Methods for reducing respiratory infections

Abstract

The present disclosure relates to methods for reducing infections, particularly respiratory viral infections, for example in subjects with COPD, the methods comprising the use of IL-33 antagonists, particularly oxIL-33 antagonists.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 323,742, filed March 25, 2022, which is incorporated by reference in its entirety for all purposes.

[0002] REFERENCE TO ELECTRONICALLY SUBMITTED SEQUENCE LISTING This application incorporates by reference the sequence listing submitted herewith in computer readable format (CRF) as a text file entitled "IL33-207-US-PSP-SequenceListing", created on February 17, 2023, and having a size of 19,729 bytes.

[0003] The present disclosure relates to methods for reducing or preventing infection, specifically respiratory tract viral infection (RTVI), in a subject with an IL-33 mediated respiratory disorder, such as in a subject with COPD. [Background technology]

[0004] Chronic obstructive pulmonary disease (COPD) exacerbations are a major source of patient suffering and a major cause of morbidity and mortality, as well as hospitalization. Respiratory tract infections, specifically viral infections, appear to be responsible for approximately half of all COPD exacerbations. Only one quarter of exacerbations appear to be unrelated to infection. Summary of the Invention [Problem to be solved by the invention]

[0005] Preventing COPD exacerbations is an important part of COPD management. Therefore, strategies to prevent infections that lead to exacerbations are desirable. These not only have a significant effect on the morbidity of COPD, but can also improve the quality of life of subjects with COPD. [Means for solving the problem]

[0006] Subjects with COPD are particularly susceptible to airway infections that lead to acute exacerbations of COPD. Club cells are an important secretory cell type in the respiratory epithelium that provides various cellular defense functions. Inhibition of club cell activity has been directly implicated in the increased susceptibility of airway epithelium to infections, such as respiratory syncytial virus (RSV) infection. RSV infection is one of many respiratory tract viral infections (RTVI) known to lead to acute exacerbations of COPD (AECOPD) (Wedzicha Proc Am Thorac Soc Vol 1. pp115-120,2004). This disclosure relates to the discovery that oxidized IL-33 (IL-33ox) attenuates club cell activity in COPD epithelium. Examples show that blocking IL-33ox activity directly restores club cell activity in COPD air-liquid interface (ALI) cultures. Thus, the present disclosure suggests that blocking the activity of oxIL-33 in subjects with COPD can enhance the activity of club cells and reduce infections that lead to acute exacerbations of COPD. Such methods in a clinical setting can have a significant impact on the morbidity of COPD and improve quality of life.

[0007] Thus, in one aspect, the disclosure provides an IL-33 antagonist for use in a method of treatment to reduce or prevent a respiratory tract infection in a subject having an IL-33-mediated respiratory disorder. In some cases, the IL-33-mediated respiratory disorder is chronic obstructive pulmonary disease (COPD). In some cases, the infection is a respiratory tract viral infection or a respiratory tract bacterial infection.

[0008] In some cases, the infection is a respiratory tract viral infection (RTVI). In some cases, the respiratory tract viral infection is caused by influenza virus (e.g., influenza virus A, influenza virus B), respiratory syncytial virus (RSV), adenovirus, metapneumovirus, cytomegalovirus, parainfluenza virus (e.g., hPIV-1, hPIV-2, hPIV-3, hPIV-4), rhinovirus, adenovirus, coxsackievirus, echovirus, coronavirus, herpes simplex virus, SARS-coronavirus, or smallpox.

[0009] In some cases, IL-33 antagonists inhibit the activity of IL-33ox, thereby increasing club cell activity in the airway epithelium.

[0010] In some cases, IL-33 antagonists inhibit the activity of IL-33ox, thereby increasing the total club cell area in the airway epithelium.

[0011] In some cases, the IL-33 antagonist inhibits the activity of oxIL-33, thereby increasing the mRNA expression level of one or more markers selected from SCGB1BA1, BPIFA1, SCGB3A1, WFDC2, MSMB, LTF, SLPI, C3, HLA-DRA, CXCL1, CD74, CXCL17, MDK, TGM2, HLA-DRB1, CXCL8, CXCL2, HLA-DRB5, CX3CL1, and HLA-DPA1 in the airway epithelium. In some cases, the one or more markers are selected from SCGB1BA1, BPIFA1, SCGB3A1, WFDC2, MSMB, and LTF. In some cases, the one or more markers include SCGB1BA1 and / or BPIFA1.

[0012] In some cases, IL-33 antagonists inhibit the activity of oxIL-33, thereby inhibiting the expression of CCSP, SCGB3A1, WFDC2, beta-microseminoprotein, lactotransferrin, SPLUNC1, secretory leukocyte protease inhibitor (SLPI), complement C3, HLA-DR alpha chain, CXC motif chemokine ligand 1 (CXCL1), cluster of differentiation 74 (CD74), CXC motif chemokine 17 (CXCL17), midkine (MDK), gamma-glutamine protein-1 (GGG-1) in airway epithelia. The method further comprises increasing the protein expression level of one or more markers selected from transmembrane tyrosine kinase 2 (TGM2), HLA class II histocompatibility antigen, DRB1 beta chain (HLA-DRB1), chemokine (CXC motif) ligand 8 (CXCL8), chemokine (CXC motif) ligand 2 (CXCL2), HLA class II histocompatibility antigen, DRB5 beta chain (HLA-DRB5), chemokine (C-X3-C motif) ligand 1 (CX3CL1), and major histocompatibility complex class II, DP alpha 1 (HLA-DPA1). In some cases, the one or more markers are selected from CCSP, SCGB3A1, WFDC2, beta-microseminoprotein, lactotransferrin, and SPLUNC1. In some cases, the one or more markers include CCSP and / or SPLUNC1.

[0013] In some cases, the respiratory epithelium includes lower respiratory tract epithelium, such as cuboidal epithelium or squamous epithelium. In some cases, the respiratory epithelium includes upper respiratory tract epithelium, such as ciliated pseudostratified columnar epithelium.

[0014] In some cases, the IL-33 antagonist inhibits the activity of IL-33ox, thereby increasing the defensive function of club cells in the airway epithelium. In some cases, increasing the defensive function of club cells in the airway epithelium includes increasing the activity of one or more proteins selected from CCSP, SCGB3A1, WFDC2, beta-microseminoprotein, lactotransferrin, SPLUNC1, SLPI, C3, HLA-DRA, CXCL1, CD74, CXCL17, MDK, TGM2, HLA-DRB1, CXCL8, CXCL2, HLA-DRB5, CX3CL1, and HLA-DPA1. These proteins have been implicated in the defensive function of the epithelium. Thus, increasing their expression by inhibiting the activity of oxIL-33 is likely to improve resistance to respiratory infections.

[0015] In some cases, the method reduces the annual exacerbation rate in the subject. In some cases, the method reduces the frequency of acute exacerbations of COPD (AECOPD) in the subject.

[0016] In some cases, the IL-33 antagonist is an IL-33ox antagonist.

[0017] In some cases, the antagonist is an antibody or antigen-binding fragment thereof. In some cases, the antibody or antigen-binding fragment thereof specifically binds to reduced IL-33 (redIL-33).

[0018] In some cases, the anti-IL-33 antibody or antigen-binding fragment thereof comprises a VH domain comprising an HCDR1 having the sequence set forth in SEQ ID NO: 1, an HCDR2 having the sequence set forth in SEQ ID NO: 2, and an HCDR3 having the sequence set forth in SEQ ID NO: 3, and a VL domain comprising an LCDR1 having the sequence set forth in SEQ ID NO: 5, an LCDR2 having the sequence set forth in SEQ ID NO: 6, and an LCDR3 having the sequence set forth in SEQ ID NO: 7. In some cases, the anti-IL-33 antibody is tozolaximab.

[0019] In another aspect, the disclosure provides a composition comprising an IL-33 antagonist disclosed herein for use in the therapeutic methods disclosed herein.

[0020] In another aspect, the disclosure provides a therapeutic method for reducing or preventing a respiratory infection in a subject having an IL-33-mediated respiratory disorder, the method comprising administering to the subject a therapeutically effective amount of an IL-33 antagonist.

[0021] In another aspect, the disclosure provides the use of an IL-33 antagonist for use in the manufacture of a medicament for the treatment of reducing or preventing a respiratory infection in a subject having an IL-33-mediated respiratory disorder.

[0022] In another aspect, the disclosure provides an IL-33 antagonist for use in reducing AECOPD in a subject having COPD, the IL-33 antagonist inhibiting the activity of IL-33ox, thereby reducing respiratory tract infection and AECOPD in the subject. [Brief description of the drawings]

[0023] [Figure 1] Schematic representation of ALI cultures and endpoint assays. [Diagram 2] Volcano plot representing differential expression of genes from bulk RNA sequencing in ALI cultures treated with IL-33ox versus untreated controls. [Diagram 3] A visual representation of the changes in the proportions of cell types in ALI cultures following treatment with IL-33ox compared to untreated controls. [Figure 4] Heatmap showing the mean expression levels of scale-normalized genes associated with mucin production or protection in the secretory state in ALI cultures treated with IL-33ox or untreated controls. [Diagram 5]Representative immunohistochemistry of COPD ALI cultures after treatment with IL-33 neutralizing antibody (tozolaximab) or hIgG1 isotype control antibody: MUC5AC / AB for goblet cells (yellow), acetylated α-tubulin for ciliated cells (cyan), and p63 for basal cells (purple) (scale bar = 70 μm). [Figure 6] Flow cytometric analysis of intracellular MUC5AC to quantify the percentage of MUC5AC single-positive goblet cells in dissociated healthy or COPD ALI cultures following treatment with tozolaximab, ST2 neutralizing, or relevant isotype control antibodies, or untreated controls (n=6 healthy, n=5 COPD). [Figure 7] ELISA of secreted MUC5AC in the apical region of healthy or COPD ALI cultures incubated with tozolaximab, ST2 neutralization, or relevant isotype control, or untreated controls (n=7 healthy, n=6 COPD). [Figure 8] Volcano plot representing differential expression of genes from bulk RNA sequencing in COPD ALI cultures treated with IL-33 neutralizing antibody (tozoraximab). [Figure 9] Heatmap showing changes in gene expression levels in COPD ALI cultures following treatment with hIgG1 isotype control antibody or tozolaximab by gene family. [Figure 10] [Figure 10A] Heatmap showing the mean expression levels of scale-normalized genes associated with mucin production or protection in the secretory state in ALI cultures of COPD treated with tozolaximab (MEDI3506) or untreated controls. [Figure 10B] Heatmap showing the mean expression levels of scale-normalized additional genes associated with protection in the secretory state in ALI cultures of COPD treated with tozolaximab (MEDI3506) or untreated controls. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] General definition "Abnormal", as used herein, refers to a difference in function compared to the function in a healthy subject, typically an increase or decrease in function compared to the function in a healthy subject.

[0025] "Abnormal epithelial physiology" as used herein means any abnormality in the function of epithelium in the human body. The functions of epithelium in the human body include: functioning as a barrier to protect the tissue underneath; regulation and exchange of chemicals between tissues and cavities; secretion of chemicals into cavities; and sensation. Abnormality in any of these functions can have devastating physiological effects. Because epithelia are present in a wide range of tissues in the body, including the skin, respiratory tract, gastrointestinal tract, reproductive tract, urinary tract, exocrine glands, and endocrine glands, abnormalities in epithelium can be involved in a wide range of diseases or pathologies. In some cases, the epithelium is an airway epithelium, and the abnormal epithelial physiology is abnormal airway epithelial physiology.

[0026] "Antibody" is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0027] "Antigen-binding fragment" and "binding fragment" refer to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', F(ab')2, Fab'-SH, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antigen-binding fragments. For a review of specific antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthuen, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994). See also WO 93 / 16185, and U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments that contain salvage receptor binding epitope residues and have increased in vivo half-lives, see U.S. Patent No. 5,869,046. Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003). Single domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain, or all or part of the light chain variable domain, of an antibody.Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies as well as production by recombinant host cells (eg, E. coli or phages).

[0028] "Club cells", also known as bronchiolar exocrine cells and formerly known as Clara cells, are low columnar / cuboidal cells with short microvilli found primarily in the small airways (bronchioles) of the lungs. Club cells are found in simple ciliated epithelium. One of the main functions of club cells is to protect the bronchiolar epithelium, which they do by, for example, secreting club cell secreted protein (CCSP, also known as uteroglobin, CC10, or CC16: UniProtKB accession number P11684). CCSP is presumed to have an anti-inflammatory function, which has been strongly implicated in regulating the inflammatory response to infection. Wang et al. demonstrated that CCSP null mice infected with RSV had increased viral persistence, pulmonary inflammation, and airway responsiveness (Wang et al The Journal of Immunology, 2003, 171:1051-1060). Therefore, increasing CCSP expression in the small airways may reduce or prevent viral infections, such as RSV infection. CCSP is encoded by the gene SCGB1A1.

