156440. For example, a person skilled in the art can label the recombinant antibodies in Table 1 with donor fluorophores and mix multiple concentrations with a fixed-concentration sample of redIL-33 labeled with receptor fluorophores. The binding characteristics can then be confirmed by measuring the fluorescence resonance energy transfer between the donor and receptor fluorophores in each sample. To elucidate competitively binding molecules, those skilled in the art can first mix test binding molecules at various concentrations with labeled antibodies at fixed concentrations as shown in Table 1. A decrease in the FRET signal when the mixture is incubated with labeled IL-33, compared to a positive control with labeled antibody alone, indicates competitive binding to IL-33. The binding molecule or fragment thereof can be said to competitively inhibit the 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 conjugating molecule is one of the following anti-IL-33 antibodies: 33_640087-7B (as described in International Publication No. 2016 / 156440), ANB020 known as etokimab (as described in International Publication No. 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 International Publication No. 2018 / 081075), IL33-158 (as described in U.S. Patent Application Publication No. 2018 / 0037644), 10C12.38.H6.87Y.581 Either IgG4 (as described in International Publication No. 2016 / 077381) or its conjugated fragments are selected, and each of the relevant literatures 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 containing a complementarity-determining region (CDR) of a pair of variable heavy chain domains (VH) and variable light chain domains (VL) selected from Table 1. Pair 1 corresponds to the VH and VL domain sequences of 33_640087-7B described in International Publication 2016 / 156440. Pairs 2-7 correspond to the VH and VL domain sequences of antibodies described in U.S. Patent Application Publication 2014 / 0271658. Pairs 8-12 correspond to the VH and VL domain sequences of antibodies described in U.S. Patent Application Publication 2017 / 0283494. Pair 13 corresponds to the VH and VL domain sequences of ANB020 described in International Publication 2015 / 106080. Pairs 14-16 correspond to the VH and VL domain sequences of antibodies described in International Publication Brochure 2018 / 081075. Pair 17 corresponds to the VH and VL domain sequences of IL33-158 described in U.S. Patent Application Publication 2018 / 0037644. Pair 18 corresponds to the VH and VL domain sequences of 10C12.38.H6.87Y.581 lgG4 described in International Publication Brochure 2016 / 077381.

[0063] [Table 1]

[0064] [Table 2]

[0065] [Table 3]

[0066] [Table 4]

[0067] [Table 5]

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

[0069] Preferably, the IL-33 antagonist is an antibody or antigen-binding fragment comprising a complementation-determining region (CDR) of the heavy chain variable region (HCVR) containing the sequence of SEQ ID NO: 7 and a complementation-determining region (CDR) of the light chain variable region (LCVR) containing 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, incorporated herein by reference.

[0070] Preferably, the IL-33 antagonist is an antibody or antigen-binding fragment comprising a complementation-determining region (CDR) of the heavy chain variable region (HCVR) containing the sequence of SEQ ID NO: 11 and a complementation-determining region (CDR) of the light chain variable region (LCVR) containing the sequence of SEQ ID NO: 29. These CDRs correspond to those derived from the antibody A25-3H04, which 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 complementation-determining region (CDR) of the heavy chain variable region (HCVR) containing the sequence of SEQ ID NO: 13 and a complementation-determining region (CDR) of the light chain variable region (LCVR) containing the sequence of SEQ ID NO: 31. These CDRs correspond to those derived from the antibody ANB020. ANB020 is fully described in International Publication No. 2015 / 106080, which is incorporated herein by reference.

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

[0073] Preferably, the IL-33 antagonist is an antibody or antigen-binding fragment comprising a complementarity-determining region (CDR) of the heavy chain variable region (HCVR) containing the sequence of SEQ ID NO: 17 and a complementarity-determining region (CDR) of the light chain variable region (LCVR) containing the sequence of SEQ ID NO: 35. These CDRs correspond to those derived from the antibody IL33-158, which 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 complementation-determining region (CDR) of the heavy chain variable region (HCVR) containing the sequence of SEQ ID NO: 18 and a complementation-determining region (CDR) of the light chain variable region (LCVR) containing the sequence of SEQ ID NO: 36. These CDRs correspond to those derived from the antibody 10C12.38.H6.87Y.581 lgG4. 10C12.38.H6.87Y.581 lgG4 is fully described in International Publication No. 2016 / 077381, which is incorporated herein by reference.

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

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

[0077] Preferably, the IL33 antibody or its antigen-binding fragment includes the VH domain of the sequence of SEQ ID NO: 1 and the VL domain of the sequence of SEQ ID NO: 19.

[0078] Preferably, the IL33 antibody or its antigen-binding fragment includes the VH domain of the sequence of SEQ ID NO: 7 and the VL domain of the sequence of SEQ ID NO: 25.

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

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

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

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

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

[0084] Preferably, the IL-33 antagonist is a binding molecule containing three CDRs in the heavy chain variable region according to Sequence ID No. 1.

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

[0086] Preferably, the IL-33 antagonist is a binding molecule containing three CDRs in the light chain variable region according to Sequence ID No. 19.

[0087] Therefore, preferably, the IL-33 antagonist is a binding molecule that may include, 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, for example, SEQ ID NOs: 19, 25, 29, 31, 34, 35, and 36.

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

[0089] Therefore, preferably, the IL-33 antagonist is a binding molecule that may contain a variable heavy chain domain (VH) and a variable light chain domain (VL) having VH CDRs 1-3 having sequences of SEQ ID NOs. 37, 38, and 39, respectively, where one or more VHCDRs have three or fewer single amino acid substitutions, insertions, and / or deletions.

[0090] Therefore, preferably, the IL-33 antagonist is a binding molecule containing a VH domain including VHCDR1-3, as shown in 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 containing a VH domain including VHCDR1-3, which are represented by SEQ ID NOs. 37, 38, and 39, respectively.

[0092] Therefore, preferably, the IL-33 antagonist is a binding molecule that may contain a variable heavy chain domain (VH) and a variable light chain domain (VL), each having VLCDR1-3 having sequences 40, 41, and 42, respectively, where one or more VLCDRs have three or fewer single amino acid substitutions, insertions, and / or deletions.

[0093] Therefore, preferably, the IL-33 antagonist is a binding molecule containing a VL domain including 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 containing a VL domain including VLCDR1-3, which are represented by SEQ ID NOs. 40, 41, and 42, respectively.

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

[0096] Therefore, preferably, the IL-33 antagonist is an antibody or its conjugated fragment comprising VH and VL, where VH has at least 90%, for example, amino acid sequences 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, identical to VH according to SEQ ID NOs. 1, 7, 11, 13, 16, 17, and 18.

[0097] Therefore, preferably, the IL-33 antagonist is an antibody or its conjugated fragment comprising VH and VL, where VH has at least 90%, for example, amino acid sequences 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to VH according to SEQ ID NO: 1.

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

[0099] Therefore, preferably, the IL-33 antagonist is an antibody or its conjugated fragment comprising VH and VL, where VL has at least 90%, for example, amino acid sequences 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, identical to VL according to SEQ ID NOs. 19, 25, 29, 31, 34, 35, and 36.

[0100] Therefore, preferably, the IL-33 antagonist is an antibody or its conjugated fragment comprising VH and VL, where VL has at least 90%, for example, amino acid sequences 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to VL according to SEQ ID NO: 19.

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

[0102] Therefore, preferably, the IL-33 antagonist is an antibody or its conjugated fragment comprising VH and VL, where VH has at least 90%, for example, amino acid sequences 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to VH according to SEQ ID NOs: 1, 7, 11, 13, 16, 17, and 18, and VL has at least 90%, for example, amino acid sequences 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to VL according to SEQ ID NOs: 19, 25, 29, 31, 34, 35, and 36.

[0103] Therefore, preferably, the IL-33 antagonist is an antibody or its conjugated fragment containing VH and VL, where VH has the amino acid sequence of SEQ ID NOs: 1, 7, 11, 13, 16, 17, and 18, and VL has the amino acid sequence of SEQ ID NOs: 19, 25, 29, 31, 34, 35, and 36.

[0104] Therefore, preferably, the IL-33 antagonist is an antibody or its conjugated fragment containing VH and VL, where VH has the amino acid sequence of SEQ ID NO: 1 and VL has the amino acid sequence of SEQ ID NO: 19.

[0105] Composition and administration The IL-33 antagonists described herein in medical use and methods may be administered to patients in the form of pharmaceutical compositions.

[0106] Preferably, the term "IL-33 antagonist" as used herein may also refer to a pharmaceutical composition comprising an IL-33 antagonist. Preferably, the pharmaceutical composition may comprise one or more IL-33 antagonists.

[0107] Preferably, IL-33 antagonists may be administered in pharmaceutically effective doses for the in vivo treatment of abnormal epithelial physiological function, or EGFR-mediated diseases, or respiratory diseases as defined by the methods of medical use and therapeutic embodiments herein.

[0108] Preferably, the "pharmaceutically effective amount" or "therapeutically effective amount" of IL-33 antagonist should be held in an amount sufficient to achieve effective binding with IL-33 and to achieve benefits, such as improving the symptoms of a disease or condition as described herein in the medical uses / methods.

[0109] Preferably, an IL-33 antagonist or a pharmaceutical composition thereof may be administered to humans or other animals in an amount sufficient to produce a therapeutic effect, in accordance with the therapeutic method / medical use described above.

[0110] Preferably, IL-33 antagonists or their pharmaceutical compositions can be administered to such humans or other animals in conventional dosage forms prepared by combining an IL-33 antagonist with a conventionally pharmaceutically acceptable carrier or diluent according to known techniques.

[0111] Those skilled in the art will recognize that the form and properties of pharmaceutically acceptable carriers or diluents depend on the amount of active ingredient combined with them, the route of administration, and other well-known variability factors. Those skilled in the art will further understand that cocktails containing one or more species of IL-33 antagonists may prove particularly effective.

[0112] The amount of IL-33 antagonist that can be combined with a carrier material to create a single dosage form varies depending on the target of treatment and the detailed method of administration. Preferably, the pharmaceutical composition may be administered as a single dose, multiple doses, or over an established period of time in an infusion. Preferably, the dosage regimen may also be adjusted to achieve the desired optimal response (e.g., a therapeutic or prophylactic response).

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

[0114] Preferably, the pharmaceutical composition is formulated to include a pharmaceutically acceptable non-toxic sterile carrier, such as physiological saline, a non-toxic buffer, or a preservative.

[0115] Preferably, the pharmaceutical composition may include a pharmaceutically acceptable carrier, such as water, an ion exchanger, alumina, aluminum stearate, lecithin, serum protein, such as human serum albumin, buffering substances, such as phosphates, glycine, sorbic acid, potassium sorbate, a mixture of partial glycerides of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicic acid, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol, and lanolin.

[0116] Preferably, the pharmaceutical composition may be a sterile aqueous or non-aqueous solution, suspension, or emulsion. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Examples of aqueous carriers include water, alcohol solutions / aqueous solutions, emulsions or suspensions, such as physiological saline and buffer media.

[0117] Preferably, pharmaceutically acceptable carriers include, but are not limited to, 0.01 to 0.1 M, preferably 0.05 M phosphate buffer or 0.8% physiological saline. Other common parenteral carriers include sodium phosphate solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's dextrose, or fixative oil. Intravenous carriers include fluid and nutrient replacement solutions, electrolyte replacement solutions, such as those based on Ringer's dextrose. Preservatives and other additives, such as antibacterial agents, antioxidants, chelating agents, and inert gases, may also be present.

[0118] Preferably, pharmaceutical compositions for injection may include sterile aqueous solutions (if water-soluble) or dispersions, and sterile powders for the immediate preparation of sterile injection solutions or dispersions. In such cases, the composition must be sterile and fluid enough to allow easy passage through an injection needle. The composition must be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi. Preferably, the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by the use of a coating such as lecithin, maintaining the required particle size in the case of dispersions, and using a surfactant.

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

[0120] Preferably, prevention of microbial activity can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it is preferable to include sugars, polyhydric alcohols, sugars, and isotonic agents such as mannitol, sorbitol, or sodium chloride in the pharmaceutical composition. Including absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition can result in sustained absorption of the injectable composition.

[0121] Preferably, sterile injection solutions can be prepared by appropriately mixing the required amount of the active compound (e.g., an IL-33 antagonist, either alone or in combination with other active agents) in a suitable solvent having one or a combination of the components listed herein, followed by filtration sterilization. Generally, dispersions are prepared by mixing the active compound in a sterile medium, which includes a basic dispersion medium and other necessary components from those listed above. In the case of sterile powders for the preparation of sterile injection solutions, the preparation method may be vacuum drying and freeze-drying, thereby producing a powder of the active component + any additional desired components from the pre-sterilized filtered solution.

[0122] Methods for administering IL-33 antagonists or their pharmaceutical compositions to subjects in need are well known to those skilled in the art or can be easily determined by those skilled in the art.

[0123] Preferably, the route of administration of an IL-33 antagonist or its pharmaceutical composition may be, for example, oral, parenteral, inhalation, or topical. Preferably, as used herein, the term parenteral includes, for example, intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration.

[0124] Preferably, the IL-33 antagonist or its pharmaceutical composition may be administered orally in an acceptable dosage form, such as capsules, tablets, aqueous suspensions, or solutions.

[0125] Preferably, the IL-33 antagonist or its pharmaceutical composition may be administered by nasal aerosol or inhalation. Such compositions may be prepared as a solution in physiological saline using benzyl alcohol or other suitable preservatives, absorption enhancers to improve bioavailability, and / or other conventional solubilizers or dispersants.

[0126] Preferably, the parenteral formulation may 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, IL-33 antagonists or their pharmaceutical compositions are delivered directly to the site of disease or pathology, such as abnormal epithelial physiological function, thereby increasing the exposure of affected tissue to the therapeutic agent. Preferably, IL-33 antagonists or their pharmaceutical compositions are administered directly to the site of disease or pathology. Therefore, preferably, IL-33 antagonists or their pharmaceutical compositions are administered to the site of abnormal epithelial physiological function, EGFR-mediated disease, or respiratory disease.

[0128] In one embodiment, administration of an IL-33 antagonist or its pharmaceutical composition is by administration into the airway. Preferably, by intranasal administration. Preferably, by intranasal inhalation. Preferably, the IL-33 antagonist or its pharmaceutical composition may be provided in the form of an inhalation device. Suitable inhalation devices are well known in the art.

[0129] In one embodiment, an inhaler is provided 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] Therefore, preferably, the IL-33 antagonist or its pharmaceutical composition is formulated as a liquid composition, preferably as an aerosolizable liquid composition.

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

[0132] Preferably, the components described above for preparing the pharmaceutical compositions described herein can be packaged and sold in kit form. Such kits may preferably have labels or accompanying information indicating that the relevant pharmaceutical compositions are useful for treating subjects suffering from or susceptible to a disease or disorder.

[0133] Preferably, the components 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 can be pressurized, and preferably they may be aerosol containers. These containers may be included in the kit as described above. Preferably, the kit may further include an inhalation device. Preferably, the inhalation device is operable to include an IL-33 antagonist or pharmaceutical composition as described herein, or a container as described above that may include an IL-33 antagonist or pharmaceutical composition as described herein.

[0134] Abnormal epithelial physiological function This disclosure relates to the medical use of IL-33 antagonists for the prevention or treatment of abnormal epithelial physiological functions.