[0029] As used herein, "IL-33" protein refers to interleukin 33, specifically mammalian interleukin 33 protein, e.g., the human protein deposited under UniProt number 095760. IL-33 is not a single species, but exists as reduced and oxidized forms. Reduced IL-33 undergoes rapid oxidation in vivo, e.g., within a time frame of 5 to 40 minutes. The terms "IL-33" and "IL-33 polypeptide" are used interchangeably. In certain cases, IL-33 is full-length. In other cases, 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 forms of IL-33, including but not limited to aa 72-270, 79-270, 95-270, 99-270, 107-270, 109-270, 111-270, 112-270, may have enhanced activity (Lefrancais 2012, 2014).

[0030] "Oxidized IL-33", "oxIL-33", or "IL-33ox" refers to a form of IL-33 that binds to RAGE and initiates RAGE-EGFR mediated signaling. It has previously been shown that activation of the IL-33ox-RAGE / EGFR pathway drives pathogenic changes in lung epithelial composition (as disclosed in WO2021 / 089563, which is incorporated by reference in its entirety). Oxidized IL-33 refers to a protein that appears as a characteristic band, for example by Western blot analysis under non-reducing conditions, in particular having a mass 4 Da less than the corresponding reduced form. Specifically, it refers to a protein with one or two disulfide bonds between cysteines independently selected from cysteines 208, 227, 232 and 259.

[0031] "Ox-IL-33 / RAGE / EGFR signaling axis" or "oxIL-33 signaling axis (acis)" refers to the RAGE / EGFR signaling pathway that is activated by oxIL-33 binding to the RAGE / EGFR signaling complex on the surface of epithelial cells.

[0032] "Reduced IL-33," "redIL-33," or "IL-33red," as used herein, refers to a form of IL-33 that binds to ST2 and initiates ST2-mediated signaling. Specifically, in the reduced form, cysteines 208, 227, 232, and 259 are not disulfide bonded.

[0033] Reference to "WT IL-33" or "IL-33" can refer to either the reduced or oxidized form, or both, unless it is clear from the context in which it is used that one of the forms is intended.

[0034] "IL-33 antagonist" refers to a molecule that inhibits the interaction of an IL-33 axis binding partner with one or more of its binding partners. An IL-33 antagonist can be an IL-33red antagonist, an IL-33ox antagonist, or an antagonist that inhibits both IL-33red and IL-33ox. In addition to IL-33ox antagonists, the present disclosure also contemplates the use of "oxIL-33 signaling axis antagonists," including RAGE and EGFR antagonists, which are receptors that complex with IL-33ox to mediate oxIL-33 signaling. As a result, antagonizing the activity of RAGE and / or EGFR can also be useful in inhibiting the pathological oxIL-33 signaling mechanisms disclosed herein.

[0035] "IL-33-mediated disorder" refers to a disease or disorder in which IL-33 has been shown to have a pathological role. In the present disclosure, IL-33-mediated disorders of the respiratory tract are specifically contemplated. These may also be referred to as IL-33-mediated respiratory disorders. Particular cases relate to IL-33-mediated respiratory disorders characterized by abnormal epithelial physiology. Such disorders include COPD, asthma, COPD overlap syndrome (ACOS), chronic bronchitis, bronchiectasis, and emphysema.

[0036] "COPD exacerbation" or "COPD exacerbation" refers to an increase in the severity and / or frequency and / or duration of one or more symptoms or signs of COPD. "COPD exacerbation" also includes any decline in a subject's respiratory health that requires and / or is treatable by therapeutic intervention (e.g., steroid therapy, antibiotic therapy, inhaled corticosteroid therapy, hospitalization, etc.). In some cases, a moderate exacerbation is defined as an acute exacerbation of COPD (AECOPD) event that requires either systemic corticosteroids (such as intramuscular, intravenous, or oral) and / or antibiotic therapy. In some cases, a severe exacerbation is defined as an AECOPD event that requires hospitalization, emergency medical visit, or results in death. In some cases, the annualized rate of moderate-severe acute exacerbations of COPD (AECOPD) includes moderate exacerbations and severe exacerbations.

[0037] "Reducing the frequency" of COPD exacerbations means that a subject administered an IL-33 antagonist disclosed herein experiences fewer COPD exacerbations after treatment with an IL-33 antagonist disclosed herein than before treatment (i.e., at least one less exacerbation), or does not experience a COPD exacerbation for at least four weeks (e.g., 4, 6, 8, 12, 14, or more weeks) from the start of treatment.

[0038] "Reduced infections" or "prevention of infections" means that a subject administered an IL-33 antagonist disclosed herein experiences fewer infections after treatment with an IL-33 antagonist disclosed herein than before treatment (i.e., at least one less infection), or does not experience an infection for at least 4 weeks (e.g., 4, 6, 8, 12, 14, or more weeks) from the start of treatment. Considering that more than 50% of COPD exacerbations are caused by infections, such as respiratory tract viral infections, a reduction in COPD exacerbations can be a useful surrogate for determining a reduction in infection rates. If infections are reduced, a concomitant reduction in the number of exacerbations is expected.

[0039] An "effective amount" or "therapeutically effective amount" of an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or prophylactic result.

[0040] The term "subject" refers to an animal, human, or non-human to which treatment according to the method of the present invention is provided. Veterinary and non-veterinary applications are contemplated. The term includes, but is not limited to, mammals, such as humans, other primates, pigs, rodents such as mice and rats, rabbits, guinea pigs, hamsters, cows, horses, cats, dogs, sheep, and goats. Typical subjects include humans, livestock, and household pets such as cats and dogs. A preferred subject is a human.

[0041] Treatment method The present disclosure provides a method for reducing or preventing an infection, particularly a respiratory infection, such as a respiratory viral infection or a respiratory bacterial infection. The method is particularly useful in subjects with IL-33-mediated respiratory disorders, particularly subjects with abnormal epithelial physiology. Abnormal epithelial physiology may generally be characterized by an imbalance of cell types that make up the respiratory epithelium.

[0042] This disclosure provides evidence that oxidized IL-33 (IL-33ox) inhibits club cell activity in airway epithelium. Club cells are a type of secretory epithelial cell with multiple cellular defense functions. Inhibiting IL-33ox activity restores club cell activity to the epithelium, including increased expression of club cell-associated genes with defense functions. This likely improves the epithelium's defense against infections, such as viral or bacterial infections, that lead to exacerbation events in diseases such as COPD.

[0043] Thus, in some cases, the disclosure provides an IL-33 antagonist for use, a method of treatment comprising administration of said IL-33 antagonist, and a use of said IL-33 antagonist in the manufacture of a medicament for reducing or preventing a respiratory infection in a subject having an IL-33-mediated respiratory disorder. It is understood that for each instance disclosing an "IL-33 antagonist for use," a corresponding "method of treatment" or "use" of said IL-33 antagonist is contemplated.

[0044] In some cases, the IL-33-mediated respiratory disorder is selected from asthma, chronic obstructive pulmonary disease (COPD), asthma-COPD overlap syndrome (ACOS), chronic bronchitis, or emphysema. In some cases, the IL-33-mediated disorder is COPD. These disorders may represent abnormal epithelial physiology in which club cell activity may be reduced. For example, the examples show that air-liquid interface (ALI) cultures of COPD epithelia exhibit reduced total club cell area and reduced mRNA and protein expression levels of club cell markers compared to ALI of healthy controls. The examples show that this dysfunction is mediated, at least in part, by IL-33ox. Total club cell area and mRNA and protein expression levels of club cell markers are restored upon treatment with an IL-33 antagonist that inhibits the activity of IL-33ox. Thus, the Examples show that IL-33 antagonists may be useful in restoring club cell activity in the airway epithelium of subjects with IL-33-mediated respiratory disorders such as COPD.

[0045] In some cases, the epithelium is selected from squamous epithelium, cuboidal epithelium, columnar epithelium, and pseudostratified epithelium. In some cases, the epithelium is ciliated pseudostratified columnar epithelium. In some cases, the epithelium is cuboidal epithelium. In some cases, the epithelium is squamous epithelium.

[0046] In some cases, the IL-33-mediated respiratory disorder is COPD. COPD is a chronic inflammatory lung disease that causes airflow obstruction from the lungs. Multiple evidences suggest that IL-33 is a driver of chronic inflammation observed in the lungs of COPD subjects. Several ongoing clinical trials using IL-33 antagonists attempt to limit chronic inflammation observed in COPD subjects. However, it has not been known that IL-33 directly affects the function of club cells in epithelium. Thus, this disclosure is the first to identify that IL-33 antagonists can be used to directly affect the physiology of airway epithelium to increase the function of club cells, thereby improving defense against respiratory tract infections.

[0047] In some cases, a subject is identified as having "mild," "moderate," "severe," or "very severe" COPD if the subject is diagnosed by a physician with such COPD in accordance with the Global Initiative for Chronic Obstructive Lung Disease (GOLD) (Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease (2017 report) (available at: goldcopd.org / wp-content / uploads / 2016 / 12 / wms-GOLD-2017-Pocket-Guide.pdf). In such cases, the subject's COPD is classified based on the severity of airway limitation as tested using a post-bronchodilator FEV1. If the subject's FEV1 is 25-80% or greater than the predicted FEV1, the subject's COPD is classified as "mild" using the GOLD classification system. The predicted FEV1 is the same as that of a person with healthy lungs. The FEV1 is based on the FEV1 values ​​of an average person of similar age, race, height, and sex. If the subject's FEV1 is 50% or more of predicted FEV1 but less than 80% of predicted FEV1, the subject's COPD is classified as "moderate" on the GOLD classification system. If the subject's FEV1 is 30% or more of predicted FEV1 but less than 50% of predicted FEV1, the subject's COPD is classified as "severe" on the GOLD classification system. If the subject's FEV1 is less than 30% of predicted FEV1, the subject's COPD is classified as "very severe" on the GOLD classification system.

[0048] In some cases, the IL-33 antagonist is for use in preventing or reducing respiratory tract infections. As used herein, "respiratory tract infection", "respiratory tract infection" and "RTI" have the same meaning. RTI is an infection of the parts of the body involved in breathing, such as the sinuses, throat, airways, or lungs.

[0049] In some cases, the IL-33 antagonist is for use in reducing or preventing a pulmonary respiratory infection (also referred to herein as a "pulmonary respiratory infection").

[0050] In some cases, the IL-33 antagonist is for use in reducing or preventing respiratory infections of the airways (also referred to herein as "airway respiratory infections").

[0051] In some cases, the IL-33 antagonist is for use in reducing or preventing small airway respiratory infections (also referred to herein as "small airway respiratory infections").

[0052] Because club cells are primarily located in the bronchioles of the lungs, the IL-33 antagonists disclosed herein may be particularly beneficial in reducing infections manifested at sites where club cells are primarily located.

[0053] In some cases, the IL-33 antagonist may be for use in reducing or preventing infections caused by viruses (also referred to herein as "respiratory viral infections," "respiratory tract viral infections," or "RTVI").

[0054] In some cases, the IL-33 antagonist may be for use in reducing or preventing RTVI caused by influenza virus (e.g., influenza virus A, influenza virus B), respiratory syncytial virus (RSV), adenovirus, metapneumovirus, cytomegalovirus, parainfluenza virus (e.g., hPIV-1, hPIV-2, hPIV-3, hPIV-4), rhinovirus, adenovirus, coxsackievirus, echovirus, coronavirus, herpes simplex virus, SARS-coronavirus, or smallpox.

[0055] In some cases, the IL-33 antagonist can be for use in reducing an infection caused by a bacterium (also referred to herein as a "respiratory bacterial infection" or "respiratory tract bacterial infection"). In some cases, the IL-33 antagonist can be for use in reducing an infection caused by Chlamydia pneumoniae or Mycoplasma pneumoniae.

[0056] In some cases, reducing or preventing infection includes increasing total club cell area in the respiratory epithelium. In some cases, the IL-33 antagonist inhibits or reduces activity of IL-33ox, thereby increasing total club cell area in the respiratory epithelium. In some cases, the respiratory epithelium is an upper respiratory tract epithelium. In some cases, the upper respiratory tract epithelium is ciliated pseudostratified columnar epithelium. In some cases, the epithelium is a lower respiratory tract epithelium. In some cases, the lower respiratory tract epithelium is cuboidal epithelium. In some cases, the lower respiratory tract epithelium is squamous epithelium.