[0135] When used herein, “abnormal epithelial physiological function” means any abnormality of the function of epithelium in the human body. Functions of epithelium 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 sensing. Abnormalities in any of these functions can have devastating physiological effects. Epithelium is present in a wide range of tissues throughout the body, including the skin, respiratory tract, gastrointestinal tract, reproductive tract, urinary tract, exocrine glands, and endocrine glands; and for such reasons, abnormalities within epithelium can be involved in a wide range of diseases or pathological conditions. Preferably, epithelium is respiratory epithelium, and abnormal epithelial physiological function is abnormal respiratory epithelial physiological function.

[0136] As used herein, "abnormal" means a difference in function compared to the function in a healthy subject, and typically means an increase or decrease in function compared to the function in a healthy subject.

[0137] Preferably, the epithelium is selected from: flattened, cubic, columnar, and pseudostratified. Preferably, the epithelium is columnar.

[0138] Preferably, the epithelium is ciliated. Preferably, the epithelium is ciliated columnar. Preferably, the abnormal epithelial physiological function is an abnormal ciliated columnar epithelial physiological function.

[0139] Preferably, abnormal epithelial physiological functions may include abnormal epithelial cell migration. Preferably, abnormal epithelial physiological functions may include a decrease in epithelial cell migration. Preferably, abnormal epithelial physiological functions may include abnormal epithelial cell proliferation. Preferably, abnormal epithelial physiological functions may include a decrease in epithelial cell proliferation.

[0140] Preferably, reduced epithelial cell migration leads to impaired epithelial ability to repair wounds. Preferably, abnormal epithelial physiological function includes impaired wound repair. Impaired wound repair may include impaired wound closure and decreased wound cell density.

[0141] Preferably, treatment of abnormal epithelial physiological function may include increasing or improving epithelial cell migration. Preferably, treatment of abnormal epithelial physiological function may include increasing or improving epithelial wound repair. Preferably, treatment of abnormal epithelial physiological function may include increasing or improving wound closure. Preferably, treatment of abnormal epithelial physiological function may include increasing or improving wound cell density.

[0142] Preferably, the abnormal epithelial physiological function is an abnormal mucociliary physiological function.

[0143] When used herein, "abnormal mucosal ciliary function" means any abnormality in the function of the mucosal ciliary function, particularly in epithelium. Abnormal function of the mucosal ciliary function in epithelium may be caused by abnormalities in the function of ciliated columnar cells and / or goblet cells, which are key to mucosal ciliary function. Preferably, abnormal mucosal ciliary function is caused by abnormal function of goblet cells.

[0144] As used herein, “mucosal ciliary body” refers to the function of ciliated columnar cells and goblet columnar cells in the epithelium that secrete and move mucus. The roles of the mucosal ciliary body in the epithelium may include: proliferation of goblet cells; differentiation of goblet cells; secretion of mucus; regulation of mucus composition; and / or mucus movement or clearance.

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

[0146] One embodiment provides a method for preventing or treating abnormal mucociliary physiological functions, such as abnormal mucociliary physiological function of the epithelium in a patient, comprising: administering an effective amount of IL-33 antagonist to a patient in need thereof.

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

[0148] Preferably, abnormal mucus production includes abnormal MUC5AC production. Preferably, abnormal goblet cell differentiation includes abnormal MUC5AC+ goblet cell differentiation. Preferably, abnormal goblet cell proliferation includes abnormal MUC5AC+ goblet cell proliferation. Preferably, abnormal epithelial thickness is an abnormal amount of MUC5AC in the total tissue area of the epithelium + including goblet cells.

[0149] Preferably, abnormal mucociliary physiological functions include: an increase in the number of goblet cells; an increase in mucus production; an increase in goblet cell differentiation; an increase in epithelial thickness; and / or a decrease in mucus clearance.

[0150] Preferably, an increase in mucus production includes an increase in MUC5AC production. Preferably, an increase in goblet cell differentiation includes an increase in MUC5AC+ goblet cell differentiation. Preferably, an increase in goblet cell proliferation includes an increase in MUC5AC+ goblet cell proliferation. Preferably, an increase in epithelial thickness is an increased amount of MUC5AC in the total tissue area of the epithelium + including goblet cells.

[0151] Preferably, an increase in MUC5AC production is caused by an increase in MUC5AC gene expression. Preferably, abnormal mucociliary physiological functions include an increase in MUC5AC gene expression in epithelial cells. Preferably, abnormal mucociliary physiological functions include an increase in the expression of MUC5AC in epithelial goblet cells.

[0152] Preferably, abnormal mucociliary physiological functions include changes in mucus composition. Such changes can 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 consistency of the mucus.

[0153] Changes in mucus composition may include increases or decreases in the ratios of different mucins, such as an increase or decrease in the ratio of mucin MUC5AC to MUC5B.

[0154] Changes in mucus composition may include an increase or decrease in mucin concentration. Preferably, changes in mucus composition include a decrease in mucin 5AC concentration. Preferably, changes in mucus composition include a decrease in the number of goblet cells having upregulated MUC5AC expression.

[0155] Such changes in mucin contained in mucus can be measured and calculated as described in International Publication No. 2018 / 204598, which is incorporated herein by reference.

[0156] Preferably, the abnormal mucus composition includes an increase in the MUC5AC:MUC5B ratio. Preferably, the abnormal mucus composition includes an increase in the MUC5AC contained in the mucus. Preferably, the abnormal mucus composition includes an increase in the viscosity of the mucus.

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

[0158] Preferably, abnormal epithelial physiological functions include abnormal tissue remodeling, such as abnormal epithelial remodeling. Preferably, abnormal epithelial physiological functions include increased tissue remodeling. Preferably, abnormal epithelial physiological functions include increased epithelial remodeling.

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

[0160] Treatment or prevention of abnormal epithelial physiological function, or treatment or prevention of abnormal mucociliary physiological function: Improvement or increase in mucociliary clearance; Reduction or inhibition of mucus production; Inhibition of abnormal mucus composition; Reduction or inhibition of epithelial remodeling; and / or This may include reducing or inhibiting the differentiation and / or proliferation of goblet cells.

[0161] Preferably, the reduction or inhibition of mucus production includes the reduction or inhibition of MUC5AC production. Therefore, preferably, treatment or prevention involves reducing or inhibiting MUC5AC production.

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

[0163] Preferably, the reduction or inhibition of goblet cell differentiation and / or proliferation is achieved by MUC5AC + This includes reducing or inhibiting the differentiation or proliferation of goblet cells. Therefore, preferably, treatment or prevention involves MUC5AC + It reduces or inhibits goblet cell differentiation or proliferation.

[0164] Preferably, reducing or inhibiting epithelial remodeling includes reducing the thickness of the respiratory epithelium. Therefore, preferably, treatment or prevention reduces the thickness of the respiratory epithelium.

[0165] Preferably, reduction or inhibition of epithelial remodeling is achieved by MUC5AC in the entire tissue region of the epithelium. + This includes reducing the amount of goblet cells. Therefore, preferably, treatment or prevention involves MUC5AC in the entire tissue area of ​​the epithelium. + It reduces or inhibits the quantity of goblet cells.

[0166] Improvement or increase in mucociliary clearance includes improvement or increase in mucociliary movement. Therefore, preferably, treatment or prevention involves improving or increasing mucociliary movement.

[0167] Preferably, the epithelium is respiratory epithelium. Preferably, the abnormal epithelial physiological function is an abnormal epithelial physiological function in respiratory epithelium.

[0168] In one embodiment, an IL-33 antagonist is provided for use in treating abnormal epithelial physiological function in respiratory diseases.

[0169] One embodiment provides a method for preventing or treating abnormal epithelial physiological function in a patient with a respiratory disease, comprising administering an effective amount of IL-33 antagonist to the patient in need.

[0170] Suitable respiratory diseases are defined elsewhere in this specification.

[0171] Preferably, the abnormal epithelial physiological function is an abnormal mucosal ciliary function in the respiratory epithelium.

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

[0173] One embodiment provides a method for preventing or treating abnormal mucosal ciliary physiological function of the respiratory epithelium of a patient, comprising: administering an effective amount of IL-33 antagonist to a patient in need thereof.

[0174] Preferably, the abnormal epithelial physiological function is the abnormal mucociliary physiological function in respiratory diseases.

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

[0176] One embodiment provides a method for preventing or treating abnormal mucociliary physiological function in patients with respiratory disease, comprising administering an effective amount of IL-33 antagonist to a patient in need.

[0177] Preferably, the abnormal epithelial physiological function is present in the airway. Preferably, the abnormal epithelial physiological function is an abnormal epithelial physiological function of the airway. Preferably, the abnormal epithelial physiological function is an abnormal mucociliary physiological function of the airway.

[0178] The airways include the upper and lower airways. Typically, the upper airways include the nasal cavity, sinuses, pharynx, and larynx. Typically, the lower airways include the trachea, bronchi, bronchioles, alveolar ducts, and alveoli.

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

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

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

[0182] One embodiment provides a method for preventing or treating abnormal mucociliary physiological function of the lower respiratory tract in a patient, comprising: administering an effective amount of IL-33 antagonist to a patient in need thereof.

[0183] In one embodiment, an IL-33 antagonist is provided for use in the prevention or treatment of abnormal mucosal ciliary body physiological function of the bronchi.

[0184] One embodiment provides a method for preventing or treating abnormal mucosal ciliary function of a patient's bronchi, comprising: administering an effective amount of IL-33 antagonist to a patient in need thereof.

[0185] EGFR signaling This disclosure is based on the discovery that oxidized IL-33 binds to RAGE, and RAGE subsequently complexes with EGFR, acting to disrupt epithelial homeostasis. The use of IL-33 antagonists may inhibit oxidized IL-33 signaling, thereby inhibiting RAGE activation and RAGE-EGFR complex formation. The data disclosed herein demonstrate that by preventing the formation of the RAGE-EGFR complex, IL-33-mediated EGFR signaling is disrupted, and normal epithelial physiology is restored.

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

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

[0191] Preferably, the IL-33 antagonist inhibits the RAGE-EGFR mediated effect. Preferably, the IL-33 antagonist inhibits the effect mediated by the RAGE-EGFR complex. Preferably, the IL-33 antagonist inhibits the 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] Preferably, the IL-33 antagonist inhibits the binding of oxidized IL-33 to RAGE, thereby inhibiting RAGE-EGFR complex formation, and thereby inhibiting RAGE-EGFR-mediated effects such as downstream signal transduction.

[0194] Preferably, IL-33 antagonists inhibit IL-33-mediated EGFR effects. Preferably, IL-33 antagonists inhibit IL-33-mediated EGFR signaling. Preferably, IL-33 antagonists inhibit oxidized IL-33-mediated EGFR effects. Preferably, IL-33 antagonists inhibit oxidized IL-33-mediated EGFR signaling. Preferably, IL-33 antagonists inhibit oxidized IL-33-mediated RAGE-EGFR effects. Preferably, IL-33 antagonists inhibit oxidized IL-33-mediated RAGE-EGFR signaling.

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

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

[0199] Therefore, preferably, IL-33 antagonists inhibit EGFR-mediated phosphorylation and / or chemokine release.

[0200] Therefore, preferably, IL-33 antagonists inhibit the phosphorylation of components of the EGFR pathway. Preferably, IL-33 antagonists inhibit the phosphorylation of one of the following: EGFR, PLC, JNK, MAPK / ERK1 / 2, p38, and STAT5. Preferably, IL-33 antagonists inhibit the EGFR-mediated phosphorylation of one of the following: EGFR, PLC, JNK, MAPK / ERK1 / 2, p38, and STAT5. Preferably, IL-33 antagonists inhibit the phosphorylation of tyrosine kinases. Preferably, IL-33 antagonists inhibit the phosphorylation of a tyrosine kinase selected from JNK, MAPK / ERK, and p38. Preferably, IL-33 antagonists inhibit the EGFR-mediated phosphorylation of a tyrosine kinase selected from JNK, MAPK / ERK, and p38.

[0201] Therefore, preferably, IL-33 antagonists inhibit the release of chemokines. Preferably, IL-33 antagonists inhibit the release of IL-8. Preferably, IL-33 antagonists inhibit the EGFR-mediated release of chemokines. Preferably, IL-33 antagonists inhibit the EGFR-mediated release of IL-8.

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

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

[0204] Furthermore, IL-33 antagonists may be used for the prevention or treatment of abnormal epithelial physiological function in EGFR-mediated diseases.

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

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

[0210] Preferably, IL-33 antagonists inhibit the RAGE-EGFR-mediated effect. Preferably, IL-33 antagonists treat or prevent diseases or conditions by inhibiting the RAGE-EGFR-mediated effect.

[0211] As used herein, “RAGE-EGFR-mediated effects” refer to any physiological effects caused by the complexation of RAGE with EGFR in the cell membrane and the resulting abnormal EGFR activity. RAGE-EGFR-mediated effects may also be expressed herein as “RAGE-EGFR signaling,” optionally “RAGE-EGFR-mediated signaling,” and / or as such. Such RAGE-EGFR-mediated effects are typically observed in epithelium and manifest as abnormal epithelial physiological functions. Abnormal epithelial physiological functions are 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, RAGE-EGFR-mediated disease and / or effect is characterized by abnormal EGFR activity. Preferably, RAGE-EGFR-mediated disease and / or effect is characterized by abnormal RAGE-EGFR signaling. Preferably, RAGE-EGFR-mediated effect and / or RAGE-EGFR signaling is characterized by RAGE-EGFR-mediated disease.

[0213] Preferably, RAGE-EGFR-mediated diseases may be diseases characterized by abnormal epithelial physiological function.

[0214] Preferably, RAGE-EGFR-mediated diseases may be diseases characterized by abnormal epithelial physiological function in the respiratory epithelium.

[0215] Preferably, RAGE-EGFR-mediated diseases may be diseases characterized by abnormal mucociliary physiological function.

[0216] Preferably, RAGE-EGFR-mediated diseases may be diseases characterized by abnormal mucosal ciliary function in the respiratory epithelium.

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

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

[0219] Preferably, abnormal epithelial physiological function may be a symptom of respiratory disease. Therefore, preferably, IL-33 antagonists may be used for the treatment or prevention of respiratory diseases characterized by abnormal epithelial physiological function.

[0220] As defined in further embodiments, IL-33 antagonists are provided for use in the prevention or treatment of respiratory diseases by improving epithelial physiological function.

[0221] Abnormal epithelial physiological function is defined elsewhere in this specification.

[0222] The improvement of desirable epithelial physiological function may include the improvement of abnormal epithelial physiological function.

[0223] Preferred means for improving abnormal epithelial physiological function are described above in this specification.

[0224] Preferably, treatment of respiratory diseases by improving abnormal epithelial physiological function is: Improvement or increase in mucociliary clearance; Reduction or inhibition of mucus production; Inhibition of abnormal mucus composition; Reduction or inhibition of abnormal 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 connection with the treatment or prevention of abnormal epithelial physiological function and may be used herein in combination with the treatment of respiratory diseases.

[0226] Preferably, abnormal EGFR activity may be a symptom of respiratory disease. Therefore, preferably, IL-33 antagonists may be used for the treatment or prevention of respiratory diseases characterized by abnormal EGFR activity.

[0227] As defined in further embodiments, IL-33 antagonists are provided for use in the prevention or treatment of respiratory diseases by inhibiting EGFR-mediated effects.

[0228] The EGFR-mediated effect is defined elsewhere in this specification.

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

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

[0231] Preferably, the respiratory disease is COPD. Preferably, the respiratory disease is bronchitis-induced COPD. Bronchitis-induced COPD is a specific form of COPD in which chronic bronchitis is present in patients with COPD. Bronchitis-induced COPD has a higher mortality rate than COPD patients because of the rapid decline in lung function, the increasing severity of symptoms, and the increased risk of secondary infections. In particular, patients with bronchitis-induced COPD have high total mucin concentrations and excessive mucus secretion. Therefore, patients with bronchitis-induced COPD may particularly benefit from treatment with IL-33 antagonists, as described herein, due to the discovery that IL-33 antagonists inhibit EGFR activity and improve mucociliary function.

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

[0233] In one embodiment, an IL-33 antagonist is provided for the prevention or treatment of bronchitis-induced COPD.

[0234] One embodiment provides a method for preventing or treating bronchitis-induced COPD in a patient, comprising administering an effective amount of IL-33 antagonist to the patient in need.

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

[0236] Preferably, IL-33 antagonists are used for the prevention and treatment of abnormal epithelial physiological functions by modulating EGFR-mediated and ST2-mediated effects. Preferably, IL-33 antagonists are used for the prevention and treatment of abnormal epithelial physiological functions by inhibiting EGFR-mediated and ST2-mediated effects. Preferably, IL-33 antagonists are used for the prevention and treatment of abnormal epithelial physiological functions by inhibiting RAGE / EGFR-mediated and ST2-mediated effects. Abnormal epithelial physiological functions are as defined elsewhere in this specification.

[0237] Preferably, IL-33 antagonists are used for the prevention and treatment of EGFR-mediated diseases by modulating EGFR-mediated and ST2-mediated effects. Preferably, IL-33 antagonists are used for the prevention and treatment of EGFR-mediated diseases by inhibiting EGFR-mediated and ST2-mediated effects. Preferably, IL-33 antagonists are used for the prevention and treatment of EGFR-mediated diseases by inhibiting RAGE / EGFR-mediated and ST2-mediated effects. EGFR-mediated diseases are defined elsewhere in this specification.

[0238] Preferably, the ST2-mediated effect includes inflammation. Therefore, preferably, IL-33 antagonists are intended for use in the prevention and treatment of ST2-mediated diseases by modulating EGFR-mediated and ST2-mediated effects. Preferably, IL-33 antagonists are intended for use in the prevention and treatment of ST2-mediated diseases by inhibiting EGFR-mediated and ST2-mediated effects. Preferably, IL-33 antagonists are intended for use in the prevention and treatment of ST2-mediated diseases by inhibiting RAGE / EGFR-mediated 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 diseases or inflammatory diseases include: COPD; allergic diseases such as asthma, chronic rhinitis, food allergy, eczema, and dermatitis; fibrotic diseases such as pulmonary fibrosis; pulmonary eosinophilia; pleural malignant lesions; rheumatoid arthritis; collagen vascular diseases; atherosclerotic vascular diseases; urticaria; inflammatory bowel diseases; Crohn's disease; celiac disease; systemic lupus erythematosus; progressive systemic sclerosis; Wegener's granulomatosis; septic shock; and Behcet's disease.

[0240] Preferably, the ST2-mediated disease or inflammatory disease is respiratory. Preferably, the ST2-mediated disease or inflammatory disease is present in the airway 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, the ST2-mediated effect and the EGFR-mediated effect, preferably the RAGE-EGFR-mediated effect, contribute to the disease pathology. Advantageously, the medical use of a single IL-33 antagonist is thought to have the potential to inhibit the activation of both RAGE and ST2 by IL-33. Therefore, a single IL-33 antagonist can treat both RAGE-EGFR-mediated diseases and ST2-mediated diseases simultaneously.

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

[0244] Suitably, abnormal epithelial physiology and inflammation may be symptoms of a respiratory disease. Accordingly, descriptions regarding the treatment and prevention of these symptoms may be in the context of a respiratory disease and may preferably include the treatment or prevention of abnormal epithelial physiology and inflammation in a respiratory disease.

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

[0246] In one embodiment, 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 an IL-33 antagonist is provided.

[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 in combination therapy to inhibit the activation of both RAGE and ST2 by IL-33. Accordingly, combinations of IL-33 antagonists are envisioned to treat both RAGE-EGFR-mediated diseases and ST2-mediated diseases simultaneously.

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

[0251] Thus, preferably, in one embodiment, 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 are provided in combination.

[0252] Therefore, preferably, in one embodiment, a method is provided for preventing or treating abnormal epithelial physiological function 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] Therefore, preferably, in one embodiment, a first IL-33 antagonist for use in the prevention or treatment of EGFR-mediated diseases and a second IL-33 antagonist for use in the prevention or treatment of ST2-mediated diseases are provided in combination.

[0254] Therefore, preferably, in one embodiment, a method is provided for preventing or treating EGFR-mediated and ST2-mediated diseases 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.

[0255] Preferably, the first IL-33 antagonist is for the prevention or treatment of abnormal epithelial physiological function and / or EGFR-mediated diseases.

[0256] Preferably, the second IL-33 antagonist is for the prevention and treatment of 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 the ST2-mediated effect. Preferably, the second IL-33 antagonist is also as defined above.

[0259] Preferably, the first antagonist may be a reducing or oxidizing IL-33 antagonist. Preferably, the second IL-33 antagonist is a reducing 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 simultaneously or at different times in combination. A suitable dosing schedule may be determined by a medical professional.

[0262] These descriptions equally apply to the above-mentioned medical use / treatment method in which ST-2 mediated diseases can also be prevented or treated.

[0263] Alternatively, in a further embodiment, the IL-33 antagonist may be administered in combination with an ST2 inhibitor. Preferably, the ST2 inhibitor may not be an IL-33 antagonist, but may inhibit the ST2 receptor by other means. Preferably, the ST2 inhibitor may function to treat or prevent ST2 mediated diseases as described above.

[0264] Thus, in one embodiment, an IL-33 antagonist for use in treating or preventing abnormal epithelial physiological function and an ST2 inhibitor for use in treating or preventing inflammation are provided in combination.

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

[0266] Preferably, abnormal epithelial physiological function and inflammation may be symptoms of respiratory disease. Therefore, descriptions of the treatment and prevention of these symptoms may be in the context of respiratory disease, and preferably include the treatment or prevention of abnormal epithelial physiological function and inflammation in respiratory disease.

[0267] Therefore, in one embodiment, an IL-33 antagonist for use in the prevention or treatment of EGFR-mediated diseases and an ST2 inhibitor for use in the treatment or prevention of ST2-mediated diseases are provided in combination.

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

[0269] Preferably, the IL-33 antagonist is as defined elsewhere in this specification. Preferred EGFR-mediated and ST2-mediated diseases are as defined elsewhere in this specification.

[0270] Preferably, the ST2 inhibitor may be any such inhibitor known in the art, e.g., GSK3772847 (described in International Publication No. 2013 / 165894) and RG6149 (International Publication No. 2013 / 173761), both of which are incorporated herein by reference.

[0271] Preferably, IL-33 antagonists and ST2 inhibitors may be administered in combination. Preferably, IL-33 antagonists and ST2 inhibitors may be administered simultaneously or at different times. A suitable dosing regimen may be determined by a healthcare professional.

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

[0273] Preferably, the patient may be a human being. The patient may be receiving medical treatment or the individual may be requesting medical treatment. Preferably, the patient may be male or female. Preferably, the patient may be an adult or a child.

[0274] Preferably, in the method described herein, a suitable patient may be a person believed to have abnormal epithelial physiological function, or EGFR-mediated disease, or 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 of developing abnormal epithelial physiological function, or EGFR-mediated disease, or respiratory disease. For example, such patients may have been in contact with individuals suffering from such conditions, may suffer from related conditions, or may meet risk factors associated with the above conditions, such as smoking, old age, or allergies.

[0276] Embodiment Parts of this 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 physiological function 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-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-induced 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 the binding of oxidized IL-33 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 its antigen-binding fragment, preferably an anti-reduced IL-33 antibody or its antigen-binding fragment. Embodiment 12 describes an IL-33 antagonist for use in accordance with Embodiment 11, wherein the anti-IL-33 antibody or its antigen-binding fragment comprises a complementarity-determining region (CDR) of a pair of variable heavy chain domains (VH) and variable light chain domains (VL) selected from Table 1. Embodiment 13 describes an IL-33 antagonist for use in accordance with Embodiment 12, wherein the anti-IL-33 antibody or its antigen-binding fragment comprises a pair of variable heavy chain domains (VH) and variable light chain domains (VL) 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 its antigen-binding fragment comprises VHCDR1 having the sequence of SEQ ID NO: 37, VHCDR2 having the sequence of SEQ ID NO: 38, VHCDR3 having the sequence of SEQ ID NO: 39, VLCDR1 having the sequence of SEQ ID NO: 40, VLCDR2 having the sequence of SEQ ID NO: 41, and VLCDR3 having the sequence of SEQ ID NO: 42. 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 IL-33 antibody or its antigen-binding fragment, comprising the VH domain of the sequence of SEQ ID NO: 1 and the VL domain of the sequence of SEQ ID NO: 19.