[0057] The examples show that blocking the activity of IL-33ox increases the proportion of club cells in COPD ALI cultures and restores the total club cell area to a similar level as that seen in healthy controls. As the club cell area is restored, the expression of club cell defense genes increases. Thus, increasing the club cell area and defense function of club cells is likely to reduce or prevent infections in subjects with IL-33-mediated disorders who were previously prone to RTVI.

[0058] The total club cell area can be measured by measuring markers from a relevant biological sample obtained from a subject. In some cases, the biological sample can be a biopsy, such as a respiratory epithelium biopsy, a bronchial scraping, bronchoalveolar lavage fluid (BALF), sputum, serum, plasma, or nasal mucosal lining fluid. In some cases, the biological sample is obtained from the respiratory epithelium. If an increase in the concentration of the marker is detected in the subject after treatment, this indicates that the treatment has successfully increased the total club cell area in the respiratory epithelium.

[0059] In some cases, the marker can be the mRNA expression level of SCGB1A1. In some cases, the marker can be the mRNA expression level of SCGB3A1. In some cases, the marker can be the mRNA expression level of WFDC2. In some cases, the marker can be the mRNA expression level of MSMB. In some cases, the marker can be the mRNA expression level of BPIFA1. In some cases, an increase in the mRNA expression level of one or more of SCGB1A1, SCGB3A1, WFDC2, MSMB, and BPIFA1 after treatment compared to a reference expression level for one or both markers indicates an increase in the total club cell area.

[0060] In some cases, the marker can be the protein expression level of CCSP. In some cases, the marker can be the protein expression level of SPLUNC1. In some cases, the marker can be the protein expression level of secretoglobin family 3A member 1 (SCGB3A1). In some cases, the marker can be the protein expression level of WAP4-disulfide core domain protein 2 (WFDC2). In some cases, the marker can be the protein expression level of beta-microseminoprotein. In some cases, an increase in the protein expression level of one or more of CCSP, SCGB3A1, WFDC2, beta-microseminoprotein, lactotransferrin, and SPLUNC1 after treatment compared to the reference expression level for one or both markers indicates an increase in the total club cell area.

[0061] In some cases, the reference expression level is a level determined in a biological sample obtained from the subject prior to treatment with an IL-33 antagonist. In some cases, the mRNA expression level is measured by qRT-PCR. In some cases, the protein expression level is measured by enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), immunofluorescence, flow cytometry, or Western blotting.

[0062] In some cases, reducing or preventing infection includes increasing the mRNA expression level of SCGB1A1. In some cases, the IL-33 antagonist inhibits or reduces the activity of IL-33ox, thereby increasing the mRNA expression level of SCGB1A1. In some cases, the increased mRNA expression level is in epithelium. In some cases, the increased expression is in airway epithelium. In some cases, the increased expression is in upper airway epithelium. In some cases, the increased expression is in ciliated pseudostratified columnar epithelium. In some cases, the increased expression is in lower airway epithelium. In some cases, the increased expression is in peripheral airway epithelium. In some cases, the increased mRNA expression level is in cuboidal epithelium. In some cases, the increased mRNA expression level is in squamous epithelium. SCGB1A1 encodes CCSP as described elsewhere herein, is secreted by club cells, and has been shown to regulate pulmonary inflammatory and immune responses to RSV infection (Wang et al The Journal of Immunology, 2003, 171:1051-1060). The examples show that treatment with IL-33 antagonists increases the expression of SCGB1A1 from COPD epithelium, thereby increasing anti-inflammatory and immune response activity against infectious agents. In some cases, the increased expression is in club 1 cells, club 2 cells, club 3 cells, or club 4 cells.

[0063] Suitable samples and methods for measuring and determining an increase in the mRNA expression level of SCGB1A1 are disclosed elsewhere herein.

[0064] In some cases, reducing or preventing infection includes increasing the protein expression level of CCSP. In some cases, the IL-33 antagonist inhibits or reduces the activity of IL-33ox, thereby increasing the protein expression level of CCSP. In some cases, the increased protein expression level is in epithelium. In some cases, the increased expression is in airway epithelium. In some cases, the increased protein expression level is in upper airway epithelium. In some cases, the increased protein expression level is in ciliated pseudostratified columnar epithelium. In some cases, the increased protein expression level is in lower airway epithelium. In some cases, the increased protein expression level is in peripheral airway epithelium. In some cases, the increased protein expression level is in cuboidal epithelium. In some cases, the increased protein expression level is in squamous epithelium.

[0065] Suitable samples and methods for measuring and determining increased protein expression levels of CCSP are disclosed elsewhere herein.

[0066] In some cases, reducing or preventing infection includes increasing the activity of CCSP. In some cases, the IL-33 antagonist inhibits or reduces the activity of IL-33ox, thereby increasing the activity of CCSP. In some cases, the increased activity of CCSP is in epithelium. In some cases, the increased activity of CCSP is in airway epithelium. In some cases, the increased activity of CCSP is in upper airway epithelium. In some cases, the increased activity of CCSP is in ciliated pseudostratified columnar epithelium. In some cases, the increased activity of CCSP is in lower airway epithelium. In some cases, the increased activity of CCSP is in peripheral airway epithelium. In some cases, the increased activity of CCSP is in cuboidal epithelium. In some cases, the increased activity of CCSP is in squamous epithelium.

[0067] In some cases, reducing or preventing infection includes increasing the mRNA expression level of BPIFA1. In some cases, the IL-33 antagonist inhibits or reduces the activity of IL-33ox, thereby increasing the mRNA expression level of BPIFA1. In some cases, the increased mRNA expression level is in epithelium. In some cases, the increased expression is in airway epithelium. In some cases, the increased expression is in upper airway epithelium. In some cases, the increased expression is in ciliated pseudostratified columnar epithelium. In some cases, the increased expression is in lower airway epithelium. In some cases, the increased expression is in small airway epithelium. In some cases, the increased mRNA expression level is in cuboidal epithelium. In some cases, the increased mRNA expression level is in squamous epithelium. BPIFA1 encodes BPI-fold containing family A member 1 (BPIFA1, also known as SPLUNC1), which has been shown to play a role in the innate immune response of the upper airway. Sayyed et al. show that BPIFIA1 protects the host from bacterial infection with Pseudomonas aeruginosa in the upper respiratory tract (Sayeed et al Infect.Immun.81:285-291(2013)). In some cases, the increased expression is in club 1 cells, club 2 cells, club 3 cells, or club 4 cells.

[0068] Suitable samples and methods for measuring and determining increased levels of BPFIA mRNA expression are disclosed elsewhere herein.

[0069] In some cases, reducing or preventing infection includes increasing the protein expression level of SPLUNC1. In some cases, the IL-33 antagonist inhibits or reduces the activity of IL-33ox, thereby increasing the protein expression level of SPLUNC1. In some cases, the increased protein expression level is in epithelium. In some cases, the increased expression is in airway epithelium. In some cases, the increased protein expression level is in upper airway epithelium. In some cases, the increased protein expression level is in ciliated pseudostratified columnar epithelium. In some cases, the increased protein expression level is in lower airway epithelium. In some cases, the increased protein expression level is in peripheral airway epithelium. In some cases, the increased protein expression level is in cuboidal epithelium. In some cases, the increased protein expression level is in squamous epithelium.

[0070] Suitable samples and methods for measuring and determining increased protein expression levels of SPLUNC1 are disclosed elsewhere herein.

[0071] In some cases, reducing or preventing infection includes increasing activity of SPLUNC1. In some cases, the IL-33 antagonist inhibits or reduces activity of IL-33ox, thereby increasing activity of SPLUNC1. In some cases, the increased activity of SPLUNC1 is in epithelium. In some cases, the increased activity of SPLUNC1 is in airway epithelium. In some cases, the increased activity of SPLUNC1 is in upper airway epithelium. In some cases, the increased activity of SPLUNC1 is in ciliated pseudostratified columnar epithelium. In some cases, the increased activity of SPLUNC1 is in lower airway epithelium. In some cases, the increased activity of SPLUNC1 is in peripheral airway epithelium. In some cases, the increased activity of SPLUNC1 is in cuboidal epithelium. In some cases, the increased activity of SPLUNC1 is in squamous epithelium.

[0072] In some cases, reducing or preventing infection includes increasing the mRNA expression level of SCGB3A1. In some cases, the IL-33 antagonist inhibits or reduces the activity of IL-33ox, thereby increasing the mRNA expression level of SCGB3A1. In some cases, the increased mRNA expression level is in epithelium. In some cases, the increased expression is in airway epithelium. In some cases, the increased expression is in upper airway epithelium. In some cases, the increased expression is in ciliated pseudostratified columnar epithelium. In some cases, the increased expression is in lower airway epithelium. In some cases, the increased expression is in peripheral airway epithelium. In some cases, the increased expression is in cuboidal epithelium. In some cases, the increased expression is in squamous epithelium. SCGB3A1 encodes SCGB3A1, a cytokine-like protein secreted by club cells that has been shown to inhibit cell proliferation in vitro (Krop et al PNAS, 2001, 98:9796-9801; Zuo et al Am J Respir Crit Care Med, 2018, 198:1375-1388). The examples show that treatment with an IL-33 antagonist increases expression of SCGB3A1 from COPD epithelia. In some cases, the increased expression is in club 1 cells, club 2 cells, club 3 cells, or club 4 cells. In some cases, the increased expression is in club 1 cells, club 2 cells, or club 3 cells.

[0073] Suitable samples and methods for measuring and determining an increase in the mRNA expression level of SCGB3A1 are disclosed elsewhere herein.

[0074] In some cases, reducing or preventing infection includes increasing the protein expression level of SCGB3A1. In some cases, the IL-33 antagonist inhibits or reduces the activity of IL-33ox, thereby increasing the protein expression level of SCGB3A1. In some cases, the increased protein expression level is in epithelium. In some cases, the increased expression is in airway epithelium. In some cases, the increased protein expression level is in upper airway epithelium. In some cases, the increased protein expression level is in ciliated pseudostratified columnar epithelium. In some cases, the increased protein expression level is in lower airway epithelium. In some cases, the increased protein expression level is in peripheral airway epithelium. In some cases, the increased protein expression level is in cuboidal epithelium. In some cases, the increased protein expression level is in squamous epithelium.

[0075] Suitable samples and methods for measuring and determining an increase in the protein expression level of SCGB3A1 are disclosed elsewhere herein.

[0076] In some cases, reducing or preventing infection includes increasing the activity of SCGB3A1. In some cases, the IL-33 antagonist inhibits or reduces the activity of IL-33ox, thereby increasing the activity of SCGB3A1. In some cases, the increase in the activity of SCGB3A1 is in epithelium. In some cases, the increase in the activity of SCGB3A1 is in airway epithelium. In some cases, the increase in the activity of SCGB3A1 is in upper airway epithelium. In some cases, the increase in the activity of SCGB3A1 is in ciliated pseudostratified columnar epithelium. In some cases, the increase in the activity of SCGB3A1 is in lower airway epithelium. In some cases, the increase in the activity of SCGB3A1 is in peripheral airway epithelium. In some cases, the increase in the activity of SCGB3A1 is in cuboidal epithelium. In some cases, the increase in the activity of SCGB3A1 is in squamous epithelium.

[0077] In some cases, reducing or preventing infection includes increasing the mRNA expression level of WFDC2. In some cases, the IL-33 antagonist inhibits or reduces the activity of IL-33ox, thereby increasing the mRNA expression level of WFDC2. In some cases, the increased mRNA expression level is in epithelium. In some cases, the increased expression is in airway epithelium. In some cases, the increased expression is in upper airway epithelium. In some cases, the increased expression is in ciliated pseudostratified columnar epithelium. In some cases, the increased expression is in lower airway epithelium. In some cases, the increased expression is in peripheral airway epithelium. In some cases, the increased mRNA expression level is in cuboidal epithelium. In some cases, the increased mRNA expression level is in squamous epithelium. WFDC2 encodes WAP4-disulfide core domain 2 (WFDC2), an antiproteolytic enzyme that is expressed by club cells and functions to defend host cells (Zuo et al Am J Respir Crit Care Med, 2018, 198:1375-1388). The examples show that treatment with an IL-33 antagonist increases expression of WFDC2 from COPD epithelia. In some cases, the increased expression is in club 1 cells, club 2 cells, club 3 cells, or club 4 cells. In some cases, the increased expression is in club 3 cells or club 4 cells.

[0078] Suitable samples and methods for measuring and determining an increase in WFDC2 mRNA expression levels are disclosed elsewhere herein.