[0277] Embodiments of this disclosure are further described in the following sections: [Section 1] An IL-33 antagonist for use in the prevention or treatment of abnormal epithelial physiological function by modulating or inhibiting the RAGE-EGFR-mediated effect. [Section 2] An IL-33 antagonist for use as described in item 1 above, wherein the abnormal epithelial physiological function is an abnormal mucociliary physiological function, preferably an abnormal mucociliary physiological function of the respiratory epithelium. [Section 3] An IL-33 antagonist for use as described in item 2 above, wherein the abnormal mucosal-ciliary body physiological function is selected from: abnormal mucus production; abnormal goblet cell differentiation; abnormal goblet cell proliferation; abnormal epithelial thickness; abnormal mucus clearance; and / or abnormal mucus composition. [Item 4] Abnormal mucus production includes abnormal MUC5AC production; and / or abnormal goblet cell differentiation includes abnormal MUC5AC+ goblet cell differentiation; and / or abnormal goblet cell proliferation includes abnormal MUC5AC+ goblet cell proliferation; and / or the abnormal epithelial thickness is an abnormal amount of MUC5AC in the entire tissue area of the epithelium + An IL-33 antagonist for use as described in item 3 above, including goblet cells. [Item 5] An IL-33 antagonist for use as described in item 2, 3, or 4 above, wherein the abnormal mucosal-ciliary body physiological function includes: increased mucus production; increased goblet cell differentiation; increased goblet cell proliferation; increased thickness of the epithelium; and / or decreased mucus clearance. [Item 6] Increased mucus production includes increased MUC5AC production; and / or increased goblet cell differentiation includes increased MUC5AC+ goblet cell differentiation; and / or increased goblet cell proliferation includes increased MUC5AC+ goblet cell proliferation; and / or the increased thickness of the epithelium is an increased amount of MUC5AC in the entire tissue area of the epithelium + An IL-33 antagonist for use as described in item 5 above, including goblet cells. [Item 7] An IL-33 antagonist for use as described in item 3 above, wherein the abnormal mucus composition includes an increase or decrease in the ratio of various mucus compounds contained in the mucus; an increase or decrease in one or more mucus compounds; and / or an increase or decrease in the concentration or consistency of the mucus. [Item 8] An IL-33 antagonist for use as described in item 7 above, wherein the abnormal mucus composition includes an increase in the ratio of MUC5AC:MUC5B; and / or the abnormal mucus composition includes an increase in MUC5AC contained in the mucus; and / or the abnormal mucus composition includes an increase in the consistency of the mucus. [Item 9] An IL-33 antagonist for use as described in item 1 above, wherein the aforementioned abnormal epithelial physiological function is abnormal epithelial remodeling. [Section 10] An IL-33 antagonist for use as described in any one of items 1 to 9 above, wherein the abnormal epithelial physiological function is the airway, preferably the abnormal mucociliary body physiological function of the airway. [Section 11] An IL-33 antagonist for use as described in item 10 above, wherein the airway is the lower airway, preferably the bronchi. [Section 12] An IL-33 antagonist for use in the prevention or treatment of EGFR-mediated diseases. [Section 13] An IL-33 antagonist for use as described in item 12 above, wherein the EGFR-mediated disease is a RAGE-EGFR-mediated disease. [Section 14] An IL-33 antagonist for use as described in item 12 or 13 above, wherein the EGFR-mediated disease is characterized by abnormal EGFR activity. [Section 15] An IL-33 antagonist for use as described in any one of items 12 to 14 above, wherein the EGFR-mediated disease is characterized by abnormal epithelial physiological function. [Section 16] An IL-33 antagonist for use in the prevention or treatment of diseases by improving epithelial physiological function. [Section 17] An IL-33 antagonist for use in the prevention or treatment of diseases by inhibiting EGFR-mediated effects. [Section 18] An IL-33 antagonist for use as described in item 17 above, wherein the EGFR-mediated effect is EGFR signaling. [Section 19] An IL-33 antagonist for use as described in item 17 or 18 above, wherein the EGFR-mediated effect is a RAGE-EGFR-mediated effect. [Section 20] An IL-33 antagonist for use as described in any one of items 17 to 19 above, wherein the EGFR-mediated effect is RAGE-EGFR-mediated signaling. [Section 21] An IL-33 antagonist for use as described in any one of items 16 to 20 above, wherein the disease is a respiratory disease. [Section 22] An IL-33 antagonist for use as described in item 21 above, wherein the aforementioned respiratory disease is characterized by abnormal epithelial physiological function and / or abnormal EGFR activity. [Section 23] An IL-33 antagonist for use as described in item 21 or 22 above, wherein the respiratory disease is a lower respiratory disease, preferably a bronchial respiratory disease. [Section 24] An IL-33 antagonist for use as described in any one of paragraphs 21 to 23 above, wherein the respiratory disease is selected from: COPD, bronchitis, emphysema, bronchiectasis such as CF-bronchiectasis or CF-bronchiectasis, asthma, or overlap of asthma and COPD (ACO). [Section 25] An IL-33 antagonist for use as described in any one of the above paragraphs 21 to 24, wherein the respiratory disease is COPD, preferably bronchitis-induced COPD. [Section 26] An IL-33 antagonist for use as described in any one of items 21 to 24 above, wherein the respiratory disease is asthma, preferably bronchial asthma. [Section 27] An IL-33 antagonist for use as described in any one of paragraphs 16 to 26 above, wherein the aforementioned prevention or treatment improves mucus clearance. [Section 28] An IL-33 antagonist for use as described in any one of paragraphs 16 to 27 above, wherein the prevention or treatment inhibits or reduces abnormal mucus production. [Section 29] An IL-33 antagonist for use as described in paragraph 28 above, wherein the aforementioned prevention or treatment inhibits or reduces MUC5AC production. [Section 30] An IL-33 antagonist for use according to any one of paragraphs 16 to 29 above, wherein the aforementioned prevention or treatment inhibits abnormal mucus composition. [Section 31] An IL-33 antagonist for use as described in paragraph 30 above, wherein the prevention or treatment inhibits or reduces the MUC5AC:MUC5B ratio; and / or the prevention or treatment inhibits or reduces MUC5AC in mucus; and / or the prevention or treatment reduces the concentration of mucus. [Section 32] An IL-33 antagonist for use as described in any one of paragraphs 16 to 31 above, wherein the aforementioned prevention or treatment inhibits abnormal epithelial remodeling. [Section 33] An IL-33 antagonist for use as described in any one of paragraphs 16 to 32 above, wherein the aforementioned prevention or treatment inhibits abnormal goblet cell differentiation or proliferation. [Section 34] The aforementioned abnormal goblet cell differentiation or proliferation is abnormal MUC5AC + An IL-33 antagonist for use as described in item 33 above, for goblet cell differentiation or proliferation. [Section 35] An IL-33 antagonist for use as described in any one of paragraphs 16 to 34 above, wherein the aforementioned prevention or treatment reduces the thickness of the respiratory epithelium. [Section 36] The aforementioned prevention or treatment is MUC5AC in the entire tissue region of the epithelium. + An IL-33 antagonist for use as described in item 35 above, for reducing the amount of goblet cells. [Section 37] An IL-33 antagonist for use as described in any one of the above items 1 to 36, wherein the IL-33 antagonist inhibits the activity of oxidized IL-33. [Section 38] An IL-33 antagonist for use as described in any one of the above paragraphs 1 to 37, wherein the IL-33 antagonist inhibits the binding of oxidized IL-33 to RAGE, thereby inhibiting RAGE-EGFR signaling. [Section 39] An IL-33 antagonist for use as described in any one of the above paragraphs 1 to 38, wherein the IL-33 antagonist downregulates or inhibits RAGE-EGFR-dependent signaling and / or RAGE-EGFR-mediated effects. [Section 40] An IL-33 antagonist for use as described in any one of the above items 1 to 39, wherein the IL-33 antagonist is a binding molecule or fragment thereof that binds to IL-33, preferably reduced IL-33 or oxidized IL-33, preferably reduced IL-33. [Section 41] An IL-33 antagonist for use as described in any one of the above items 1 to 40, wherein the IL-33 antagonist is an antibody or an antigen-binding fragment thereof, preferably an anti-IL-33 antibody or an antigen-binding fragment thereof, preferably an anti-reduced IL-33 antibody or an antigen-binding fragment thereof. [Section 42] An IL-33 antagonist for use as described in any one of the above sections 1 to 41, wherein the IL-33 antagonist is a binding molecule containing a complementarity-determining region (CDR) of a pair of variable heavy-chain domains (VH) and variable light-chain domains (VL) selected from Table 1. [Section 43] An IL-33 antagonist for use as described in any one of sections 1 to 42 above, wherein the IL-33 antagonist is a binding molecule containing a pair of variable heavy-chain domains (VH) and variable light-chain domains (VL) selected from Table 1. [Section 44] The IL-33 antagonist according to any one of items 1 to 43 above, wherein the IL-33 antagonist is a binding molecule comprising VHCDR1 having the sequence of SEQ ID NO: 37, VHCDR2 having the sequence of SEQ ID NO: 38, VHCDR3 having the sequence of SEQ ID NO: 39, VLCDR1 having the sequence of SEQ ID NO: 40, VLCDR2 having the sequence of SEQ ID NO: 41, and VLCDR3 having the sequence of SEQ ID NO: 42. [Section 45] A method for preventing or treating abnormal epithelial physiological function in a subject by administering a therapeutically effective dose of an IL-33 antagonist to modulate or inhibit the RAGE-EGFR-mediated effect. [Section 46] The method according to item 45, wherein the abnormal epithelial physiological function is an abnormal mucociliary physiological function, preferably an abnormal mucociliary physiological function of the respiratory epithelium. [Section 47] The method according to item 46 above, wherein the abnormal mucociliary physiological function is selected from: abnormal mucus production; abnormal goblet cell differentiation; abnormal goblet cell proliferation; abnormal epithelial thickness; abnormal mucus clearance; and / or abnormal mucus composition. [Section 48] Abnormal mucus production includes abnormal MUC5AC production; and / or abnormal goblet cell differentiation includes abnormal MUC5AC+ goblet cell differentiation; and / or abnormal goblet cell proliferation includes abnormal MUC5AC+ goblet cell proliferation; and / or abnormal epithelial thickness includes an abnormal amount of MUC5AC in the entire tissue region of the epithelium. + The method described in item 47 above, comprising goblet cells. [Section 49] The method according to item 47 or 48, wherein abnormal mucosal ciliary physiological function includes: increased mucus production; increased goblet cell differentiation; increased goblet cell proliferation; increased thickness of the epithelium; and / or decreased mucus clearance. [Section 50] Increased mucus production includes increased MUC5AC production; and / or increased goblet cell differentiation includes increased MUC5AC + increased goblet cell differentiation; and / or increased goblet cell proliferation includes increased MUC5AC + increased goblet cell proliferation; and / or increased thickness of the epithelium includes an increased amount of MUC5AC in the entire tissue region of the epithelium. +The method described in item 49 above, comprising goblet cells. [Section 51] The method according to item 47, wherein the abnormal mucus composition includes an increase or decrease in the ratio of various mucus compounds contained in the mucus; an increase or decrease in one or more mucus compounds; and / or an increase or decrease in the concentration or density of the mucus. [Section 52] The method according to item 51, wherein the abnormal mucus composition includes an increase in the ratio of MUC5AC:MUC5B; and / or the abnormal mucus composition includes an increase in the amount of MUC5AC contained in the mucus; and / or the abnormal mucus composition includes an increase in the concentration of the mucus. [Section 53] The method according to item 45, wherein the abnormal epithelial physiological function is abnormal epithelial remodeling. [Section 54] The method according to any one of items 45 to 53, wherein the abnormal epithelial physiological function is the airway, preferably the abnormal mucociliary body physiological function of the airway. [Section 55] The method according to item 54, wherein the airway is a lower airway, preferably a bronchus. [Section 56] A method for the prevention or treatment of EGFR-mediated disease by administering a therapeutically effective dose of an IL-33 antagonist. [Section 57] The method according to item 56 above, wherein the EGFR-mediated disease is a RAGE-EGFR-mediated disease. [Section 58] The method according to item 56 or 57, wherein the EGFR-mediated disease is characterized by abnormal EGFR activity. [Section 59] The method according to any one of items 56 to 58 above, wherein the EGFR-mediated disease is characterized by abnormal epithelial physiological function. [Section 60] A method for preventing or treating disease by administering a therapeutically effective dose of an IL-33 antagonist to improve epithelial physiological function. [Section 61] A method for preventing or treating a disease by administering a therapeutically effective dose of an IL-33 antagonist to inhibit the EGFR-mediated effect. [Section 62] The method according to item 61, wherein the EGFR-mediated effect is EGFR signaling. [Section 63] The method according to item 61 or 62, wherein the EGFR-mediated effect is a RAGE-EGFR-mediated effect. [Section 64] The method according to any one of items 61 to 63, wherein the EGFR-mediated effect is RAGE-EGFR-mediated signaling. [Section 65] The method according to any one of items 60 to 64 above, wherein the disease is a respiratory disease. [Section 66] The method according to item 65, wherein the respiratory disease is characterized by abnormal epithelial physiological function and / or abnormal EGFR activity. [Section 67] The method according to item 65 or 66, wherein the respiratory disease is a lower respiratory disease, preferably a bronchial respiratory disease. [Section 68] The method according to any one of items 65 to 67 above, wherein the respiratory disease is selected from: COPD, bronchitis, emphysema, bronchiectasis such as CF-bronchiectasis or CF-bronchiectasis, asthma, or overlap of asthma and COPD (ACO). [Section 69] The method according to any one of items 65 to 68, wherein the respiratory disease is COPD, preferably bronchitis-induced COPD. [Section 70] The method according to any one of items 65 to 69, wherein the respiratory disease is asthma, preferably bronchial asthma. [Section 71] The method according to any one of items 45 to 70 above, wherein the method improves mucus clearance. [Section 72] The method according to any one of items 45 to 71, wherein the method inhibits or reduces abnormal mucus production. [Section 73] The method according to item 72, wherein the abnormal mucus production is an increase in MUC5AC production. [Section 74] The method according to any one of items 45 to 73, wherein the method inhibits abnormal mucus composition. [Section 75] The method according to item 74, wherein the method inhibits or reduces the MUC5AC:MUC5B ratio; and / or the method inhibits or reduces MUC5AC in the mucus; and / or the method inhibits or reduces the concentration of the mucus. [Section 76] The method according to any one of items 45 to 75, wherein the method inhibits abnormal epithelial remodeling. [Section 77] The method according to any one of items 45 to 76, wherein the method inhibits or reduces abnormal goblet cell differentiation or proliferation. [Section 78] The above method, abnormal MUC5AC + The method described in item 77 above, which inhibits or reduces goblet cell differentiation or proliferation. [Section 79] The method according to any one of items 45 to 78, wherein the method reduces the thickness of the respiratory epithelium. [Section 80] The above method is used in the entire tissue region of the respiratory epithelium, and MUC5AC + The method described in item 79 above for reducing the amount of goblet cells. [Section 81] The method according to any one of items 45 to 80 above, wherein the IL-33 antagonist inhibits the activity of oxidized IL-33. [Section 82] The method according to any one of items 45 to 81, wherein the IL-33 antagonist prevents the binding of oxidized IL-33 to RAGE, thereby inhibiting RAGE-EGFR signaling. [Section 83] The method according to any one of items 45 to 82, wherein the IL-33 antagonist downregulates or inhibits RAGE-EGFR-dependent signaling and / or RAGE-EGFR-mediated effects. [Section 84] The method according to any one of items 45 to 83, wherein the IL-33 antagonist is a binding molecule or fragment thereof that binds to IL-33, preferably reduced IL-33 or oxidized IL-33, preferably reduced IL-33. [Section 85] The method according to any one of items 45 to 84, wherein the IL-33 antagonist is an antibody or an antigen-binding fragment thereof, preferably an anti-IL-33 antibody or an antigen-binding fragment thereof, preferably an anti-reduced IL-33 antibody or an antigen-binding fragment thereof. [Section 86] The method according to any one of items 45 to 85, wherein the IL-33 antagonist is a binding molecule containing a complementarity-determining region (CDR) of a pair of variable heavy-chain domains (VH) and variable light-chain domains (VL) selected from Table 1. [Section 87] The method according to any one of items 45 to 86 above, wherein the IL-33 antagonist is a binding molecule comprising a pair of variable heavy chain domains (VH) and variable light chain domains (VL) selected from Table 1. [Section 88] The method according to any one of items 45 to 87, wherein the IL-33 antagonist is a binding molecule comprising VHCDR1 having the sequence of SEQ ID NO: 37, VHCDR2 having the sequence of SEQ ID NO: 38, VHCDR3 having the sequence of SEQ ID NO: 39, VLCDR1 having the sequence of SEQ ID NO: 40, VLCDR2 having the sequence of SEQ ID NO: 41, and VLCDR3 having the sequence of SEQ ID NO: 42. [Section 89] An IL-33 antagonist for use as described in any one of paragraphs 45 to 88, or as described in any one of paragraphs 1 to 44, wherein the IL-33 antagonist is an anti-IL33 antibody or its antigen-binding fragment comprising the VH domain of the sequence of SEQ ID NO: 1 and the VL domain of the sequence of SEQ ID NO: 19. Embodiments will be described as illustrative examples with reference to the following drawings. [Brief explanation of the drawing]