[0079] In some cases, reducing or preventing infection includes increasing the protein expression level of WFDC2. In some cases, the IL-33 antagonist inhibits or reduces the activity of IL-33ox, thereby increasing the protein expression level of WFDC2. In some cases, the increased protein expression level is in epithelium. In some cases, the increased expression is in airway epithelium. In some cases, the increased protein expression level is in upper airway epithelium. In some cases, the increased protein expression level is in ciliated pseudostratified columnar epithelium. In some cases, the increased protein expression level is in lower airway epithelium. In some cases, the increased protein expression level is in peripheral airway epithelium. In some cases, the increased protein expression level is in cuboidal epithelium. In some cases, the increased protein expression level is in squamous epithelium.

[0080] Suitable samples and methods for measuring and determining increased protein expression levels of WFDC2 are disclosed elsewhere herein.

[0081] In some cases, reducing or preventing infection includes increasing activity of WFDC2. In some cases, the IL-33 antagonist inhibits or reduces activity of IL-33ox, thereby increasing activity of WFDC2. In some cases, the increased activity of WFDC2 is in epithelium. In some cases, the increased activity of WFDC2 is in airway epithelium. In some cases, the increased activity of WFDC2 is in upper airway epithelium. In some cases, the increased activity of WFDC2 is in ciliated pseudostratified columnar epithelium. In some cases, the increased activity of WFDC2 is in lower airway epithelium. In some cases, the increased activity of WFDC2 is in peripheral airway epithelium. In some cases, the increased activity of WFDC2 is in cuboidal epithelium. In some cases, the increased activity of WFDC2 is in squamous epithelium.

[0082] In some cases, reducing or preventing infection includes increasing the mRNA expression level of MSMB. In some cases, the IL-33 antagonist inhibits or reduces the activity of IL-33ox, thereby increasing the mRNA expression level of MSMB. In some cases, the increased mRNA expression level is in epithelium. In some cases, the increased expression is in airway epithelium. In some cases, the increased expression is in upper airway epithelium. In some cases, the increased expression is in ciliated pseudostratified columnar epithelium. In some cases, the increased expression is in lower airway epithelium. In some cases, the increased expression is in peripheral airway epithelium. In some cases, the increased mRNA expression level is in cuboidal epithelium. In some cases, the increased mRNA expression level is in squamous epithelium. MSMB encodes beta-microseminoprotein secreted by club cells (Zuo et al Am J Respir Crit Care Med, 2018, 198:1375-1388). The examples show that treatment with an IL-33 antagonist increases expression of beta-microseminoprotein from COPD epithelium. In some cases, the increased expression is in club 1 cells, club 2 cells, club 3 cells, or club 4 cells. In some cases, the increased expression is in club 1 cells, club 2, or club 3 cells.

[0083] Suitable samples and methods for measuring and determining an increase in the mRNA expression level of MSMB are disclosed elsewhere herein.

[0084] In some cases, reducing or preventing infection includes increasing the protein expression level of beta-microseminoprotein. In some cases, the IL-33 antagonist inhibits or reduces the activity of IL-33ox, thereby increasing the protein expression level of beta-microseminoprotein. In some cases, the increased protein expression level is in epithelium. In some cases, the increased expression is in airway epithelium. In some cases, the increased protein expression level is in upper airway epithelium. In some cases, the increased protein expression level is in ciliated pseudostratified columnar epithelium. In some cases, the increased protein expression level is in lower airway epithelium. In some cases, the increased protein expression level is in peripheral airway epithelium. In some cases, the increased protein expression level is in cuboidal epithelium. In some cases, the increased protein expression level is in squamous epithelium.

[0085] Suitable samples and methods for measuring and determining an increase in the protein expression level of beta-microseminoprotein are disclosed elsewhere herein.

[0086] In some cases, reducing or preventing infection includes increasing the activity of beta-microseminoprotein. In some cases, the IL-33 antagonist inhibits or reduces the activity of IL-33ox, thereby increasing the activity of beta-microseminoprotein. In some cases, the increased activity of beta-microseminoprotein is in epithelium. In some cases, the increased activity of beta-microseminoprotein is in airway epithelium. In some cases, the increased activity of beta-microseminoprotein is in upper airway epithelium. In some cases, the increased activity of beta-microseminoprotein is in ciliated pseudostratified columnar epithelium. In some cases, the increased activity of beta-microseminoprotein is in lower airway epithelium. In some cases, the increased activity of beta-microseminoprotein is in peripheral airway epithelium. In some cases, the increased activity of beta-microseminoprotein is in cuboidal epithelium. In some cases, the increased activity of beta-microseminoprotein is in squamous epithelium.

[0087] In some cases, reducing or preventing infection includes increasing the mRNA expression level of LTF. In some cases, the IL-33 antagonist inhibits or reduces the activity of IL-33ox, thereby increasing the mRNA expression level of LTF. In some cases, the increased mRNA expression level is in epithelium. In some cases, the increased expression is in airway epithelium. In some cases, the increased expression is in upper airway epithelium. In some cases, the increased expression is in ciliated pseudostratified columnar epithelium. In some cases, the increased expression is in lower airway epithelium. In some cases, the increased expression is in peripheral airway epithelium. In some cases, the increased mRNA expression level is in cuboidal epithelium. In some cases, the increased mRNA expression level is in squamous epithelium. LTF encodes lactotransferrin, an antimicrobial protein with various host cell defense functions. It is known to be expressed in airway epithelia, e.g., submucosal glands and surface epithelia (Dubin et al Am J Physiol Lung Cell Mol Physiol 286:L750-L755,2004). The examples show that treatment with an IL-33 antagonist increases expression of LTF in COPD epithelia. In some cases, the increased expression is in club 1 cells, club 2 cells, club 3 cells, or club 4 cells. In some cases, the increased expression is in club 4 cells.

[0088] Suitable samples and methods for measuring and determining increased levels of LTF mRNA expression are disclosed elsewhere herein.

[0089] In some cases, reducing or preventing infection includes increasing the protein expression level of lactotransferrin. In some cases, the IL-33 antagonist inhibits or reduces the activity of IL-33ox, thereby increasing the protein expression level of lactotransferrin. In some cases, the increased protein expression level is in epithelium. In some cases, the increased expression is in airway epithelium. In some cases, the increased protein expression level is in upper airway epithelium. In some cases, the increased protein expression level is in ciliated pseudostratified columnar epithelium. In some cases, the increased protein expression level is in lower airway epithelium. In some cases, the increased protein expression level is in peripheral airway epithelium. In some cases, the increased protein expression level is in cuboidal epithelium. In some cases, the increased protein expression level is in squamous epithelium.

[0090] Suitable samples and methods for measuring and determining increased protein expression levels of lactotransferrin are disclosed elsewhere herein.

[0091] In some cases, reducing or preventing infection includes increasing lactotransferrin activity. In some cases, the IL-33 antagonist inhibits or reduces IL-33ox activity, thereby increasing lactotransferrin activity in respiratory epithelium. In some cases, the increased lactotransferrin activity is in epithelium. In some cases, the increased lactotransferrin activity is in airway epithelium. In some cases, the increased lactotransferrin activity is in upper airway epithelium. In some cases, the increased lactotransferrin activity is in ciliated pseudostratified columnar epithelium. In some cases, the increased lactotransferrin activity is in lower airway epithelium. In some cases, the increased lactotransferrin activity is in peripheral airway epithelium. In some cases, the increased lactotransferrin activity is in cuboidal epithelium. In some cases, the increased lactotransferrin activity is in squamous epithelium.

[0092] In some cases, the IL-33 antagonist reduces or prevents infection, thereby increasing the defensive function of club cells in the airway epithelium. In some cases, increasing the defensive function of club cells comprises increasing the mRNA expression level of one or more club cell defense genes. In some cases, the one or more club cell defense genes are selected from the following list: SCGB1BA1, BPIFA1, SCGB3A1, WFDC2, MSMB, LTF, SLPI, C3, HLA-DRA, CXCL1, CD74, CXCL17, MDK, TGM2, HLA-DRB1, CXCL8, CXCL2, HLA-DRB5, CX3CL1, and HLA-DPA1. In some cases, the one or more club cell defense genes are selected from SCGB1BA1, BPIFA1, SCGB3A1, WFDC2, MSMB, and LTF. In some cases, the one or more club cell defense genes are selected from SCGB1BA1 and BPIFA1. In some cases, increasing the defensive function of club cells comprises increasing the protein expression level of one or more proteins that provide a defensive function of club cells selected from the following list: CCSP, SCGB3A1, WFDC2, beta-microseminoprotein, lactotransferrin, SPLUNC1, SLPI, C3, HLA-DRA, CXCL1, CD74, CXCL17, MDK, TGM2, HLA-DRB1, CXCL8, CXCL2, HLA-DRB5, CX3CL1, and HLA-DPA1. In some cases, the one or more proteins that provide a defensive function of club cells are selected from CCSP, SCGB3A1, WFDC2, beta-microseminoprotein, lactotransferrin, and SPLUNC1. In some cases, the one or more proteins that provide a defensive function of club cells are selected from CCSP and SPLUNC1. In some cases, the club cell defensive genes include SCGB1A1, LTF, and / or BPIFA1.

[0093] In some cases, reducing or preventing infection comprises increasing the mRNA expression level of one or more of the markers selected from the following list: SLPI, C3, HLA-DRA, CXCL1, CD74, CXCL17, MDK, TGM2, HLA-DRB1, CXCL8, CXCL2, HLA-DRB5, CX3CL1, and HLA-DPA1.

[0094] In some cases, reducing or preventing infection comprises increasing the protein expression level of one or more of the following markers: secretory leukocyte protease inhibitor (SLPI), complement C3, HLA-DR alpha chain, C-X-C motif chemokine ligand 1 (CXCL1), cluster of differentiation 74 (CD74), C-X-C motif chemokine 17 (CXCL17), midkine (MDK), protein-glutamine gamma-glutamyltransferase 2 (TGM2), HLA class II histocompatibility complex, DRB1 beta chain (HLA-DRB1), chemokine (C-X-C motif) ligand 8 (CXCL8), chemokine (C-X-C motif) ligand 2 (CXCL2), HLA class II histocompatibility complex, DRB5 beta chain (HLA-DRB5), chemokine (C-X3-C motif) ligand 1 (CX3CL1), and major histocompatibility complex class II, DP alpha 1 (HLA-DPA1).

[0095] In some cases, reducing or preventing infection includes increasing the activity of one or more of SLPI, C3, HLA-DRA, CXCL1, CD74, CXCL17, MDK, TGM2, HLA-DRB1, CXCL8, CXCL2, HLA-DRB5, CX3CL1, and HLA-DPA1.

[0096] In some cases, reducing or preventing infection comprises increasing the mRNA expression level of one or more markers selected from the list consisting of SLPI, C3, HLA-DRA, CXCL1, CD74, CXCL17, MDK, TGM2, HLA-DRB1, CXCL8, CXCL2, HLA-DRB5, CX3CL1, and HLA-DPA1.

[0097] In some cases, reducing or preventing infection comprises increasing the protein expression level of one or more markers selected from the list consisting of SLPI, C3, HLA-DRA, CXCL1, CD74, CXCL17, MDK, TGM2, HLA-DRB1, CXCL8, CXCL2, HLA-DRB5, CX3CL1, and HLA-DPA1.

[0098] In some cases, reducing or preventing infection comprises increasing the activity of one or more markers selected from the list consisting of SLPI, C3, HLA-DRA, CXCL1, CD74, CXCL17, MDK, TGM2, HLA-DRB1, CXCL8, CXCL2, HLA-DRB5, CX3CL1, and HLA-DPA1.

[0099] In some cases, reducing or preventing infection reduces the annual exacerbation rate of COPD.More than 50% of exacerbations are caused by respiratory tract viral infections.Therefore, improving the activity of club cells, thereby reducing the frequency of RTVI in subjects with COPD, is likely to reduce the annual exacerbation rate in subjects.

[0100] In some cases, reducing or preventing infections reduces the frequency of acute exacerbations of COPD (AECOPD).

[0101] In another aspect, the present disclosure provides a composition comprising an IL-33 antagonist for use in a method of treatment for preventing or reducing respiratory tract infection in a subject with COPD. This is achieved by increasing the activity of club cells in the airway epithelium by inhibiting the activity of IL-33ox. The respiratory tract infection may be any of those described elsewhere herein. In a specific example, the respiratory tract infection may be a respiratory tract viral infection. The reduction of respiratory tract infection can be determined by monitoring the frequency of acute exacerbations of COPD (AECOPD) in the subject. If the number of AECOPD over a period of time in the subject after treatment is statistically lower compared to the number of AECOPD over the same period of time before treatment, this indicates that the treatment has reduced respiratory tract infection in the subject. This is because more than 50% of AECOPD is caused by respiratory tract infection in COPD.

[0102] In some cases, the period is greater than 6 months. In some cases, the period is greater than 12 months. In some cases, the period is between 12 and 24 months. In some cases, the period is 18 months, 20 months, 22 months, or 24 months. In some cases, the period is 24 months.

[0103] In another aspect, the disclosure provides an IL-33 antagonist for use in a method of treatment for reducing AECOPD in a subject with COPD, the IL-33 antagonist attenuating or inhibiting the activity of IL-33ox, thereby reducing respiratory tract infection in the subject.

[0104] In some cases, attenuating or inhibiting the activity of IL-33ox increases the defensive function of club cells, thereby reducing respiratory tract infections in a subject.

[0105] In some cases, attenuating or inhibiting the activity of IL-33ox increases the defensive function of club cells, thereby reducing respiratory tract infections in a subject.

[0106] In some cases, attenuating or inhibiting the activity of IL-33ox increases the mRNA expression levels of one or more of the markers described elsewhere herein, thereby reducing respiratory tract infections in the subject.

[0107] In some cases, attenuating or inhibiting the activity of IL-33ox increases the protein expression levels of one or more of the markers described elsewhere herein, thereby reducing respiratory tract infections in the subject.

[0108] In some cases, attenuating or inhibiting the activity of IL-33ox increases the activity of one or more proteins described herein that provide the defensive function of club cells, thereby reducing respiratory tract infections in a subject.

[0109] In some cases, attenuating or inhibiting the activity of IL-33ox increases the total club cell area in the epithelium of a subject, thereby reducing respiratory tract infections in the subject. In some cases, the epithelium is airway epithelium. In some cases, the epithelium is upper respiratory tract epithelium. In some cases, the epithelium is ciliated pseudostratified columnar epithelium. In some cases, the epithelium is lower respiratory tract epithelium. In some cases, the epithelium is peripheral airway epithelium. In some cases, the epithelium is cuboidal epithelium. In some cases, the epithelium is squamous epithelium.

[0110] IL-33 antagonists The methods described herein include using an IL-33 antagonist.

[0111] In some cases, the IL-33 antagonist is a binding molecule. In some cases, the binding molecule specifically binds to IL33. Such a binding molecule is also referred to as an "IL-33 binding molecule" or an "anti-IL-33 binding molecule." In some cases, the binding molecule specifically binds to IL-33 and inhibits or attenuates an activity of IL-33.

[0112] In some cases, the IL-33 antagonist is an antibody or antigen-binding fragment thereof. It is contemplated that antibodies or antigen-binding fragments thereof that specifically bind to and inhibit components of the oxIL-33 / RAGE / EGFR signaling axis may be useful in the methods disclosed herein.

[0113] In some cases, the binding molecule is an antibody. In some cases, the antibody can be monoclonal (mAb), recombinant, chimeric, humanized, e.g., complementarity determining region (CDR) grafted, human, antibody variants including single chain, and / or bispecific, as well as antigen-binding fragments, variants, or derivatives thereof. Antigen-binding fragments include the portion of an antibody that binds to an epitope on a polypeptide of interest. Examples of such antigen-binding fragments include Fab and F(ab') fragments generated by enzymatic cleavage of full-length antibodies. Other antigen-binding fragments include those generated by recombinant DNA techniques, such as expression of recombinant plasmids containing nucleic acid sequences encoding antibody variable regions.

[0114] As used herein, "monoclonal antibody" or "monoclonal antibody composition" refers to polypeptides, including antibodies, bispecific antibodies, and the like, having substantially identical amino acid sequences or derived from the same genetic source. The term also includes preparations of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0115] A "chimeric" antibody refers to an antibody in which a portion of the heavy (H) and / or light (L) chain is identical or homologous to a corresponding sequence in an antibody from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to a corresponding sequence in an antibody from another species or belonging to another antibody class or subclass. Fragments of such antibodies are also included so long as they exhibit the desired biological activity. See U.S. Pat. No. 4,816,567; Morrison et al., 1985, Proc. Natl. Acad. Sci. 81:6851-55.

[0116] In some cases, the monoclonal antibody is a "humanized" antibody. Methods for humanizing non-human antibodies are well known in the art. See U.S. Patent Nos. 5,585,089 and 5,693,762. Usually, a humanized antibody has one or more amino acid residues introduced from a non-human source. Humanization can be performed, for example, by replacing at least a portion of a rodent complementarity determining region with the corresponding region of a human antibody using methods described in the art (Jones et al., 1986, Nature 321:522-25; Riechmann et al., 1998, Nature 332:323-27; Verhoeyen et al., 1988, Science 239:1534-36).

[0117] Human antibodies or antigen-binding fragments thereof that bind IL-33 are also contemplated. Using transgenic animals (e.g., mice) capable of producing a repertoire of human antibodies in the absence of endogenous immunoglobulin production, such antibodies are produced by immunization with a polypeptide antigen (i.e., having at least six consecutive amino acids), optionally conjugated to a carrier. See, e.g., Jakobovits et al., 1993, Proc. Natl. Acad. Sci. 90:2551-55; Jakobovits et al., 1993, Nature 362:255-58; Bruggermann et al., 1993, Year in Immuno. 7:33. See also PCT Application Nos. PCT / US96 / 05928 and PCT / US93 / 06926. Further methods are described in U.S. Patent No. 5,545,807, PCT Application Nos. PCT / US91 / 245 and PCT / GB89 / 01207, as well as European Patent No. 54607381 and European Patent Publication No. 546073A1. Human antibodies can also be produced by the expression of recombinant DNA in host cells or by expression in hybridoma cells, as described herein.

[0118] Chimeric, CDR-grafted, and humanized antibodies and / or antibody variants are typically produced by recombinant methods. Nucleic acids encoding the antibodies are introduced into and expressed in host cells using the materials and procedures described herein. In some cases, the antibodies are produced in mammalian host cells, such as CHO cells. Monoclonal (e.g., human) antibodies can be produced by expression of recombinant DNA in host cells or by expression in hybridoma cells, as described herein.

[0119] Antibodies and antigen-binding fragments thereof useful in the methods herein may comprise: (a) a heavy chain variable region comprising an HCDR1 having the sequence set forth in SEQ ID NO:1, a VHCDR2 having the sequence of SEQ ID NO:2, and a VHCDR3 having the sequence of SEQ ID NO:3; and (b) a light chain variable region comprising a VLCDR1 having the sequence of SEQ ID NO:5, a VLCDR2 having the sequence of SEQ ID NO:6, and a VLCDR3 having the sequence of SEQ ID NO:7.

[0120] In some cases, the IL-33 antibody, or antigen-binding fragment thereof, comprises a VH domain comprising VHCDRs 1-3 of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively.

[0121] In some cases, the IL-33 antibody, or antigen-binding fragment thereof, comprises a VH domain comprising VH CDRs 1-3 consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively.

[0122] In some cases, the anti-IL-33 antibody, or antigen-binding fragment thereof, comprises the HCDR1, HCDR2, and HCDR3 sequences of a VH domain having the sequence set forth in SEQ ID NO:4.

[0123] In some cases, the IL-33 antibody or antigen-binding fragment thereof comprises a variable heavy domain (VH) and a variable light domain (VL) having VLCDR1-3 having the sequences of SEQ ID NOs: 5, 6, and 7, respectively, wherein one or more of the VLCDRs have no more than three single amino acid substitutions, insertions, and / or deletions.

[0124] In some cases, the IL-33 antibody, or antigen-binding fragment thereof, comprises a VL domain comprising VHCDRs 1-3 of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively.

[0125] In some cases, the IL-33 antibody, or antigen-binding fragment thereof, comprises a VL domain comprising VHCDR1-3 consisting of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively.

[0126] In some cases, the anti-IL-33 antibody, or antigen-binding fragment thereof, comprises the LCDR1, LCDR2, and LCDR3 sequences of a VL domain having the sequence set forth in SEQ ID NO:8.

[0127] Also contemplated for use in the methods disclosed herein are anti-IL-33 antibodies or antigen-binding fragments thereof that include a heavy chain variable region (VH) domain at least 95%, 90%, or 85% identical to the sequence set forth in SEQ ID NO: 4. In some cases, the anti-IL-33 antibody or antigen-binding fragment thereof includes a light chain variable region (VL) domain at least 95%, 90%, or 85% identical to the sequence set forth in SEQ ID NO: 8. In some cases, the anti-IL-33 antibody or antigen-binding fragment thereof includes (a) a heavy chain variable region (VH) at least 95%, 90%, or 85% identical to the sequence set forth in SEQ ID NO: 4, and (b) a light chain variable region (VL) at least 95%, 90%, or 85% identical to the sequence set forth in SEQ ID NO: 8. In some cases, the anti-IL-33 antibody is 33_640087_7B, as disclosed in WO 2016 / 156440, which is incorporated herein by reference. 33_640087_7B, also referred to in the art as MEDI3506 or tozolaximab, is an anti-IL-33 antibody that binds with high affinity to the reduced form of IL-33 (redIL-33). 33_640087_7B also inhibits the conversion of redIL-33 to the oxidized form (oxIL-33), which has been shown to induce signaling through RAGE and induce epithelial cell proliferation. A Phase III clinical trial investigating the efficacy and safety of tozolaximab (MEDI3506) in symptomatic chronic obstructive pulmonary disease with a history of exacerbations is currently underway (NCT05166889).

[0128] 33_640087_7B is an exemplary anti-IL-33 antibody having (a) a heavy chain variable region comprising an HCDR1 having the sequence set forth in SEQ ID NO:1, a VHCDR2 having the sequence of SEQ ID NO:2, a VHCDR3 having the sequence of SEQ ID NO:3; and (b) a light chain variable region comprising a VLCDR1 having the sequence of SEQ ID NO:5, a VLCDR2 having the sequence of SEQ ID NO:6, and a VLCDR3 having the sequence of SEQ ID NO:7.

[0129] 33_640087_7B also comprises a VH domain having the amino acid sequence set forth in SEQ ID NO:4, and a VL domain having the amino acid sequence set forth in SEQ ID NO:8.

[0130] 33_640087_7B is an IgG1 antibody, and the sequences of the full-length light and heavy chains of 33_640087_7B, including the IgG1 chain, are set forth in SEQ ID NOs: 9 and 10, respectively.

[0131] Other exemplary IL-33 binding antagonists include anti-IL-33 antibodies or antigen-binding fragments thereof, including ANB020, also known as etokimab (described in WO 2015 / 106080), itepekimab, 9675P (described in US Patent Application Publication No. 2014 / 0271658), A25-3H04 (described in US Patent Application Publication No. 2017 / 0283494), Ab43 (described in WO 2018 / 081075), IL33-158 (described in US Patent Application Publication No. 2018 / 0037644), 10C12.38.H6.87Y.581 IgG4 (described in WO 2016 / 077381), or binding fragments thereof. Other exemplary anti-IL-33 antibodies or antigen-binding fragments thereof include any of the other anti-IL-33 antibodies described in WO 2016 / 156440, WO 2015 / 106080, US 2014 / 0271658, US 2017 / 0283494, WO 2018 / 081075, US 2018 / 0037644, or WO 2016 / 077381, all of which are incorporated herein by reference.

[0132] In some cases, the anti-IL-33 antibody or antigen-binding fragment thereof has similar or the same pharmacokinetic (pK) characteristics as 33_670087_7B in humans.

[0133] In some cases, the IL-33 binding molecule specifically binds to reduced IL-33 (IL-33red), oxidized IL-33 (IL-33ox), or both IL-33red and IL-33ox.

[0134] In some cases, the IL-33 binding molecule can attenuate or inhibit the activity of IL-33 by binding to reduced or oxidized IL-33. In some cases, where the binding molecule inhibits or attenuates the activity of reduced IL-33 and the activity of oxidized IL-33, this is accomplished by binding to the reduced form of IL-33 (i.e., by binding to reduced IL-33). In such cases, the binding molecule can bind to IL-33red and prevent it from converting to IL-33ox.

[0135] In some cases, the binding molecules are greater than 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 In some cases, the binding affinity for redIL-33 is less than 5×10 -14M (i.e., less than 0.05 pM). In some cases, the binding affinity is measured using Kinetic Exclusion Assays (KinExA) or BIACORE™. In some cases using KinExA, protocols such as those described in WO 2016 / 156440, the entire contents of which are incorporated by reference herein (see, e.g., Example 11) are used. Binding molecules that bind redIL-33 with this binding affinity have been found to bind tightly enough to prevent dissociation of the binding molecule / redIL-33 complex within biologically relevant time scales. Without wishing to be bound by theory, it is believed that this binding strength prevents release of the antigen prior to degradation of the binding molecule / antigen complex in vivo, minimizing any IL-33-dependent activity associated with release of IL-33 from the binding complex.