[0278] [Figure 1] Figure 1 shows grayscale heatmaps of kinase phosphorylation doubling compared to untreated controls for each detection assay of the MAP kinase phosphorylation antibody array. Reduced IL-33 (IL-33-01 and IL-33-16, respectively) did not produce 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 of the receptor tyrosine kinase (RTK) activity array. In each case, oxIL-33 induced a positive signal in the RTK array corresponding to the epidermal growth factor receptor (EGFR), while reduced IL33-01 and IL33-16 did not. Dot intensity correlates with receptor tyrosine kinase phosphorylation. [Figure 3A] Figure 3A shows pEGFR (Tyr1068) activity in normal human bronchial epithelial (NHBE) cells stimulated with increased concentrations of IL-33 or EGFR ligand. oxIL-33 promoted EGFR phosphorylation, similar to EGF, HB-EGF, and TGFα, but reduced IL-33 (IL33-01) did not. [Figure 3B] Figure 3B shows pEGFR(Tyr1068) activity in A549 cells stimulated with increased concentrations of IL-33 or EGFR ligand. Oxylated IL-33 (IL-33-01) promoted EGFR phosphorylation in a pattern similar to that observed in NHBE cells, as well as EGF, HB-EGF, and TGFα, but reduced IL-33 (IL-33-01) did not. [Figure 3C] Figure 3C shows pEGFR(Tyr1068) activity in A549 cells stimulated with increased concentrations of IL-33, EGFR ligand, or RAGE ligand. oxIL-33 promoted EGFR phosphorylation, similar to EGF, but wild-type (WT) IL-33 (IL-33-01), C->S mutant (mut) IL-33 (IL-33-16), or RAGE ligand did not. [Figure 4]Figure 4 shows that, as analyzed by Western blotting, oxidized IL-33 induces phosphorylation of several molecules involved in the EGFR pathway (EGFR, PLC, AKT, JNK, ERK 1 / 2, p38). [Figure 5] Figure 5 shows that increasing the dose of anti-EGFR antibody reduces oxIL-33-01-induced STAT5 phosphorylation compared to isotype controls. [Figure 6] Figure 6 shows immunoprecipitation with anti-EGFR and subsequent detection of EGFR, RAGE, or IL-33 by Western blotting. IL-33 and RAGE co-precipitate with EGFR and form a complex after NHBE stimulation with oxIL-33. RAGE appears to be specific to the oxIL-33 signaling complex compared to EGF. [Figure 7A] Figure 7A shows that oxIL-33 directly binds 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 directly bind to EGFR (although it does bind to the known EGFR ligand, EGF). However, when RAGE is added to this assay in combination with oxIL-33, EGFR binding is observed. [Figure 8] Figure 8 shows Western blots of EGFR, RAGE, and IL-33 in wild-type and RAGE-deficient A549 cells after immunoprecipitation with anti-EGFR or anti-RAGE, followed by activation with oxIL-33 at the indicated time points. [Figure 9] Figure 9 shows that STAT5 phosphorylation induced by oxIL-33-01 is reduced by anti-RAGE antibodies but not by anti-ST2 antibodies. [Figure 10]Figure 10 shows that EGF and oxidized IL-33 (oxIL-33) induce EGFR clustering and internalization in EGFR-GFP A549 cells. Representative images are shown after 5 minutes of stimulation. The histograms show EGFR depletion in the non-clustered regions of cells treated with EGF and oxIL-33 (left shift of the bell-shaped peak in the histogram), and an increase in the number of saturated pixels (intensity 255) in these cells caused by clustering. [Figure 11] Figure 11 shows the doubling of IL-8 secretion by NHBES and DHBE 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. The bar graphs show the mean and SEM from four NHBE and three DHBE donors. [Figure 12A] Figure 12A shows the relative wound healing density of A549 cells after treatment with reduced IL-33, oxIL-33, or EGF. The bar graphs show the mean and SEM results from six technical replications for each condition. [Figure 12B] Figure 12B shows the relative wound healing density of NHBE cells after treatment with reduced IL-33, oxIL-33, or EGF. The bar graphs show the mean and SEM values ​​from six technical replications for each condition. [Figure 13] Figure 13 shows the scratch wound closure rates 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. The bar graphs show the mean and SEM results from six technical replications for each condition. [Figure 14] Figure 14 shows the relative wound healing density in human bronchial epithelial cells from healthy subjects, smokers, and COPD patients, with and without stimulation by oxidized IL-33. [Figure 15]Figure 15 shows the 24-hour wound closure (%) of DHBE COPD cells (n=5 donors) and NHBE cells (n=5 donors) compared to DHBE cells treated with IgG1 control, anti-IL-33 (33_640087-7B), anti-RAGE (M4F4), and anti-ST2. The bar graph shows the mean and SEM from each of the n=5 individual donors. [Figure 16A] Figure 16A shows typical 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 healthy donors. [Figure 16B] Figure 16B shows the quantification of immunohistochemistry of various epithelial cell types using HALO software after 7 days of treatment with anti-IL-33 (33_640087-7B) or isotype control antibody. The data shown are mean and SEM from n=2–3 individual donors. [Figure 16C] Figure 16C shows the quantification of goblet cells using HALO software after treatment with anti-IL-33 (33_640087-7B) or isotype control antibody for 7 days. The data shown are the mean and SEM from individual donors (n=2–3). [Figure 17] Figure 17 shows examples of staining of individual mucins (mucin 5AC and mucin 5B) in ALI cultures derived from healthy (heathy) (1 donor) or COPD (1 donor), and the reduction in mucin staining in COPD cultures after 7 days of treatment with anti-IL-33 (33_640087-7B). [Figure 18] Figure 18 shows a tSNE plot illustrating the different proportions of cell subtypes observed in COPD ALI cultures from individual donors treated with anti-IL-33 compared to untreated cultures. [Figure 19A]Figure 19A shows a representative flow cytometry contour plot for 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 protein 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. The 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), representing 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, similar to IL-13. The violin plot shows all data points and medians. [Figure 19C] Figure 19C shows combined flow cytometry data from ALI cultures from normal donors (n=6) exhibiting Muc5AC single-positive goblet cells. Reduced IL-33 (IL-33[C->S]) did not increase goblet cells above baseline. Oxy IL-33 (oxidized IL-33-01) increased the percentage of goblet cells, similar to IL-13. The violin plot shows all data points and medians. [Figure 19D]Figure 19D shows combined RT-qPCR data from ALI cultures from normal donors (n=4) showing a magnification change in MUC5AC mRNA. Reduced IL-33 (IL-33[C->S]) did not increase MUC5AC mRNA. Oxygenated IL-33 (oxidized IL-33-01) increased MUC5AC mRNA, similar to IL-13. The violin plot shows all data points and medians. [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. Oxygenated IL-33 (oxidized IL-33-01) caused a visual increase in goblet cells. [Figure 20B] Figure 20B shows the quantification of mucin 5ac + mucin 5b area (% 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 21A] Figure 21A shows a representative flow cytometry contour plot for detecting goblet cells in ALI cultures from COPD donors. Muc5B is plotted on the x-axis and Muc5AC on the y-axis. ALI cultures were treated with antibody for 7 days. Anti-IL-33 (33_640087-7B) treatment reduced the total number of goblet cells. The 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) representing total goblet cells (combined Muc5AC single-positive, Muc5B single-positive, and Muc5AC and Muc5B double-positive goblet cells). ALI cultures were treated with antibody for 7 days. Anti-IL-33 (33_640087-7B) treatment reduced the total goblet cell count. The violin plot shows all data points and medians. [Figure 21C] Figure 21C shows combined flow cytometry data derived from ALI cultures from COPD donors (n=6) exhibiting 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. The violin plot shows all data points and medians. [Figure 21D] Figure 21D shows combined RT-qPCR data from ALI cultures derived from COPD donors (n=5) showing catalytic changes in MUC5AC mRNA. Anti-IL-33 (33_640087-7B) treatment reduced MUC5AC expression. The violin plot shows all data points and the median. [Figure 21E] Figure 21E shows combined flow cytometry data derived from ALI cultures from COPD donors (n=6), indicating overall survival across all treatment conditions, as determined by LD-negative cell staining. [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. Seven-day treatment with anti-IL-33 (33_640087-7B) induced a visual reduction in goblet cells. [Figure 22B]Figure 22B shows the quantification of Muc5ac+Muc5b area (% 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 23A] Figure 23A shows the quantification of Muc5AC in apical wash hydrate obtained from COPD and healthy ALI cultures. Muc5AC levels are higher in COPD cultures, as determined by Muc5AC ELISA. [Figure 23B] Figure 23B shows the quantification of Muc5AC in apical wash obtained from healthy ALI cultures. ALI cultures were treated with reduced IL-33mut16 (IL-33[C->S]), oxIL-33, and wild-type IL-33 before being measured by Muc5AC ELISA. [Figure 23C] Figure 23C shows the quantification of Muc5AC in apical wash 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. [Examples]

[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), the applicant described the discovery of an oxidized disulfide-bound form of IL-33 (DSB IL-33), showing that this form does not bind to ST2 and cannot activate ST2-dependent signaling. Subsequently (see International Publication No. 2016156440A1 pamphlet), the applicant showed that oxIL-33 binds to the receptor for advanced glycation end products (RAGE), signals RAGE-dependently, activates STAT5, and affects epithelial migration.

[0280] To further investigate the function of oxIL-33, epithelial cells were stimulated with reduced or oxidized IL-33, and the signaling pathways were examined. In this specification, we demonstrate that oxIL-33 is a novel ligand for the complex of the receptor for advanced glycation end products (RAGE) and the epidermal growth factor receptor (EGFR), and significantly influences epithelial function.

[0281] 1. Cloning and expression of human maturation and cysteine ​​variants of IL33 cDNA molecules encoding mature components 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 include 10xHis, Avitag, and a factor Xa protease cleavage site (MHHHHHHHHHHAAGLNDIFEAQKIEWHEAAIEGR SEQ ID NO: 43) at the N-terminus of the protein. By transforming Escherichia coli (E. coli) BL21 (DE3) cells, 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 prepared. The 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. The cells were resuspended in 2× DPBS containing cOmplete EDTA-free protease inhibitor cocktail tablets (Roche, 11697498001) and 50 U / mL of benzonase nuclease (Merck Millipore, 70746-3), and lysed by ultrasonic treatment. The cell lysates were clarified by centrifugation at 50,000×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 2× DPBS and 1 mM DTT at 5 mL / min. The column was washed with 2× DPBS, 1 mM DTT, 20 mM imidazole, pH 7.4 to remove impurities, and then washed with 2× DPBS and 0.1% TritonX-114 to remove endotoxin-immobilized proteins.After further washing with 2×DPBS, 1 mM DTT, 20 mM imidazole, and pH 7.4, the samples were eluted with 2×DPBS, 1 mM DTT, 400 mM imidazole, and 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 with 2×DPBS. Peak fractions were analyzed by SDS-PAGE. The fractions containing pure IL-33 were pooled, and their concentration was measured by absorbance at 280 nm. The final samples were analyzed by SDS-PAGE.

[0282] To prepare untagged IL-33, N-terminally tagged His10 / Avitag IL33 was incubated with 10 units of factor Xa per 1 mg of protein in 2× DPBS buffer (GE Healthcare, 27084901) at room temperature for 1 hour. Untagged IL-33 was purified by SEC chromatography in 2× DPBS using a HiLoad Superdex 75pg column (GE Healthcare, 28989333) at a flow rate of 1 mL / min.

[0283] 2. Preparation and purification of oxidized IL-33 (oxIL-33) Reduced IL-33-01 was oxidized by diluting it to a final concentration of 0.5 mg / mL in 60% IMDM medium (without phenol red) and 40% DPBS, and incubating at 37°C for 18 hours. Aggregates formed during the oxidation process were removed from the sample by loading it onto a HiTrap Capto Q ImpRes anion exchange column (GE Healthcare, 17547055). Before loading, the sample was prepared by adding 1 M Tris, pH 9.0 until the pH reached 8.3, and then adding 5 M NaCl to a final concentration of 125 mM. Under these loading conditions, aggregates and monomer oxIL-33 bound to the column flowed and were recovered without binding. Tags were cleaved from oxIL-33 by incubating 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, a soluble human ST2S extracellular domain fused to human IgG1 Fc-His6 was incubated with the sample at 22°C for 30 minutes to conjugate the reduced IL-33. The sample was concentrated in a 3,000 Da cutoff centrifuge 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. The fraction containing pure oxIL-33 was 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 evaluated by SDS-PAGE, HP-SEC, and RP-HPLC.

[0284] 3. Cloning, expression, and purification of human ST2ECD cDNA encoding the naturally occurring ST2S soluble isoform of ST2 (UniProt accession Q01638-2), which lacks the endogenous signal peptide (amino acid residues 19-328), was amplified by PCR using a primer encoding extension compatible with Gibson assembly and a CD33 signal peptide fused to the N-terminus of the ST2S coding sequence. The coding sequence of human IgG1Fc with a His6 tag at the C-terminus was similarly amplified. The ST2S cDNA and IgG1Fc-His6 cDNA were assembled using Gibson assembly with pDEST12.2OriP, a mammalian CMV promoter-driven expression vector with an OriP replication origin from EBV, enabling episome maintenance of cell lines expressing EBNA-1 protein. For protein expression, the plasmid was primary transformed into a suspension culture of CHO cells overexpressing EBNA-1 using polyethyleneimine as the transfection reagent. Conditional 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 2× DPBS, and the protein was eluted with 25 mM sodium acetate, pH 3.6. The fraction containing ST2S-Fc-His6 was pooled and loaded onto a HiLoad Superdex 200 26 / 600pg column (GE Healthcare, 28989336) equilibrated with 2× DPBS at 2 mL / min. The fraction containing pure ST2S-Fc-His6 protein was pooled, and its concentration was measured 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 P07306), which lacks a cytoplasmic domain and transmembrane domain (amino acid residues 62-291), was chemically synthesized in Geneart using a CD33 signal peptide fused to the N-terminus of the subsequent ECD domain with a His10_Avi tag sequence. The construct was directly cloned into pDEST12.2OriP, a mammalian CMV promoter-driven expression vector with an OriP replication origin from EBV, enabling episome maintenance in cell lines expressing EBNA-1 protein. For protein expression, the plasmid was primary transformed into a suspension culture medium of HEK Freestyle 293F cells using 293-fectin as the transfection reagent. Conditional media containing the secreted HisAVi_hASGPR ECD fusion protein were collected 7 days after transfection by immobilized metal affinity chromatography and loaded onto a HisTrap Excel column (GE Healthcare, 17371205) equilibrated with 2×DPBS at 4 mL / min. The column was washed with 2×DPBS, 40 mM imidazole, pH 7.4 to remove impurities, and the sample was eluted with 2×DPBS, 400 mM imidazole, pH 7.4. 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 with 2×DPBS. Peak fractions were analyzed by SDS-PAGE. Fractions containing pure monomer ASGPR were pooled, and their concentration was measured 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 was harvested with oxidase (PAA, #L1 1-007) and 1 × 10⁶ cells were added to 6 well dishes (Corning Costar, 3516) of culture medium [BEGM (Lonza CC-3171) and supplement kit (Lonza CC-4175)]. 6 Cells were seeded in 2 mL of medium. The cells were incubated at 37°C in 5% CO2 for 18–24 hours. After this period, the medium was aspirationed, the cells were washed twice with 1 mL of PBS, and then starvation medium (BEGM (Lonza CC-3171) supplemented with 1% penicillin / streptomycin) was added. The plates were then incubated for a further 18–24 hours at 37°C in 5% CO2 before stimulation.

[0287] The MAP kinase phosphorylation antibody array kit (ab211061) was purchased from Abcam, and the experiment was performed according to the manufacturer's instructions. Six well dishes of NHBE, starved for 18–24 hours, were left untreated or treated with 30 ng / mL of reduced IL-33, IL-33-16, or oxidized IL-33, and then returned to an incubator at 37°C and 5% CO2 for 10 minutes (see Table 2 for the activators used in this assay). After removing the plates from the incubator, the cells were washed with ice-cold PBS, and 100 μL of 1× lysis buffer provided in the kit was added per well. Protein extracts were transferred to 1.5 mL tubes and clarified at 14,000 rpm at 4°C. Protein concentration was measured using the BCA method (Thermo, 23225), with 250 μg of total protein per array membrane. All subsequent steps were performed according to the manufacturer's instructions. The membrane was visualized using 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 IL-33 (IL33-01 and IL33-16, respectively), oxidized IL33-01 (oxIL-33) activated several key signaling molecules consistent with receptor tyrosine kinase (RTK)-mediated pathways (Figure 1).

[0290] 6. Oxidized IL-33 activates the epidermal growth factor receptor (EGFR). Screening was performed using a 71RTK array to identify receptor tyrosine kinases (RTKs) activated by oxIL-33. The RTK phosphorylation antibody array kit (ab193662) was purchased from Abcam, and the experiment was performed according to the manufacturer's instructions. NHBE was cultured and 1 × 10⁶ cells were placed in a 6-well plate (Corning Costar, 3516) of culture medium [BEGM (Lonza CC-3171) and supplement kit (Lonza CC-4175)]. 6 Seeds were seeded in 2 mL of solution. Cells were incubated at 37°C in 5% CO2 for 18-24 hours. After this time, the culture medium was aspirationed, the cells were washed twice with 1 mL of PBS, and then starvation medium (BEGM (Lonza CC-3171) without supplementation kit) was added. The plates were then incubated for a further 18–24 hours at 37°C and 5% CO2 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 carried out according to the manufacturer's instructions. The membranes were visualized with LiCor C-digit and quantified using Image Lite Studio. No response was detected for 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) induced 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 further confirmed. After activation, EGFR was phosphorylated with Tyr1068, and this phospho-EGFR could be detected using a homogeneous FRET (fluorescence resonance energy transfer) HTRF® (homogeneous time-resolved fluorescence, Cisbio International) assay (Cisbio kit #64EG1PEH). In summary, NHBE was detected in 5 × 10⁶ wells of culture medium [BEGM (Lonza CC-3171) and supplement kit (Lonza CC-4175)] in a 96-well plate (Corning Costar, 3598). 5 Plates were plated with 100 μL of medium. Plates were incubated at 37°C and 5% CO2 for 18–24 hours. After this time, the medium was aspirationed, the cells were washed twice with 0.2 mL of PBS, and then starvation medium (BEGM (Lonza CC-3171) without supplement kit) was added. The plates were then incubated for a further 18–24 hours at 37°C and 5% CO2, stimulated with increasing concentrations of IL-33-01, IL-33-16, and oxIL-33 (oxidized IL-33-01), as well as EGFR ligands (Tables 2 and 3), and returned to the incubator at 37°C and 5% CO2 for 10 minutes. The medium was aspirationed 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). EnVision plate reader (Perkin Time-resolved fluorescence was read at emission wavelengths of 620 nm and 665 nm using Elmer. The data was analyzed by calculating the 665 / 620 nm ratio, and the EC50 value was determined by curve fitting using the four-parameter logistic equation with GraphPad Prism software.

[0292] [Table 7]

[0293] Similarly, EGFR phosphorylation was evaluated in epithelial cell line A549 using the HTRF assay, as described above in this section. In summary, A549 cells were obtained from ATCC and cultured in RPMI GlutaMax medium supplemented with 1% penicillin / streptomycin and 10% FBS. Cells were harvested with acutase (PAA,#L1 1-007) and 5 × 10⁶ cells were collected. 5 Cells were seeded in 96-well plates at 100 μL 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 incubated 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 ligand, and RAGE ligand (Tables 2 and 3), and then returned to the incubator at 37°C and 5% CO2 for 10 minutes. The medium was aspirationed 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 emission wavelengths of 620 nm and 665 nm using an EnVision plate reader (Perkin Elmer). The data was analyzed by calculating the 665 / 620 nm ratio, and the EC50 value was determined by curve fitting using a four-parameter logistic equation with GraphPad Prism software.