[0136] In some cases, the binding molecule 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 For example, the binding molecules of the present disclosure may specifically bind to redIL-33 with an on rate (k(on)) of 10 or more. 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 The antibody may bind to redIL-33, or a fragment or variant thereof, with an on rate (k(on)) of at least 10. In some cases, the k(on) rate is at least 10. 7M -1 seconds -1 In some cases, the binding molecules are greater than or equal to 5×10 -1 seconds -1 , 10 -1 seconds -1 , 5×10 -2 seconds -1 , 10 -2 seconds -1 , 5×10 -3 seconds -1 , or 10 -3 seconds -1 For example, a binding molecule of the present disclosure may specifically bind redIL-33 with an off-rate (k(off)) of 5×10 -4 seconds -1 , 10 -4 seconds -1 , 5×10 -5 seconds -1 , or 10 -5 seconds -1 , 5×10 -6 seconds -1 , 10 -6 seconds -1 , 5×10 -7 seconds -1 Or 10 -7 seconds -1 It may be considered that the antibody binds to redIL-33, or a fragment or variant thereof, with an off-rate (k(off)) of 10 or less. In some cases, the k(off) rate is 10 or less. -3 seconds -1The following. IL-33 is an alarmin cytokine that is released rapidly and in high concentrations in response to inflammatory stimuli. RedIL-33 is converted to its oxidized state approximately 5-45 minutes after release into the extracellular environment (Cohen et al Nat Commun 6,8327(2015)). Without wishing to be bound by theory, binding to redIL-33 at these k(on) and / or k(off) rates can minimize exposure to redIL-33 prior to conversion from reduced to oxIL-33. Furthermore, the k(off) rate can prevent release of IL-33 from the binding molecule / antigen complex prior to degradation of the complex in vivo. These binding kinetics can also act to prevent conversion of redIL-33 to oxIL-33, thus preventing pathological signaling of oxidized IL-33 via RAGE / EGFR (as described in WO2021 / 089563, incorporated herein by reference).

[0137] In some cases, an IL-33 antibody or antigen-binding fragment thereof may competitively inhibit the binding of IL-33 to 33_640087-7B (described in WO 2016 / 156440). WO 2016 / 156440 discloses that 33_640087-7B binds to redIL-33 with particularly high affinity, attenuating both ST-2 and RAGE-dependent IL-33 signaling. An antibody or antigen-binding fragment thereof is considered to competitively inhibit the binding of a reference antibody to a given epitope if it specifically binds to that epitope to the extent that it blocks the binding of the reference antibody to that epitope to some extent. Competitive inhibition can be determined by any method known in the art, for example, solid-phase assays such as competitive ELISA assays, dissociation-promoted lanthanide fluorescent immunoassays (DELFIA®, Perkin Elmer), and radioligand binding assays. For example, one skilled in the art can determine whether antibodies or antigen-binding fragments thereof compete for binding to IL-33 by using an in vitro competitive binding assay, such as the HTRF assay described in paragraphs 881-886 of WO 2016 / 156440, which is incorporated herein by reference. For example, one skilled in the art can label 33_640087-7B with a donor fluorophore and mix multiple concentrations with a fixed concentration sample of acceptor fluorophore-labeled redIL-33. Fluorescence resonance energy transfer between the donor fluorophore and the acceptor fluorophore in each sample can then be measured to confirm binding characteristics. To elucidate competitively binding antibody molecules, one skilled in the art can first mix various concentrations of test binding molecules with a fixed concentration of labeled 33_640087-7B antibody. A reduction in FRET signal when the mixture is incubated with labeled IL-33 compared to a positive control of labeled antibody only indicates competitive binding to IL-33. An antibody or antigen-binding fragment thereof can be said to competitively inhibit binding of a reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0138] In various cases, the anti-IL-33 antibody or antigen-binding fragment thereof is selected from a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a recombinant antibody, an antigen-binding antibody fragment, a single chain antibody, a monomeric antibody, a diabody, a triabody, a tetrabody, a Fab fragment, an lgG1 antibody, an lgG2 antibody, an lgG3 antibody, and an lgG4 antibody. In some cases, the anti-IL-33 antibody or antigen-binding fragment is selected from the group consisting of a diabody, a triabody, a tetrabody, a Fab fragment, a single domain antibody, an scFv, and the dose is adjusted so that the binding sites are equimolar to those administered by the bivalent antibody.

[0139] In some cases, the anti-IL-33 antibody, or antigen-binding fragment thereof, binds to an IL-33 comprising the amino acid sequence of SEQ ID NO: 11. In various cases, the anti-IL-33 antibody, or antigen-binding fragment thereof, can bind to a mature form of a full-length IL-33 protein comprising the amino acid sequence of SEQ ID NO: 11. In various cases, the anti-IL-33 antibody, or antigen-binding fragment thereof, can bind to an IL-33 protein fragment comprising amino acids 72-270, 79-270, 95-270, 99-270, 107-270, 109-270, 111-270, or 112-270 of SEQ ID NO:11.

[0140] In various cases, the anti-IL-33 antibody or antigen-binding fragment thereof can bind to the reduced form (red-IL-33) and / or the oxidized form (ox-IL-33) of IL-33. In some cases, the anti-IL-33 antibody or antigen-binding fragment thereof can preferentially bind to the reduced form (red-IL-33) and / or the oxidized form (ox-IL-33) of IL-33.

[0141] In various cases, the anti-IL-33 antibody or antigen-binding fragment thereof may be an inhibitory antibody capable of inhibiting IL-33 or a fragment thereof as defined herein. In various cases, the inhibitory antibody may inhibit the association of IL-33 or a fragment thereof with the IL-33 receptor.

[0142] In some cases, the anti-IL-33 antibody comprises a light chain sequence set forth in SEQ ID NO:9 and a heavy chain sequence set forth in SEQ ID NO:10.

[0143] In some cases, the anti-IL-33 antibody comprises a light chain having the sequence set forth in SEQ ID NO:9 and a heavy chain having the sequence set forth in SEQ ID NO:10.

[0144] In some cases, the anti-IL-33 antibody comprises a light chain consisting of the sequence set forth in SEQ ID NO:9 and a heavy chain consisting of the sequence set forth in SEQ ID NO:10.

[0145] In some cases, the binding molecule inhibits the activity of IL-33ox. In some cases, the binding molecule inhibits the binding of IL-33ox to the RAGE / EGFR complex.

[0146] Composition and Administration The IL-33 antagonists in the medical uses and methods described herein may be administered to a patient in the form of a pharmaceutical composition.

[0147] Suitably, reference herein to an "IL-33 antagonist" may also refer to a pharmaceutical composition comprising an IL-33 antagonist. Suitably, a pharmaceutical composition may comprise one or more IL-33 antagonists.

[0148] Suitably, the IL-33 antagonists may be administered in a pharma- ceutical effective amount for the in vivo treatments described herein.

[0149] Suitably, the IL-33 antagonist or pharmaceutical composition thereof may be administered to humans or other animals in accordance with the above-mentioned methods of treatment / medical uses in an amount sufficient to produce a therapeutic effect.

[0150] Suitably, the IL-33 antagonist or pharmaceutical composition thereof may be administered to such humans or other animals in conventional dosage forms prepared by combining the IL-33 antagonist with conventional pharma- ceutically acceptable carriers or diluents in accordance with known techniques.

[0151] One of ordinary skill in the art will recognize that the form and nature of the pharma- ceutically acceptable carrier or diluent will depend on the amount of active ingredient with which it is to be combined, the route of administration, and other well-known variables. One of ordinary skill in the art will further appreciate that cocktails comprising one or more species of IL-33 antagonists may prove to be particularly effective.

[0152] The amount of IL-33 antagonist that can be combined with carrier materials to produce a single dosage form varies depending on the subject of treatment and the particular method of administration. Suitably, the pharmaceutical composition can be administered as a single dose, multiple doses, or over an established period of time in an infusion. Suitably, the dosing regimen can also be adjusted to provide the optimum response desired (e.g., a therapeutic or prophylactic response).

[0153] Preferably, the IL-33 antagonist is formulated for ease of administration and to promote stability of the IL-33 antagonist.

[0154] Preferably, the pharmaceutical compositions are formulated to contain a pharma- ceutically acceptable, non-toxic, sterile carrier, such as physiological saline, non-toxic buffers, preservatives, and the like.

[0155] Suitably, the pharmaceutical compositions may comprise a pharma- ceutically acceptable carrier, a sterile aqueous or non-aqueous solution, suspension, and / or emulsion.

[0156] Preferably, pharmaceutical compositions for injectable use may include sterile aqueous solutions (if water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In such cases, the compositions must be sterile and fluid to the extent that easy syringability exists. The compositions must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0157] Suitable formulations for use in the therapeutic methods disclosed herein are described in Remington's Pharmaceutical Sciences (Mack Publishing Co.), 16th ed. (1980).

[0158] Preferably, the prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents. In many cases, it will be preferable to include an isotonic agent in the pharmaceutical composition. Prolonged absorption of the injectable composition can be achieved by including an agent that delays absorption in the composition.

[0159] Preferably, sterile injection solution can be prepared by incorporating the required amount of IL-33 antagonist in a suitable solvent with one or a combination of the components listed herein as required, followed by filtration sterilization.Generally, dispersion is prepared by incorporating the active compound in a sterile medium, which contains a basic dispersion medium and other necessary components from those listed above.In the case of sterile powder for preparing sterile injection solution, the preparation method can be vacuum drying and freeze-drying, which can obtain a powder of the active ingredient and any additional desired ingredients from the solution previously sterile-filtered.

[0160] Methods for administering an IL-33 antagonist or a pharmaceutical composition thereof to a subject in need thereof can be readily determined by one of skill in the art.

[0161] Suitably, the route of administration of the IL-33 antagonist or pharmaceutical composition thereof may be, for example, oral, parenteral, by inhalation or topical. Suitably, the term parenteral as used herein includes, for example, intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or intravaginal administration.

[0162] Suitably, the IL-33 antagonist or pharmaceutical composition thereof may be administered orally in any acceptable dosage form including, for example, a capsule, tablet, aqueous suspension, or solution.

[0163] Suitably, parenteral formulations can be a single bolus dose, an infusion, or a loading bolus dose followed by a maintenance dose. These compositions can be administered at specific fixed or variable intervals, for example, once a day, or "as needed."

[0164] Suitably, the components recited herein above for preparing the pharmaceutical compositions described herein may be packaged and sold in kit form, which may bear a label or package insert indicating that the associated pharmaceutical composition is useful for treating a subject suffering from or susceptible to a disease or disorder. EXAMPLES

[0165] The airway epithelium plays a central role in the initiation and development of chronic airway diseases (Carlier et al Front. Physiol. 12, 691227 (2021)). Sustained exposure to pathogens and noxious stimuli can alter the structure and composition of the airway epithelium, leading to irreversible changes such as those that occur in chronic airway diseases (Carlier et al Front. Physiol. 12, 691227 (2021); Hogg et al Annu. Rev. Pathol. 4, 435-459 (2009)). Genetic analyses suggest that IL-33 drives the pathology of chronic airway diseases such as asthma and COPD. Rare loss-of-function mutations in IL-33 reduce the risk of asthma and COPD, whereas gain-of-function mutations are associated with an increased risk of COPD (Rabe et al Lancet Respir. Med. 9, 1288-1298 (2021); Smith et al PLoS Genet. 13, e1006659 (2017)). IL-33 binds to IL-1 receptor-like 1 (IL1RL1, also known as ST2) on the cell surface and activates the NF-κB inflammatory signaling pathway, leading to chronic airway inflammation (Liew et al Nat. Rev. Immunol. 16, 676-689 (2016)).

[0166] The effects of IL-33 on immune cells are well established. However, more recent evidence has emerged that IL-33 has direct effects on epithelial cells. In vitro epithelial injury assays and validated IL-33 pathway blocking reagents indicate that oxidized IL-33 (oxIL-33 or IL-33ox) signals through a newly described RAGE / EGFR signaling pathway that alters the functional dynamics of airway epithelia (as disclosed in WO2021 / 089563, which is incorporated herein by reference).

[0167] In the studies herein, we investigated the transcriptional dynamics of airway epithelia to more fully delineate the role of the RAGE / EGFR complex in health and disease.

[0168] IL-33ox Reverses epithelial cell fate IL-33 ox To investigate transcriptional changes in the lung epithelium driven by NKG2, normal human bronchial epithelial (NHBE) cells from healthy donors were differentiated into air-liquid interface (ALI) cultures, modeling the physiology of human epithelia (Zscheppang et al. Biotechnol. J. 13, 1700341 (2018)) (Figure 1).

[0169] IL-33 ox Treatment induced a plethora of transcriptional changes that contrasted with no treatment ( Fig. 2 ).

[0170] IL-33 ox reduced the expression of genes associated with epithelial cell differentiation and increased the expression of genes associated with the negative regulation of wound closure (data not shown). Genes associated with mitochondrial organization, ATP metabolism, endoplasmic reticulum / Golgi vesicle trafficking, and cellular stress markers were also upregulated (data not shown).

[0171] Next, we investigated the effect of IL-33 in ALI cultures from healthy donors. ox We investigated transcriptional changes induced by IL-33 at the single cell level (Hewitt et al Nat. Rev. Immunol. 21, 347-362 (2021)). We identified 15 cellular states in healthy, untreated ALI cultures that represent the major airway epithelial cell types and are consistent with the complex cellular heterogeneity observed in vivo (Jackson et al Cell Rep. 32, 107872 (2020); Ruiz Garcia et al Development 146, dev177428 (2019)) (Table 1). ox The percentage of secretory and basal cells increased, whereas the percentage of ciliated cells and rare cell types decreased, in healthy ALI cultures after treatment with IL-33 (Figure 3). Within the secretory cell population, IL-33 oxFollowing treatment, the proportion of mucus-producing cells increased, while the proportion of mature club cells (club 3, involved in epithelial defense and expressing high levels of SCGB1A1 and SCGB3A1 (Zuo et al Crit. Care Med. 198, 1375-1388 (2018)) decreased.

[0172] [Table 1]

[0173] [Table 2]

[0174] Differential gene expression analysis showed that club-related genes involved in epithelial defense functions (e.g., SCGB1A1, SCGB3A1, BPIFA1, WFDC2, and MSMB (Zuo et al Crit. Care Med. 198, 1375-1388 (2018); Akram et al Mucosal Immunol. 11, 71-81 (2018); Goldfarbmuren et al Nat. Commun. 11, 2485 (2020)) were downregulated in all secretory cells (Figure 4). Notably, the most highly differentially expressed genes in the single-cell data were consistent with the data from bulk RNA sequencing (data not shown). Taken together, these data suggest that IL-33 is downregulated in the secretory cells. ox Our results suggest that COPD may drive epithelial remodeling at the expense of club and ciliated cell status, reducing the intrinsic epithelial defense against infection. Such a phenotype may confer an increased risk of respiratory tract infections in subjects with COPD, which are the major cause of acute exacerbations of COPD.

[0175] Blocking IL-33 reverses key traits of COPD Exogenous IL-33 oxThe epithelial changes induced by long-term exposure to chlorhexidine were similar to those observed in the airway epithelium of patients with COPD (Kesimer et al N.Engl.J.Med.377,911-922(2017);Ha et al Pharmacology 97,84-100(2016);Boucher N.Engl.J.Med.380,1941-1953(2019);Kim et al Am.J.Respir.Crit.Care Med.187,228-237(2013)).

[0176] Inhibition of IL-33 with tozolaximab in COPD ALI cultures reduced the percentage of mucus-secreting cells and the amount of MUC5AC released (Figures 5-7). These effects were not observed after selective blockade of ST2 (data not shown), suggesting that IL-33 ox This suggests that may be a driving factor for this phenotype.

[0177] In addition, IL-33 ox Inhibition of signal transduction induced substantial transcriptomic changes, restoring genes associated with ciliated and club cells, thereby reversing the COPD phenotype of ALI cultures (Figures 8 and 9). We observed downregulation of genes known to be associated with goblet cell differentiation and carbohydrate biosynthesis, and upregulation of genes associated with detoxification function, club cells, and cilia organization and assembly (Figure 9).

[0178] Using single-cell transcriptomics, we investigated the effect of blocking endogenous IL-33 signaling in COPD ALI cultures (Figure 10). Although no major changes in the proportions of different cell states were observed, inhibition of IL-33 signaling upregulated genes associated with epithelial host defense function and club cell markers (e.g., SCGB1A1 and SCGB3A1) (Mootz et al Allergy https: / / doi.org / 10.1111 / all.15033,https: / / doi.org / 10.1111 / all.15033(2021)) (Figure 10).

[0179] Consideration This study identifies a previously unknown mechanism by which IL-33 alters the transcriptional and functional dynamics of airway epithelia through a newly described RAGE / EGFR signaling pathway. ox The present inventors have clarified the role of IL-33. ox We hypothesize that IL-33 plays a protective role in the lung during acute injury or infection, but excessive exposure during chronic injury disrupts normal repair processes, leading to epithelial dysfunction, mucosal hypersecretion, and pathogenesis. Recent clinical data have demonstrated clinical benefits of IL-33 inhibition in patients with COPD (Rabe et al. Based on our findings, we propose that IL-33 inhibition may be an effective treatment for COPD. ox and IL-33 red Therapies designed to inhibit both IL-33 and IL-33 signaling red We speculate that this will have a greater clinical impact than therapies that only target ST2-induced inflammation. Furthermore, our results suggest that tozolaximab may reverse the pathogenic phenotype of COPD epithelium and restore the defense mechanisms of club cells, thereby reducing infections that lead to COPD exacerbations, decreasing hospitalizations and increasing quality of life for patients.

[0180] Materials and Methods cell culture NHBE cells NHBE cells (Lonza, CC-2540) were cultured in complete BEGM (Lonza, CC-3171) with a supplement kit (Lonza, CC-4175) according to the manufacturer's protocol.

[0181] ALI culture Transwells (Corning, CLS3460 and CLS3470) containing 12 mm or 6.5 mm or 0.4 μm polyester membrane inserts were coated with CellAdhere Type I Collagen (Stemcell, 07001) diluted once in distilled H2O and incubated at 37°C for 1–16 h, followed by washing with PBS.

[0182] Lung epithelial cells from healthy controls (bronchial [Lonza, CC-2540] or small airways [Epithelix, EP61SA]) or patients with COPD (bronchial [Lonza, 195275] or small airways [Epithelix, EP66SA]) were grown in 4 T-175 flasks in Epix Medium (Propagenix, 276-201) for bronchial cells or small airway epithelial cell growth medium (PromoCell, C-21070) for small airway epithelial cells. Once confluent, passage 2 cells were cultured at 1 × 10 6 Cells were frozen at 10 cells / vial. Passage 2 cells were seeded into two T-75 flasks, grown to 80% confluence, washed and detached with 6 ml of trypsin (Lonza, CC-5034). The cell suspension was centrifuged at 1,200 RPM for 5 minutes and the cells were transferred to PneumaCult ALI medium (Stemcell, 05001) for bronchial cells or PneumaCult ALI-S medium (Stemcell, 05050) for peripheral airway cells at 8 × 10 5 The cells were resuspended at 1000 cells / ml and 0.5 ml and 0.25 ml were dispensed into 12 mm and 6.5 mm inserts, respectively, and 1 ml or 0.5 ml of ALI medium was added to the space under each insert. The cells were maintained in ALI medium until tight junctions were formed. The medium was then removed from the apical side and the cells were allowed to differentiate for 3 weeks, with medium being replaced on the basal side every 2-3 days.

[0183] Fully differentiated healthy cultures were either left untreated or treated with IL-33 ox (30ng / ml), unlabeled IL-33 C>SThe differentiated COPD cultures were either left untreated or treated with 1 μg / ml tozolaximab, 1 μg / ml NIP228 (IgG1 isotype control), 10 μg / ml mNIP228, 10 μg / ml anti-ST2, 1 μg / ml anti-RAGE (4F4), 1 μg / ml anti-EGFR (Millipore, clone LA1), or RAGE-FC (R&D Systems). In both healthy and COPD ALI cultures, treatment was provided in the basolateral side of the medium for 7 days. Medium was changed every 2–3 days. Antibodies used in this study are listed in Supplementary Table 5. Tozolaximab inhibits IL-33 red IL-33 by preventing it from interacting with ST2L red IL-33 by directly inhibiting ST2L signaling and preventing the formation of oxidized IL-33 ox -Indirectly inhibits RAGE / EGFR signaling (ESC paper in preparation).

[0184] Recombinant protein production Cloning and expression of IL-33 Wild-type (WT) human IL-33 (aa 112-270) UniProt accession number 095760 (IL-33 red ) and a variant with all four cysteine ​​residues mutated to oxidation-resistant serines (IL-33 C>S A cDNA molecule encoding IL-33 was synthesized by primer extension PCR and cloned into pJexpress 411 (DNA 2.0). WT IL-33 was stored in its reduced form (IL-33) in 2xDPBS storage buffer before being added to the culture medium. red Both sequences were modified to contain 10xhis, an Avi tag, and a factor Xa protease cleavage site (MHHHHHHHHHHAAGLNDIFEAQKIEWHEAAIEGR (SEQ ID NO: 12)) at the N-terminus. IL-33 red (N-terminally tagged His10 / Avi tag; WT, SEQ ID NO: 13) and IL-33 C>S(N-terminally tagged His10 / Avi tag; WT, SEQ ID NO: 14) was produced by transforming Escherichia coli BL21(DE3) cells, which were grown in autoinduction medium (Overnight Express Autoinduction System 1, Merck Millipore, 71300-4) at 37°C for 18 hours, harvested by centrifugation and stored at -20°C. Cells were resuspended in 2xDPBS containing Complete EDTA-free protease inhibitor cocktail tablets (Roche, 11697498001) and 50U / ml benzonase nuclease (Merck Millipore, 70746-3) and lysed by sonication. Cell lysates were centrifuged at 50,000xg for 30 minutes at 4°C. IL-33 protein was purified from the supernatant by immobilized metal affinity chromatography and further purified by size exclusion chromatography (SEC) using a HiLoad 26 / 600 Superdex 75pg column (GE Healthcare, 28989334). Peak fractions were analyzed by SDS PAGE. Fractions containing pure IL-33 were pooled and their concentration was determined by measuring absorbance at 280 nm. The final sample was analyzed by SDS-PAGE. Unlabeled IL-33 red or IL-33 C>S To generate IL-33, N-terminally labeled His10 / Avi tagged IL-33 was incubated with 10 units of Factor Xa (GE Healthcare, 27084901) per mg of protein in 2xDPBS at room temperature for 1 h. Unlabeled IL-33 was purified using SEC on a HiLoad 16 / 600 Superdex75 pg column (GE Healthcare, 28989333) at a flow rate of 1 ml / min.

[0185] IL-33 ox Preparation and purification of IL-33 red was oxidized by dilution in 60% IMDM (without phenol red) and 40% DPBS to a final concentration of 0.5 mg / ml. oxLabeling was performed by incubating with factor Xa (NEB, P8010L) at a final concentration of 1 μg per well for 120 min at 22 °C. ox The remaining IL-33 was removed from the sample. red To fully deplete, soluble human ST2 fused to human IgG1 Fc-His6 was incubated with the samples for 30 min at 22° C. Samples were concentrated and loaded onto a HiLoad 26 / 600 Superdex 75 column (GE Healthcare, 28989334) at a flow rate of 2 ml / min. Pure IL-33 ox Each fraction containing was tested for its ability to activate EGFR (homogeneous time-resolved fluorescence [HTRF] assay in A549 and NHBE cells). Active fractions were pooled, concentrated, and the final concentration of the sample was determined using UV absorbance spectroscopy at 280 nm. The quality of the final product was assessed by SDS-PAGE, high-speed SEC, and reverse-phase HPLC.

[0186] qPCR After 7 days of treatment, 4-week-old normal or COPD ALI cultures on 6.5 mm inserts were lysed for RNA analysis. Each ALI apical surface was incubated with 200 μl of PBS at 37° C. for 30 min. Direct-zol RNA Miniprep Kit (Zymo Research, R2050) was used for RNA extraction. For submerged cultures (A549 cells, HUVEC and NHBE cells), RNeasy Mini Kit (Qiagen, 74104) was used. cDNA was synthesized using High-Capacity RNA-to-cDNA Kit (Thermo, 4388950).

[0187] For RT-qPCR, 4 μl of cDNA, 5 μl of TaqMan Fast Advanced Master Mix (Thermo, 4444557), 0.5 μl of MUC5AC FAM probe (Thermo, Hs01365616_m1) or MUC2 (Thermo, Hs00894041_g1) or CST1 (Thermo, Hs00606961_m1) or ST2 long (Thermo, Hs00249389_m1) or ST2 short (Thermo, Hs01073297_m1), and 0.5 μl of GAPDH VIC probe (Thermo, Hs02786624_g1) were added to a MicroAmp EnduraPlate (Thermo, 4483273). Plates were sealed and briefly centrifuged before analysis using the QuantStudio7 Flex Real-Time PCR System (Thermo). ΔΔCT was calculated by normalizing data to untreated controls.

[0188] Bulk RNA sequencing RNA extracted from ALI cultures was processed externally by Source BioScience (Cambridge, UK). Libraries were prepared using the Illumina mRNA Standard Kit. Sequencing was performed on an Illumina NovaSeq 6000 System to generate 30M 150 base pair paired-end reads. RNA libraries were prepared according to the NEBNext Ultra II Directional RNA Sample Preparation Protocol for Illumina Paired-End Multiplexed Sequencing.