[0294] In both NHBE and A549 cells, oxIL-33 promoted EGFR phosphorylation as well as the true agonist EGF (Figure 3). This was not replicated by other RAGE ligands tested.

[0295] 7. Western blot of signaling components Western blotting experiments were performed to further investigate which elements of the EGFR signaling complex are activated in response to oxIL-33 (oxidized IL-33-01). NHBE cells 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 minutes. The medium was then aspired, the cells were washed with ice-cold PBS, and 150 μL of lysis buffer [1× 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)] was added. After the cells were placed on ice for 10 minutes, the lysate was transferred to a 1.5 mL tube and heated at 90°C for 5 minutes. The solution was transferred to a new 1.5 mL tube, and 10 μL of the sample and 5 μL of protein ladder (BioRad, 1610374) were electrophoresed on a 4–12% SDS-PAGE gel (Thermo, NW04127BOX) in MES running buffer (B0002). The gel was transferred to a PVDF membrane (BioRad, 1704156) using Transblot Turbo (BioRad). The PVDF membrane was blocked for 10 minutes with PBS-tween solution containing 5% skim milk powder (Marvel). The membrane was then incubated overnight at 4°C with primary antibody in PBS-tween containing 5% BSA. The membrane was then washed five times with PBS-tween and incubated at room temperature for 1 hour with secondary HRP-tagged antibody in PBS-tween containing 5% skim milk powder. Next, the membrane was washed five times with PBS-tween, and then ECL (BioRad, 1705062) was added to visualize the Licor C-digit.

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

[0297] 8.ox-IL-33 induces STAT-5 phosphorylation, which is blocked by EGFR neutralizing agents. Next, it was sought to determine whether oxIL33-mediated STAT5 activation could be inhibited by blocking binding to EGFR. In summary, A549 cells were cultured in RPMI GlutaMax medium supplemented with 1% penicillin / streptomycin and 10% FBS. Cells were harvested with acutase and 5 × 10⁶ cells were extracted. 5 Seeds were seeded in 100 μL of PBS into 96-well plates 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 incubated at 37°C and 5% CO2 for 18–24 hours. Anti-EGFR antibodies (clone LA1 (05-101, Millipore) or isotype control (MAB002, R&D Systems)) were added to the wells in a dose-dependent manner, and the plates were returned to the incubator for 30 minutes. The plates were then stimulated with oxidized IL-33 (30 ng / mL) for 30 minutes, lysed using phosho-STAT5 ELISA kit lysis buffer (85-86112-11, ThermoFischer Scientific), and spread according to the manufacturer's instructions, with absorbance read at 450 nM. As shown in Figure 5, cells activated with oxIL-33-01 showed phosphorylation of STAT5, which decreased in the presence of anti-EGFR antibodies (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, immunoprecipitation experiments were performed to investigate the signaling complex. First, an anti-EGFR antibody was covalently bound 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 bound according to the manufacturer's instructions. After successful coupling, the beads were resuspended in 30 mg / mL PBS and maintained at 4°C.

[0299] Obtain NHBE from Lonza (CC-2540), and freeze vials into 15cm dishes (Thermo, 157150), 1 x 10 per dish. 6The cells were directly seeded. The NHBE cells were maintained in complete BEGM medium (Lonza) for one month according to the manufacturer's protocol, with the medium changed every three days until the cells reached confluence. During this time, the plates were incubated at 37°C and 5% CO2. The day before stimulation, the plates were washed twice with 20 mL of PBS, and then 15 mL of starvation medium (BEGM (Lonza CC-3171) without supplement kit) was added. The plates were then incubated at 37°C and 5% CO2 for a further 18–24 hours, after which they were stimulated with medium alone (unstimulated control), 30 ng / mL of reduced IL-33-01, 30 ng / mL of oxidized IL-33-01, or 30 ng / mL of EGF, and returned to 37°C and 5% CO2 for 10 minutes. The medium was aspirationed, the plates were washed twice with ice-cold PBS, and then 1 mL of lysis buffer (Abcam, ab152163) containing a phosphatase and protease inhibitor (Thermo, 78440) was added per 15 cm dish. After scraping the cells into the lysis buffer, 2 mL of Protein The protein was transferred to a LoBind tube (Eppendorf, Z666513) and clarified by rotating 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 with lysis buffer. 6 mg of total protein extract was incubated with 100 μL of anti-EGFR Dynabeads (above) in a clear 2 mL LoBind tube. The tube was then placed in an inverted mixer at 4°C for 5 hours. The Dynabeads were immobilized using a magnet (BioRad, 1614916), the protein extract was aspirationed, and washed in 2 mL of wash buffer 1 (50 mM Tris-HCl pH 7.5 (Thermo, 15567027), 0.5% TritonX The beads were replaced with 100 (Sigma, X100) and 0.3 M NaCl. This was repeated at least four times. The beads were then washed 10 more times in the same manner with washing buffer 2 (50 mM Tris-HCl pH 7.5). After the final washing step, 50 μL of 1% Rapidest (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.Next, the supernatant was transferred to a new LoBind 2 mL tube. Further, 100 μL of 50 mM Tris-HCl pH 8.0 was added to the resin and mixed, then combined with the initial eluate. Next, TCEP (Sigma, 646547) was 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 in the dark at room temperature for 20 minutes. Alkylation was quenched by adding DTT (Sigma, D5545) to 10 mM. Finally, 50 mM Tris-HCl buffer pH 8.0 was 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 400 rpm shaking platform. Next, the sample was acidified with trifluoroacetic acid (Sigma, 302031) to a final concentration of 2.0% (v / v) and incubated at 37°C for 1 hour. The sample was then centrifuged at 14,000 rpm for 30 minutes, and the supernatant was transferred to a new 2 mL LoBind tube. The sample was then processed through a C18 column (Thermo, 87784) according to the manufacturer's instructions. The sample was then dried using speed-vac and stored at -20°C. The sample was then analyzed by peptide mass fingerprinting mass spectrometry (PMF-LC-MS). The results were analyzed using Scaffold software.

[0300] EGFR was detected similarly under 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 were 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 the 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 after stimulation with oxIL-33, forming complexes with EGFR, but are not detected after stimulation with reduced IL-33-01 (IL-33) or EGF. The number in parentheses indicates the number of unique peptides identified for each protein.

[0302] [Table 8]

[0303] To confirm these observations, immunoprecipitation and Western blotting were also performed on cell lysates prepared according to the protocol described above. After measuring the NHBE protein extract concentration, 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 in an inverted mixer at 4°C for 2.5 hours. Then, 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 another hour while 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% Triton X, and 0.25 M NaCl) and once with 500 μL of 10 mM Tris (pH 7.5). Next, 35 μL of LDS sample buffer (Thermo, NP0008) containing a reducing agent (Thermo, NP0004) was heated at 95°C for 5 minutes to release the protein from the magnetic beads. The solution was transferred to a new 1.5 mL tube, and 10 μL of the sample and 5 μL of protein ladder (BioRad, 1610374) were electrophoresed on a 4-12% SDS-PAGE gel (Thermo, NW04127BOX) in MES running buffer (B0002). The gel was transferred to a PVDF membrane (BioRad, 1704156) using Transblot Turbo (BioRad). The PVDF membrane was blocked for 10 minutes in PBS-tween solution containing 5% skim milk powder (Marvel). Next, the membranes were incubated overnight at 4°C 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. Then, the membranes were washed five times with PBS-tween, and subsequently incubated for 1 hour at room temperature in PBS-tween containing 5% skim milk powder with anti-rabbit secondary HRP-tagged antibody (Cell Signaling Technology, 7074) or anti-goat secondary HRP-tagged antibody (R&D Systems, HAF109).Next, the membrane was washed five times with PBS-tween, and then ECL (BioRad, 1705062) was added to visualize the Licor C-digit. Western blotting confirmed that RAGE co-precipitated with EGFR in the presence of oxIL-33, but RAGE was not detected upon EGF stimulation (Figure 6). These findings reveal that RAGE and EGFR are functional components of the oxidized IL-33 signaling complex.

[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 and contributes to downstream signaling. The experiments in this section are designed to determine whether oxIL-33 is a direct binding ligand for either RAGE or EGFR. To further understand the formation of the signaling complex and to assess whether oxIL-33 directly interacts with EGFR, we examined the binding of oxIL-33 to RAGE, ST2-Fc, and EGFR using ELISA.

[0305] Proteins and Modifications: Biotinylated proteins containing the Avitag sequence motif (GLNDIFEAQKIEWHE SEQ ID NO: 46) using the 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] A streptavidin plate (Thermo Scientific, AB-1226) was coated with 100 μL / well of biotinylated antigen (10 μg / mL in PBS) at room temperature for 1 hour. The plate was 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 plate was 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 at room temperature for 1 hour. Alternatively, 100 μL of EGFR-Fc (R&D Systems #344-ER-050) in PBS at a concentration of 10 μg / mL in PBS was added for 1 hour, either in the presence or absence of 10 μg / mL of untagged RAGE (Sino Biological, 11629-HCCH) in PBS. The plate was washed three times with 200 μL of PBS-T. Then, RAGE-Fc, ST2-Fc, and EGFR-Fc were detected at room temperature for 1 hour using 100 μL / well of anti-human IgG HRP (Sigma AO170, 5.1 mg / mL) diluted 1:10000 in blocking buffer. The plate was washed three times with PBS-T and colored with TMB, 100 μL / well (Sigma, T0440). The reaction was quenched with 0.1 M H2SO4 in 50 μL / well. The absorbance at 450 nm was read using CytationGen5 or a similar instrument. The results showed that oxIL-33 exhibited a clear interaction with RAGE (Figure 7A), but direct binding of oxIL-33 to EGFR was minimal (Figure 7B). EGFR binding to oxIL-33 was observed only by adding sRAGE to this assay (Figure 7B). This could not be replicated when oxIL-33 was used in place of the true RAGE agonist HMGB1 (Figure 7B).

[0308] The necessity of RAGE in oxIL-33-induced EGFR signaling was further confirmed using RAGE-deficient cell lines. The RAGE knockout A549 cell line was generated as follows:

[0309] A mammalian plasmid containing a red fluorescent protein (RFP) expression vector, a guide RNA targeting exon 3 of AGER (TGAGGGGATTTTCCGGTGC SEQ ID NO: 47), and Cas9 endonuclease was prepared. A549 condition medium was prepared by growing A549 cells for 2 days in a T-175 flask with F12K nut mix (Gibco, 10% FBS and 1% penicillin / streptomycin supplemented). Used medium was removed from A549, filtered, and diluted 5-fold with fresh Gibco F12K nut mix (20% FBS and 1% penicillin / streptomycin supplemented). A549 was then divided into three T-75 flasks, 2 × 10⁶. 5Cells were seeded in a total of 15 mL of medium at a rate of cells / mL and placed overnight in a 37°C, 5% CO2 incubator. 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. Next, 0.75 mL of the transfection mixture was added to each T-75 flask. The flasks were returned to the incubator for 2 days. Then, A549 cells were detached using accutase and 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 supplied with conditional medium every 3–5 days. When more than 50% of the cells were confluent, they were transferred to 24-well plates for growth. This upscaling process was continued until each successful clone was divided into T15 flasks. The cells were then divided into 12-well plates and grown until more than 50% confluent, after which genomic PCR was analyzed to determine successful knockout. Cells were lysed in 100 μL of DNA lysis buffer per well (Viagen Bitoech, 301-C, supplemented with 0.3 μg / mL proteinase K). These samples were incubated at 55°C for 4 hours, followed by incubation at 85°C for 15 minutes. RAGE PCR was performed using forward and reverse primers with the following sequences: forward - gttgcagcctcccaacttc (SEQ ID NO: 48), reverse - aatgaggccagtggaagtca (SEQ ID NO: 49). The reaction and cycles were set up 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]. The PCR reaction was performed with an initial denaturation at 98°C for 30 seconds, followed by 35 cycles of 5 seconds at 98°C, 10 seconds at 57°C, and 20 seconds at 72°C, and then a final step of 2 minutes at 72°C.4 μL of PCR product was mixed with 6 μL of nuclease-free water and 2 μL of 6× DNA loading buffer (Thermo Scientific, R0611). The sample was electrophoresed on a 1% agarose gel (1:10000 SYBR safe) at 90V for 1 hour and then visualized with a Versadoc Imager. The remainder of the PCR product was then cleaned up using the QIAquick PCR purification kit (Qiagen, 28104) according to the manufacturer's protocol. DNA-50 concentration was measured using nanodrop. Several clones (selected from the results) were sent for in-house sequencing. The results showed successful stop codon insertion in clones RAGE09 and RAGE10.

[0310] To confirm the essential role of RAGE in oxIL-33-mediated EGFR signaling, immunoprecipitation and Western blotting were then performed on A549 cells and RAGE-deficient A549 cells. In summary, cell lines were activated at various time points (0–15 mins) using oxIL-33-01. Following immunoprecipitation of EGFR or RAGE, Western blotting with anti-RAGE, anti-EGFR, and anti-IL-33 was performed according to the relevant experimental protocols detailed in Section 9. The results demonstrate the crucial role of RAGE in the formation of complexes 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, blocking antibodies were tested. In summary, A549 cells were cultured in RPMI GlutaMax medium supplemented with 1% penicillin / streptomycin and 10% FBS. Cells were harvested with acutase and 5 × 10⁶ cells were extracted. 5The cells were seeded in 100 μL of PBS into a 96-well plate 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 incubated at 37°C and 5% CO2 for 18–24 hours. Anti-RAGE (M4F4; International Publication No. 2008137552); anti-ST2 (AF532; RnD Systems); or isotype control (MAB002, R&D Systems) was added to the wells in a dose-dependent manner, and the plates were returned to the incubator for 30 minutes. Next, the plates were 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 at 450 nM was read. As shown in Figure 9, cells activated with oxIL-33-01 showed STAT5 phosphorylation, which decreased 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. Next, we investigated whether oxIL-33 induces changes in EGFR dynamics compared to EGF.

[0313] 12. Confocal experiments on EGF internalization This experiment aims to investigate the dynamics of EGFR in epithelial cells after stimulation with EGF, reduced or oxidized IL-33, using confocal imaging. 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) at a rate of 1 mL per 24-well glass-bottom plate (Greiner, 662892). EGF receptors linked to green fluorescent protein (GFP) allow 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 1:5000 dilution. Cells were briefly stained with CellMask before membrane marking and live-imaged at 1 frame / min immediately after confocal processing to record EGFR-GFP dynamics. Cells were stained at 37°C for 5 minutes, 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 / well. Confocal images were immediately acquired. 40x oil objective lens, 1 min / frame, 5 stacks at 2 μm intervals for 25 minutes (approximately 30 minutes after protein addition). The disordered (dotted line) pattern of the GFP signal indicates receptor clustering and internalization on the membrane. Pixel intensity histograms for membrane regions (masked with CellMask) and intracellular regions (masked with inverse CellMask, not shown) were created at different time points than live imaging and showed EGFR depletion in non-clustered regions (left shift of bell-shaped peaks in the histogram) and an increased number of saturated pixels caused by clustering (intensity 255). oxIL-33 induced clustering and internalization of EGF receptors, but EGF stimulation resulted in the most evident EGFR clustering. In contrast, reduced IL-33 (IL-33-16) did not show significant changes in EGFR cell 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) (DHBE; Lonza 00195275) were maintained in complete BEGM medium (Lonza) for one month according to the manufacturer's protocol, with the medium changed every three days until the cells reached confluence. Cells were harvested with accutase and placed in 5 × 10⁶ well plates (Corning 3596) in culture medium. 5 Cells were seeded in 100 μL of medium. Plates were incubated at 37°C and 5% CO2 for 18–24 hours. After this period, the medium was aspirationed, and the cells were washed twice with 100 μL of PBS. Then, starvation medium (BEGM (Lonza CC-3171) without a supplementation kit, supplemented with 1% penicillin / streptomycin) was added. The plates were then incubated at 37°C and 5% CO2 for a further 18–24 hours. After that, the plates were stimulated with medium only (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 and 5% CO2. 24 hours after stimulation, the supernatant was collected and subjected to a multiplex assay (Mesoscale Discovery). Chemokine production was evaluated using K15047D-2). As shown in Figure 11, NHBE and DHBE showed a four-fold increase in IL-8 secretion when activated with oxIL-33 compared to unstimulated cells (culture medium only). No significant differences were observed with other chemokines (TARC, MIP-1a, MIP1b, 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. In contrast to EGF, oxIL-33 impairs the closure of scratch wounds by 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 acutase (PAA, #L1 1-007) and 5 × 10⁶ cells were collected. 5 Cells were seeded in 100 μL of 96-well plates 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 incubated at 37°C and 5% CO2 for 18–24 hours. Cells were scratched using WoundMaker® (Essen Bioscience), followed by the washing of the wells twice with 200 μL of PBS, and then RPMI GlutaMax medium supplemented with 0.1% FBS (v / v) and 1% (v / v) penicillin / streptomycin, containing the indicated stimuli: medium only (unstimulated control), 30 ng / mL reduced IL-33-01, 30 ng / mL oxidized IL-33-01, or 30 ng / mL EGF, and incubated back to 37°C and 5% CO2. The plates were placed in 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.

[0316] NHBE (CC-2540) was obtained from Lonza and maintained in complete BEGM medium [BEGM (Lonza CC-3171) and supplement kit (Lonza CC-4175)] according to the manufacturer's protocol. Cells were harvested with accutase and placed in 5 × 10⁶ wells of a 96-well ImageLock plate (Sartorius, 4379) in culture medium. 5Cells were seeded in 100 μL of medium. Plates were incubated at 37°C and 5% CO2 for 18–24 hours. After this time, the medium was aspirationed, and the cells were washed twice with 100 μL of PBS, followed by the addition of BEGM (Lonza CC-3171) without a supplement kit, supplemented with 1% penicillin / streptomycin (Steptomycin). The plates were then incubated at 37°C and 5% CO2 for a further 18–24 hours before scratch wound treatment. Cells were scratched using WoundMaker® (Essen Bioscience), and the wells were washed twice with 200 μL of PBS, followed by the indicated stimuli: medium only (unstimulated control), 30 ng / mL reduced IL-33-01, 30 ng / mL oxidized IL-33-01, or 30 ng / mL BEBM medium (Lonza) supplemented with 0.1% FBS (v / v) and 1% (v / v) penicillin / streptomycin, containing EGF, was added and returned to 37°C and 5% CO2. The plates were placed in IncucyteZoom for 48 hours for imaging and analysis of wound healing. Relative wound density was calculated via the wound healing algorithm in the IncucyteZoom software. As shown in Figure 12, oxIL-33 inhibited wound healing in deep cultures of A549 cells (Figure 12A) and NHBE cells (Figure 12B), having the opposite effect to EGF, which resulted in an increase in wound cell density.

[0317] 15. Impairment of scratch wound closure by oxidized IL-33 can be prevented by antibodies that neutralize RAGE or EGFR, but not by ST2. To understand whether these functional effects of oxIL-33 are mediated via RAGE / EGFR, scratch assays were performed in 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, International Publication No. 2008137552), or anti-EGFR (clone LA1, 05-101 Millipore). oxIL-33 inhibited scratch closure, but reduced IL-33 did not. This effect of oxIL-33 was reversed by anti-RAGE and anti-EGFR, but not by anti-ST2, which also indicates 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 deep culture. 16.oxIL-33 may induce COPD-like reactions in healthy NHBE patients in scratch wound closure assays. Next, the effects of oxidized IL-33 on bronchial epithelial cells from healthy individuals, smokers, and COPD patients were investigated. NHBE (CC-2540), NHBE (CC-2540) from smokers, and DHBE (COPD, 00195275) were obtained from Lonza and maintained in complete BEGM medium (Lonza) according to the manufacturer's protocol. Scratch assays were performed as described in Section 14. Cells were treated with medium alone (unstimulated control) or with 30 ng / mL of oxidized IL-33. Bronchial epithelial cells from smokers or COPD patients showed impaired scratch closure ability similar to the impairment observed after treating healthy cells with oxIL-33, compared to cells from healthy controls (Figure 14). In contrast to healthy cells, oxIL-33 did not further impair the scratch wound closure response in smokers and COPD HBE cells (Figure 14).

[0319] 17. Blocking endogenous IL-33 via the RAGE / EGFR pathway may improve impaired scratch wound repair phenotype in COPD basal cells. Since epithelial cells are known to produce IL-33, autocrine IL-33 secretion may be responsible for the impaired scratch wound repair phenotype observed in COPD cells. To investigate this, a scratch closure assay was performed in 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 acutase and placed in 5 × 10⁶ wells of a 96-well ImageLock plate (Sartorius, 4379) in culture medium. 5Cells were seeded in 100 μL of medium. Plates were incubated at 37°C and 5% CO2 for 18-24 hours. After this time, the medium was aspirationed, and the cells were washed twice with 100 μL of PBS, followed by the addition of BEGM (Lonza CC-3171) without a supplement kit, supplemented with 1% penicillin / streptomycin (Steptomycin). Then, before scratch wounds, the plates were incubated for a further 18-24 hours at 37°C and 5% CO2. Cells were scratched using WoundMaker® (Essen Bioscience), and the wells were washed twice with 200 μL of PBS, followed by the addition of 10 μg / mL of anti-IL-33 (33_640087-7B, described in International Publication No. 2016 / 156440 brochure), anti-ST2 (AF532, R&D BEBM medium (Lonza) supplemented with 0.1% FBS (v / v) and 1% (v / v) penicillin / streptomycin, containing either anti-RAGE (Systems), anti-RAGE (M4F4, International Publication No. 2008137552), or NIP228 (IgG1 isotype control), was added and returned to 37°C and 5% CO2. Plates were placed in IncucyteZoom for 48 hours for imaging and analysis of wound healing. Relative wound density was calculated via the wound healing algorithm in the IncucyteZoom software. As previously observed, COPD cells exhibited impaired scratch closure response compared to cells derived from healthy subjects. Anti-IL-33 and anti-RAGE could 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, which is signaled via the RAGE / EGFR pathway (Figure 15), whereas anti-ST2 did not.

[0320] Example 7 - Anti-IL-33 reduces goblet cells in 3D epithelial cultures. 18. Airway basal cell culture at the gas-liquid interface (ALI) Next, the inventors attempted to determine the relevance of IL-33 signaling in gas-liquid interface cell culture ("ALI culture"). ALI culture is a method of growing basal cells on a basal surface in contact with the culture medium and an upper (apical) cell layer exposed to air. ALI culture enables the development of three-dimensional cellular structures in vitro using the mucosal ciliary phenotype of pseudostratified epithelium, similar to tracheal epithelium. Therefore, ALI culture can be used to study fundamental aspects of respiratory epithelium, such as intercellular signaling, disease modeling, and respiratory regeneration.

[0321] Cryovials of frozen lung basal cells from healthy controls or COPD patients were obtained from the University of North Carolina and the University of Pittsburgh. The cells were thawed and plated into T-75 flasks coated with Purecol Type I bovine collagen (Advanced BioMatrix, San Diego, CA) diluted 1:70 in 1×PBS (Gibco, Waltham, MA), and grown in Epix medium (276-201, Propagenix, Rockville, MD). After reaching confluence, these cells were divided into an appropriate number of T-75 flasks and then harvested for ALI culture. Transwells for ALI culture, containing 12 mm, 0.4 μM polyester membrane inserts (Costar, Corning, NY), were prepared by coating the inserts with 1:70 Purecol solution and incubating at 37°C for 1–16 hours. After removing the Purecol solution, the transwells were placed under UV light for 30 minutes and then washed with PBS. Basal cells in 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 with a ViCell counter (Beckman Coulter, Brea, CA), and spun down at 1,000 RPM for 5 minutes. Next, the cells were transferred to Pneumacult ALI medium (Stemcell Tech, Vancouver, BC) in a 3.57 × 10⁶ layer. 5The cells were resuspended at a density of / mL and 700 μL was dispensed into each transwell. 1 mL of ALI medium was added to the space below the insert. The cells were left immersed in the 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 2 weeks, with the medium changed every other day at the basal side. Fully differentiated cultures were treated for 7 days with either no antibody, 1 μg / mL anti-IL-33 (33_640087-7B), or 1 μg / mL NIP228 (IgG1 isotype control) by treatment agent in the medium supplied to the basal side of the culture. The medium was changed every other day (including any associated treatments).

[0322] 19. IHC triple staining (basal layer, cup, and ciliary layer) 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 serial 3-plex chromogenic assay using Ventana Discovery Ultra. Antigen was recovered using Cell Conditioner 1 (CC1) (catalog no. 5424569001, Roche), and endogenous peroxidase was blocked with Discovery Inhibitor (catalog no. 7017944001, Roche) for 12 minutes. Anti-p63 (clone 4A4) (catalog number 790-4509, Roche, Basel, Switzerland) was applied at 36°C for 24 minutes, visualized with mouse anti-HQ (12 mins) (catalog number 7017782001, Roche) and anti-HQ HRP (12 mins) (catalog number 7017936001, Roche), and incubated on Teal substrate (catalog number 8254338001, Roche) for 12 minutes. The slides were treated with an antibody denaturation step using Cell Conditioner 2 (CC2) (catalog number 5424542001, Roche) (24 minutes at 100°C), and then antitubulin (catalog number ab24610, Abcam, Cambridge, UK) was diluted to 0.01 μg / mL in Dako antibody diluent (catalog number S3022) for 16 minutes. This was detected using mouse OmniMap-HRP (8 minutes) (catalog number 5269652001, Roche) and visualized using Discovery Purple substrate (catalog number 7053983001, Roche) for 16 minutes. The slides were subjected to additional antibody denaturation with CC2, and then a cocktail of rabbit anti-mucin 5AC 1.1 μg / mL and rabbit anti-mucin 5B 7 μg / mL (catalog numbers ab198294 and ab87376, Abcam, respectively) was applied for 20 minutes. Visualization was performed with anti-rabbit NP (4 mins) (catalog number 7425317001, Roche), anti-NP-AP (8 mins) (catalog number 7425325001, Roche), and then with Discovery Yellow (catalog number 7698445001, Roche) for 20 minutes.Stained slides were rinsed with Dawn detergent, counterstained with hematoxylin (catalog no. 5277965001, Roche), rinsed, dehydrated with a stepwise series of ethanol and xylene, and mounted in permanent mount medium. Quantification using HALO software showed 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 mucin and improves mucus motility 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 by serial double immunofluorescence assay with Ventana Discovery Ultra. Antigens were recovered using Cell Conditioner 1 (Ultra CC1). Endogenous peroxidase was blocked with Discovery Inhibitor for 12 minutes, and for 8 minutes of that block, it was blocked with S Block (RUO) Roche Diagnostics (catalog no. 760-4212). The samples were incubated at 36C for 24 minutes with anti-mucin 5B diluted with Dako Ab Diluent, S3022 at a concentration of 7 μg / mL, and detected with anti-rabbit HQ (Roche Diagnostics catalog no. 760-4815) for 4 minutes and anti-HQ-HRP (Roche Diagnostics catalog no. 760-4820) for 8 minutes. The samples were then incubated with the tyramide conjugate Discovery FITC (Roche Diagnostics catalog no. 760-232) for 8 minutes. Dual sequencing was selected in the Discovery Ultra program, and the samples were treated with Cell Conditioner 2 (CC2) in an antibody denaturation step (100°C for 24 minutes), followed by neutralization with Discovery Inhibitor (40°C for 24 minutes), after which anti-mucin 5AC, 1.1 μg / mL, was added at 36°C for 20 minutes. 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 the 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.The samples were incubated with 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI nucleic acid staining), Thermo Fisher catalog number D1306, and diluted to 1 μg / mL with deionized water for 2 minutes. The samples were rinsed with deionized water, and coverslips were placed on ProLong Gold Antifade mount medium (Thermo Fisher, catalog number P36930) and stored in a light-shielding slide box. The stained slides were imaged using a Zeiss LSM 880 confocal microscope (Carl Zeiss Microscopy, LLC, White Plains, NY). Figure 17 shows that anti-IL-33 treatment of ALI cultures may lead to downregulation of various mucins in COPD cultures.

[0324] 21. Anti-IL-33 reverses impaired myxociliary clearance observed in COPD ALI cultures. ALI cultures from COPD donors were prepared 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. Short videos of FluoSphere movement were captured using a Zeiss LSM800 microscope, showing increased myxociliary motility after treatment with anti-IL-33 (33_640087-7B), but not with 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 filter by washing up and down with PBS pipetting and then transferred to 15 mL Falcon tubes. 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. Cell suspensions were loaded according to the standard protocol for the Chromium Single Cell 3' Kit, capturing 5000–10000 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), followed by sequencing using NextSeq500 or NovaSeq6000 instruments (Illumina). Initial data processing was performed using the Cell Ranger version 2.0 pipeline (10x Genomics). Post-processing was performed using the Seurat package to eliminate and normalize low cell quality. Each sample was analyzed as independent data to capture heterogeneity within the sample (cell subtyping). Clustering and visualization were performed using t-distribution stochastic neighbor embedding (tSNE). Identification of COPD cell clusters was guided by marker genes. For other samples, the initial cluster was manually examined, and then the Seurat label transfer algorithm was applied to identify subtypes. MUC5AC and MUC5B gene expression analysis was performed for each cluster of cells from COPD ALI cultures with and without anti-IL-33 treatment, as described in Section 18. Heatmaps and tSNE plots were created using Seurat. Figure 18 shows a tSNE plot illustrating the different proportions of cell subtypes observed in ALI cultures treated with anti-IL-33 (33_640087-7B) compared to untreated cultures.As shown in Figure 18, a decrease in MUC5B hypercellularity was observed after anti-IL-33 treatment.

[0326] Example 10 - Anti-IL-33 reduces goblet cells in COPD 3D epithelial cultures. 22. Airway basal cell culture at the gas-liquid interface (ALI) To quantify and investigate the effects of oxIL-33 in physiologically relevant gas-liquid interface (ALI) culture systems, a flow cytometry assay was developed to distinguish between goblet cell types (MUC5ac vs. MUC5b) and the remaining epithelial cells (mucin-negative).

[0327] Cryovials of frozen lung basal cells from healthy (CC-2540) controls or COPD (195275) patients were received from Lonza. One vial per donor was thawed and plated in 4xT-175 flasks of Epix medium (276-201, Propagenix, Rockville, MD). After reaching confluence, these cells were frozen at P2 at 1e6 cells per vial. The P2 cells were placed in 2xT-75 flasks of Epix medium and grown until 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 with dH2O) and incubating at 37°C for 1–16 hours. The collagen I solution was removed and the transwell was washed with PBS. The basal 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 placed in Pneumacult ALI medium (Stemcell Tech, Vancouver, BC) in 8 × 10⁶ units.5 The cells were resuspended at a density of / mL and 0.5mL was dispensed into each 12mm transwell and 0.25mL into each 6.5mm transwell. 1mL of ALI medium was added to the space below the 12mL insert and to the 0.5mL space below the 6.5mm insert. The cells were left immersed in the 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 3 weeks, with the medium being changed at the basal side every Monday, Wednesday, and Friday. Fully differentiated normal cultures were left untreated or treated for 7 days with reduced or oxidized untagged IL33-01 (30ng / mL), untagged IL33-16 (30ng / mL), IL-13 (10ng / mL), EGF (30ng / mL), or HMGB1 (30ng / mL) by using a treatment agent 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 the following agents in the culture medium supplied to the basal side: 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. Medium changes were performed every Monday, Wednesday, and Friday (including related treatments).

[0328] [Table 10]

[0329] 23. FACS analysis of goblet cells in ALI cultures Following a 7-day 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 (transwell surface) of each transwell and incubated for 30 minutes. The apical wash was stored at -80°C for mucin analysis. 150 μL of trypsin (Lonza Trypsin Subculture Pack - #CC-5034) was added to both the apical and basal outer (below the transwell) compartments. The transwells were returned to the incubator for 30 minutes. ALI was separated by gently pipetting the trypsin up and down. 150 μL of trypsin neutralization 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 the plate was centrifuged at 1200 RPM at 4°C for 5 minutes. 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 at 4°C for 5 minutes to remove the live-dead dye, and 200 μL of PBS was added to each well. The plate was centrifuged at 1200 RPM at 4°C for 5 minutes to remove the PBS, 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 at 4°C for 5 minutes, and the solution was removed. The cells were resuspended in 300 μL of 1× permeabilization solution (Thermo 00-8333). 5e4 cells from each well were added to a new 96-well U-bottom plate, centrifuged at 1200 RPM at 4°C for 5 minutes, and the cells were resuspended in 50 μL of 1× permeabilization solution. 50 μL of antibody staining cocktail (1:400 anti-Muc5AC and 1:800 anti-Muc5B) or isotype staining cocktail of the same dilution. The plate was incubated on ice in the dark for 30 minutes. The plate was centrifuged at 1200 RPM at 4°C for 5 minutes, and the solution was removed.The cells were washed with PBS, centrifuged, and then resuspended in 150 μL of PBS. Data were then acquired using BD FACSymphony® and analyzed using FlowJo software.

[0330] [Table 11]

[0331] 24. qPCR / bulk RNA sequencing analysis of ALI cultures Following a 7-day 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 wash was stored at -80°C. ALI cultures were lysed and RNA was extracted using the MagMAX®-96 Total RNA Isolation Kit (Thermo, AM1830). Then, cDNA was synthesized using the RNA with the High-Capacity RNA-to-cDNA® Kit (Thermo, 4388950). Thus, 9 μL of each RNA sample was incubated with 10 μL of 2XRT buffer mix and 1 μL of 20XRT enzyme mix in PCR tubes (Thermo, AM12230), placed in a thermocycler, and incubated at 37°C for 60 minutes. The reaction was stopped by heating at 95°C for 5 minutes and then maintaining 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 plates were sealed, briefly centrifuged, and then analyzed using a QuantStudio® 7Flex real-time PCR system (Thermo). Next, the delta-delta-ct was calculated by normalizing the data to an untreated, normal control.

[0332] oxIL-33 led to an increase in goblet cell count, particularly a subset of MUC5AC+ goblet cells, while reduced IL-33 did not (Figure 19A-C). Therefore, MUC5AC mRNA copies increased upon treatment with oxIL-33, as determined by qPCR (Figure 19D).

[0333] 25. IHC triple staining (basal, cup, 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; Airway Basal Cell Gas-Liquid Interface (ALI) Cultures: 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 by a serial triple-chromogenic assay with Ventana Discovery Ultra. Antigen recovery was performed with Cell Conditioner 1 (Ultra CC1) (catalog no. 5424569001, Roche), and endogenous peroxidase was blocked with Discovery Inhibitor (catalog no. 7017944001, Roche) for 12 minutes. Anti-p63 (clone 4A4) (catalog number 790-4509, Roche, Basel, Switzerland) was applied for 24 minutes, visualized with anti-mouse HQ (12 minutes) (catalog number 7017782001, Roche) and anti-HQ HRP (12 minutes) (catalog number 7017936001, Roche), and incubated with Discovery Purple kit (catalog number 07053983001, Roche) for 12 minutes. The slides were treated with an antibody denaturation step using Cell Conditioner 2 (Ultra CC2) (catalog number 5424542001, Roche) (92°C for 24 minutes), and then antitubulin (catalog number ab24610, Abcam, Cambridge, UK) was diluted with Dako antibody diluent (catalog number S3022) for 16 minutes (concentration on the slide: 0.003 μg / mL). This was detected using mouse OmniMap-HRP (8 min) (catalog number 5269652001, Roche) and visualized using the Discovery Teal HRP kit (catalog number 82544338001, Roche).The slides were subjected to additional antibody denaturation with CC2, and then a cocktail of rabbit anti-mucin 5AC 1.1 μg / mL (dispenser concentration) and rabbit anti-mucin 5B 7 μg / mL (dispenser concentration) (catalog numbers ab198294 and ab87376, Abcam, respectively) was applied for 20 minutes. Visualization was performed using anti-rabbit NP (4 mins) (catalog number 7425317001, Roche), anti-NP-AP (8 mins) (catalog number 7425325001, Roche), and then Discovery Yellow (catalog number 7698445001, Roche) for 20 minutes. The stained slides were counterstained with hematoxylin II (8 min) (catalog no. 5277965001, Roche) and Bluing's reagent (4 min) (catalog no. 5266769001, Roche), rinsed with dish soap, dehydrated in a stepwise series of ethanol and xylene, and mounted in permanent mount medium.

[0334] IHC images were analyzed using HALO v3.1 (Indica Labs) and initially manually annotated to exclude out-of-focus areas and tissue damage. A random forest classifier was trained to recognize epithelium and separate it from the transmembrane and glass slide backgrounds. For ciliated area quantification, a separate 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 CytoNuclear 2.0.9 algorithm was used to segment cells based on nuclear staining and further detect basal cells by counting p63-positive nuclei. All quantification methods were validated against human recognition and achieved 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 the role of oxIL-33 in promoting goblet cell differentiation within lung epithelium. This suggests that epithelium chronically exposed to ox-IL-33 evolves towards a goblet cell hyperplasia phenotype that negatively impacts lung function.

[0337] 26. Reversal of COPD goblet cell phenotype by blocking agents A key feature of COPD is excessive mucus due to increased goblet cells and mucus secretion (Gohy et al 2019 Sci Rep 9:17963). To investigate whether oxidized IL-33 can play a direct role in the goblet cell COPD phenotype, we established information by reading ALI cultures from COPD donors, 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 resulted in a decrease in MUC5AC+ goblet cell count (Figures 21A-D). No treatment affected the viability of the ALI cultures (Figure 21E), confirming that the treatment phenomena were not due to artificial substances or antibody toxicity. Consistent with previous results, anti-ST2 treatment did not result in a decrease in goblet cell count, providing further evidence that the disease phenotype is directly mediated by IL-33, primarily ox-IL-33, via the oxIL-33-RAGE-EGFR pathway. The effect of anti-IL-33 antibody (33-640087_7B) on COPD ALI cultures was further confirmed by immunohistochemical analysis, and blocking IL-33 resulted in a decrease in goblet cell count by pairwise analysis (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 the apical mucus from both healthy and COPD ALI cultures were measured using ELISA. To quantify the mucin released from ALI cultures, the apical supernatant was analyzed for MUC5AC levels by immunoassay (Novus NBP2-76703) according to the manufacturer's protocol. Samples were diluted 1:2000 with sample diluent, and concentrations were estimated from the recombinant MUC5AC protein standard curve. As shown in Figure 23, ALI cultures from COPD patients showed increased MUC5AC release levels 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 blocking with anti-IL-33 (33_640087_7B), which inhibits MUC5AC protein levels released from ALI cultures, but not with anti-ST2 or isotype mAb controls (Figure 23C).

[0340] Overall, this example highlights the role of oxidized IL-33 in dysregulation of lung epithelial cell differentiation. The results suggest that, when uncontrolled, oxidized IL-33 may be responsible for goblet cell hyperplasia and the 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 significant therapeutic effects on COPD patients by restoring normal epithelial physiological function, for example, by reducing goblet cell count and reducing excessive mucus production.

[0341] additional array In addition to the sequences listed in Table 1, the following additional CDR sequences are provided: Sequence ID 37: SYAMS Sequence ID 38: GISAIDQSTYYADSVKG Sequence ID 39: QKFMQLWGGGLRYPFGY Sequence ID 40: SGEGMGDKYAA Sequence ID 41: RDTKRPS Sequence ID 42: GVIQDNTGV N-terminal His10 / Avitag / Factor Xa protease cleavage site Sequence ID 43: MHHHHHHHHHHAAGLNDIFEAQKIEWHEAAIEGR IL-33-01 Sequence ID 44: [ka] IL-33-16 Sequence ID 45: [ka] Avitag array motif Sequence ID 46: GLNDIFEAQKIEWHE gRNA vector targeting RAGE exon 3 Sequence ID 47: TGAGGGGATTTTCCGGTGC RAGE Forward Primer Sequence ID 48: gttgcagcctcccaacttc RAGE Reverse Primer Sequence ID 49: aatgaggccagtggaagtca Human ST2S (signal peptide is underlined) Sequence ID 50: [ka] Human ST2S-huIgG1 Fc-His6 (signal peptide is underlined) Sequence ID 51: [ka] Sequence ID 52: His10 / Avitag Human ASGPR ECD (Signal peptide is underlined, tag is double underlined) [ka]

Claims

1. A pharmaceutical composition for the prevention or treatment of respiratory diseases by inhibiting RAGE-EGFR-mediated signaling, wherein the pharmaceutical composition comprises an anti-IL-33 antibody or its antigen-binding fragment, and the respiratory disease is selected from COPD, bronchitis, emphysema, bronchiectasis including CF-bronchiectasis and non-CF-bronchiectasis, asthma, or overlap of asthma and COPD (ACO), and is characterized by abnormal EGFR activity.

2. The pharmaceutical composition according to claim 1, wherein the respiratory disease is a lower respiratory disease.

3. The pharmaceutical composition according to claim 2, wherein the lower respiratory disease is a respiratory disease of the bronchi.

4. The pharmaceutical composition according to claim 1, wherein the respiratory disease is COPD or asthma.

5. The pharmaceutical composition according to claim 4, wherein the COPD is bronchitis-induced COPD, or the asthma is bronchial asthma.

6. The aforementioned prevention or treatment a. To improve mucus clearance and / or inhibit or reduce abnormal mucus production; b. Inhibit or reduce MUC5AC production; c. To inhibit abnormal mucus composition and / or abnormal epithelial remodeling; d. Inhibit abnormal goblet cell differentiation or proliferation; and / or e. Reduce the thickness of the respiratory epithelium and / or MUC5AC in the entire tissue region of the epithelium. ＋ Reduce the number of goblet cells. A pharmaceutical composition according to any one of claims 1 to 5.

7. The inhibition of the abnormal mucus composition includes reducing the MUC5AC:MUC5B ratio, inhibiting or reducing MUC5AC in the mucus, and / or reducing the mucus concentration; and / or The aforementioned inhibition of abnormal goblet cell differentiation or proliferation results in abnormal MUC5AC ＋ This includes inhibiting goblet cell differentiation or proliferation. The pharmaceutical composition according to claim 6.

8. A pharmaceutical composition for the prevention or treatment of diseases characterized by abnormal EGFR activity and abnormal epithelial physiological function, by modulating or inhibiting the RAGE-EGFR-mediated effect, comprising an anti-IL-33 antibody or its antigen-binding fragment, wherein the abnormal epithelial physiological function is an abnormal mucociliary physiological function selected from increased mucus production, increased goblet cell differentiation, increased goblet cell proliferation, increased epithelial thickness, decreased mucus clearance, and / or abnormal mucus composition.

9. The pharmaceutical composition according to claim 8, wherein the abnormal mucociliary physiological function is the abnormal mucociliary physiological function of the respiratory epithelium.

10. Increased mucus production includes increased MUC5AC production; and / or increased goblet cell differentiation includes MUC5AC + increased goblet cell differentiation; and / or increased goblet cell proliferation includes MUC5AC + increased goblet cell proliferation; and / or increased epithelial thickness includes the increased amount of MUC5AC in the entire tissue area of ​​the epithelium. ＋ The pharmaceutical composition according to claim 8, comprising goblet cells.

11. The pharmaceutical composition according to claim 8, wherein the abnormal mucus composition includes an increase or decrease in the ratio of various mucus compounds contained in the mucus; an increase or decrease in one or more mucus compounds; and / or an increase or decrease in the concentration or density of the mucus.

12. The pharmaceutical composition according to claim 11, wherein the abnormal mucus composition includes an increase in the ratio of MUC5AC:MUC5B; and / or the abnormal mucus composition includes an increase in the amount of MUC5AC contained in the mucus; and / or the abnormal mucus composition includes an increase in the concentration of the mucus.

13. The pharmaceutical composition according to claim 8, wherein the abnormal epithelial physiological function is abnormal epithelial remodeling.

14. The pharmaceutical composition according to claim 8, wherein the abnormal epithelial physiological function is an abnormal epithelial physiological function of the airway.

15. The pharmaceutical composition according to claim 14, wherein the airway is the lower airway.

16. The pharmaceutical composition according to claim 15, wherein the lower respiratory tract is a bronchus.

17. The pharmaceutical composition according to any one of claims 1 to 16, wherein the anti-IL-33 antibody or its antigen-binding fragment inhibits the activity of oxidized IL-33.

18. The pharmaceutical composition according to any one of claims 1 to 17, wherein the anti-IL-33 antibody or its antigen-binding fragment prevents the binding of oxidized IL-33 to RAGE, thereby inhibiting RAGE-EGFR signaling.

19. The pharmaceutical composition according to any one of claims 1 to 18, wherein the anti-IL-33 antibody or its antigen-binding fragment is an anti-reducing IL-33 antibody or its antigen-binding fragment.

20. The pharmaceutical composition according to any one of claims 1 to 19, wherein the antibody or antigen-binding fragment thereof comprises VHCDR1 having the sequence of SEQ ID NO: 37, VHCDR2 having the sequence of SEQ ID NO: 38, VHCDR3 having the sequence of SEQ ID NO: 39, VLCDR1 having the sequence of SEQ ID NO: 40, VLCDR2 having the sequence of SEQ ID NO: 41, and VLCDR3 having the sequence of SEQ ID NO:

42.

21. The pharmaceutical composition according to claim 20, wherein the antibody or its antigen-binding fragment comprises VH and VL, the VH having an amino acid sequence corresponding to SEQ ID NO: 1, and the VL having an amino acid sequence corresponding to SEQ ID NO: 19.

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