[0189] The quality of the sequenced libraries was evaluated using the STAR GRCh38 ensemble (v100) against the human genome. 49 Based on the alignment, MultiQC 48 The adapter trimming was confirmed using NGmerge 50 Gene expression quantification was performed using Salmon ES Cell Quantification (GRCh38 ensemble (v100) as reference).51 The bioinformatics workflow was performed using Nextflow. 52 and Bioconda software management tool 53 Differential expression analysis was performed using the "apeglm" 55 DESeq2 with fold change reduction 54 The experiment was carried out in R using the package. 56 The Benjamini-Hochberg method was used for multiple calibration of the fold changes and q values. Volcano plots showing fold changes and q values ​​were generated using Spotfire (TIBCO) data analysis software. Gene Set Variation Analysis (GSVA) 57 Using the public COPD patient gene expression dataset GSE37147 44 , GSE11784 46 , and GSE47460 45 Gene set enrichment scores were calculated per sample for the signatures generated in. Calculations were performed using the GSVA package in R. Patient groups were compared based on disease and smoking status for gene sets GSE37147 and GSE11784, and based on COPD severity by GOLD stage for GSE47460. Significance was calculated using one-way ANOVA, followed by post-hoc pairwise comparisons using Tukey's honest significant difference test performed in Prism9 (GraphPad).

[0190] Single-cell RNA sequencing Single cell suspensions of ALI cultures were generated as described in FACS analysis, washed twice with PBS / 0.04% BSA, and resuspended at 900 cells / μL. Cells were mixed with reagents from the Chromium Next GEM Single Cell 3' GEM Kit v3.1 (10x Genomics, 1000123) according to the manufacturer's instructions. Both the sample mixture and capture beads from the Chromium Next GEM Single Cell 3' Gel Bead Kit v3.1 (10x Genomics, 1000122) were loaded onto a microfluidic chip (Chromium Next GEM Chip G (10x Genomics, 2000177)) with the goal of capturing 8000 cells per sample. The chips were run through a Chromium Single Cell Controller (10x Genomics, GCG-SR-1) for single cell partitioning and barcoding, and cDNA was prepared from barcoded cells using Chromium Next GEM Single Cell 3' GEM Kit v3.1 (10x Genomics, 1000123). Data were aligned to the GRCh38-3.0.0 human reference genome using CellRanger v3.0.1 (10x Genomics). Normalization and downstream analysis were performed using Seurat v3.2.3 in R v3.6.3. 58The analysis was carried out using the package Seurat. Raw counts were normalized and scaled (default parameters) using the NormalizeData and ScaleData functions of Seurat. Uniform Manifold Approximation and Projection (UMAP) dimensionality reduction was obtained by applying the RunUMAP function of Seurat taking into account the top 2,000 most variable genes, and the first 25 principal components (PCA) were obtained using the RunPCA function of Seurat. For healthy donors (n=1), cell clustering was performed by applying the FindNeighbors and FindClusters functions of Seurat. A resolution of 0.5 was applied since it refined clusters containing goblet cell identity. Clusters that showed high similarity in the obtained dendrogram using BuildClusterTree were merged. Cell cluster markers were obtained using the FindAllMarkers function of Seurat (non-parametric Wilcoxon rank sum test) and used for annotation of the clusters. Cell types were manually annotated based on highly expressed genes in each cluster and known epithelial airway cell markers (Supplementary Fig. 1 and Supplementary Table 3). 17,20,59-61 Healthy donors were used as reference and cell annotations were projected onto COPD donors (n=3) with the TransferData function in Seurat. All differential gene expression analyses between individual treatments were performed using the FindMarkers and FindAllMarkers functions in Seurat with the non-parametric Wilcoxon rank sum test. Genes considered differentially expressed had a log fold change cutoff of 0.5 and P values ​​adjusted for multiple testing <0.001 (Bonferroni correction).

[0191] statistical analysis Statistical analysis was performed using R (v.4.0.2) or Prism 9 (GraphPad), which was also used to generate plots. Data are presented as means with standard errors of the mean and, unless otherwise indicated, one-way ANOVA followed by Tukey's test was performed for comparisons between more than two groups. A significance threshold of 0.05 was used for P values. Box plots were generated using the following parameters: the black horizontal line in each box represents the median. The boxes span from the first to the third quartile of values. The whiskers extend outwards from the box up to 1.5 × interquartile range (third quartile to first quartile). In each box, the lowest dot is the minimum value and the highest dot is the maximum value. All experiments are represented by several biological replicates or independent experiments unless otherwise stated. The number of replicates per experiment is indicated in the legend. Quantitative Venn diagrams of mass spectrometry data were generated using the Bioinformatics & Evolutionary Genomics web tool 62 All Western blots, co-immunoprecipitation experiments, FACS analyses, ELISAs, and RT-qPCR were independently replicated at least twice with similar results. No statistical methods were used to predetermine sample sizes.

[0192] Further reading 17 Jackson,NDet al.Single-cell and population transcriptomics reveal pan-epithelial remodeling in type 2-high asthma.Cell Rep.32,107872(2020). 20 Ruiz Garcia,S.et al.Novel dynamics ofHuman mucociliary differentiation revealed by single-cell RNA sequencing of nasal epithelial cultures.Development 146,dev177428(2019). 44 Steiling,K.et al.A dynamic bronchial airway gene expression signature of chronic obstructive pulmonary disease and lung function impairment.Am.J.Respir.Crit.Care Med.187,933-942(2013). 45 Tan,J.et al.Expression of RXFP1 is decreased in idiopathic pulmonary fibrosis.Implications for relaxin-based therapies.Am.J.Respir.Crit.Care Med.194,1392-1402(2016). 46 Tilley,A.E.et al.Biologic phenotyping of theHuman small airway epithelial response to cigarette smoking.PLoS One 6,e22798(2011). 48 Ewels,P.,Magnusson,M.,Lundin,S.& Kaller,M.MultiQC:summarize analysis results for multiple tools and samples in a single report.Bioinformatics 32,3047-3048(2016). 49 Dobin,A.et al.STAR:ultrafast universal RNA-seq aligner.Bioinformatics 29,15-21(2013). 50 Gaspar,J.M.NGmerge:merging paired-end reads via novel empirically-derived models of sequencing errors.BMC Bioinformatics 19,536(2018). 51 Patro,R.,Duggal,G.,Love,M.I.,Irizarry,R.A.& Kingsford,C.Salmon provides fast and bias-aware quantification of transcript expression.Nat.Methods 14,417-419(2017). 52 Di Tommaso,P.et al.Nextflow enables reproducible computational workflows.Nat.Biotechnol.35,316-319(2017). 53 Gruning,B.et al.Bioconda:sustainable and comprehensive software distribution for the life sciences.Nat.Methods 15,475-476(2018). 54 Love,M.I.,Huber,W.& Anders,S.Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.Genome Biol.15,550(2014). 55 Zhu,A.,Ibrahim,J.G.& Love,M.I.Heavy-tailed prior distributions for sequence count data:removing the noise and preserving large differences.Bioinformatics 35,2084-2092(2019). 56 Storey,J.D.& Tibshirani,R.Statistical significance for genomewide studies.Proc.Natl.Acad.Sci.USA 100,9440-9445(2003). 57 Hanzelmann, S., Castelo, R. & Guinney, J. GSVA: gene set variation analysis for microarray and RNA-seq data. BMC Bioinformatics 14, 7 (2013). 58 Stuart, T. et al. Comprehensive integration of single-cell data. Cell 177, 1888 - 1902.e1821 (2019). 59 Deprez, M. et al. A single-cell atlas of the Human Healthy airways. Am. J. Respir. Crit. Care Med. 202, 1636 - 1645 (2020). 60 Travaglini, K. J. et al. A molecular cell atlas of the Human lung from single-cell RNA sequencing. Nature 587, 619 - 625 (2020). 61 Vieira Braga, F. A. et al. A cellular census of Human lungs identifies novel cell states in Health and in asthma. Nat. Med. 25, 1153 - 1163 (2019). 62 Bioinformatics and evolutionary genomics. Https: / / bioinformatics.psb.ugent.be / cgi-bin / liste / Venn / calculate_venn.htpl

[0193] Array

[0194]

Table 3

Table 4

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Table 5

Claims

1. A pharmaceutical composition comprising an IL-33 antagonist for use in a therapeutic method for reducing or preventing respiratory tract infections in subjects with IL-33-mediated respiratory disorder, wherein the IL-33-mediated respiratory disorder is chronic obstructive pulmonary disease (COPD), the infection is a respiratory tract viral infection or a respiratory tract bacterial infection, and the respiratory tract viral infection is a respiratory tract viral infection caused by influenza virus (e.g., influenza virus A, influenza virus B), respiratory syncytial virus (RSV), adenovirus, metapneumonia virus, cytomegalovirus, parainfluenza virus (e.g., hPIV-1, hPIV-2, hPIV-3, hPIV-4), rhinovirus, coxsackievirus, echovirus, herpes simplex virus, or smallpox.

2. The pharmaceutical composition according to claim 1, wherein the infection is a respiratory tract bacterial infection caused by Chlamydia pneumoniae or Mycoplasma pneumoniae.

3. (a) The IL-33 antagonist inhibits the activity of IL-33ox, thereby increasing the activity of club cells in the airway epithelium. (b) The IL-33 antagonist inhibits the activity of IL-33ox, thereby increasing the total club cell area in the airway epithelium, and / or (c) The IL-33 antagonist inhibits the activity of IL-33ox, thereby increasing the mRNA expression level of one or more markers selected from SCGB1BA1, BPIFA1, SCGB3A1, WFDC2, MSMB, LTF, SLPI, C3, HLA-DRA, CXCL1, CD74, CXCL17, MDK, TGM2, HLA-DRB1, CXCL8, CXCL2, HLA-DRB5, CX3CL1, and HLA-DPA1 in the airway epithelium. The pharmaceutical composition according to claim 1 or 2.

4. The pharmaceutical composition according to claim 1 or 2, wherein the IL-33 antagonist inhibits the activity of IL-33ox, thereby increasing the mRNA expression level of one or more markers selected from SCGB1BA1, BPIFA1, SCGB3A1, WFDC2, MSMB, and LTF in the airway epithelium.

5. The pharmaceutical composition according to claim 4, wherein one or more markers are selected from SCGB1BA1 and BPIFA1.

6. The IL-33 antagonist inhibits the activity of IL-33ox, thereby inhibiting the activity of CCSP, SCGB3A1, WFDC2, beta-microseminoprotein, lactotransferrin, SPLUNC1, secretory leukocyte protease inhibitor (SLPI), complement C3, HLA-DR alpha chain, C-X-C motif chemokine ligand 1 (CXCL1), differentiation antigen group 74 (CD74), C-X-C motif chemokine 17 (CXCL17), midkine (MDK), protein-gamma-glutamyltransferase 2 ( The pharmaceutical composition according to claim 1 or 2, which increases the protein expression level of one or more markers selected from TGM2), HLA class II histocompatibility antigen, DRB1 beta chain (HLA-DRB1), chemokine (C-X-C motif) ligand 8 (CXCL8), chemokine (C-X-C motif) ligand 2 (CXCL2), HLA class II histocompatibility antigen, DRB5 beta chain (HLA-DRB5), chemokine (C-X3-C motif) ligand 1 (CX3CL1), and major histocompatibility complex class II, DP alpha 1 (HLA-DPA1).

7. The pharmaceutical composition according to claim 1 or 2, wherein the IL-33 antagonist inhibits the activity of IL-33ox, thereby increasing the protein expression level of one or more markers selected from CCSP, SCGB3A1, WFDC2, beta-microseminoprotein, lactotransferrin, and SPLUNC1 in the airway epithelium.

8. The pharmaceutical composition according to claim 7, wherein one or more markers are selected from CCSP and SPLUNC1.

9. The pharmaceutical composition according to claim 3, wherein the airway epithelium includes lower airway epithelium.

10. The pharmaceutical composition according to claim 9, wherein (a) the lower respiratory tract epithelium comprises cuboidal epithelium, or (b) the lower respiratory tract epithelium comprises squamous epithelium.

11. The pharmaceutical composition according to claim 3, wherein the airway epithelium includes upper airway epithelium.

12. The pharmaceutical composition according to claim 11, wherein the upper respiratory tract epithelium includes ciliated columnar epithelium.

13. The pharmaceutical composition according to claim 3, wherein the mRNA expression level is measured by qRT-PCR.

14. The pharmaceutical composition according to claim 6, wherein the protein expression level is measured by enzyme immunoassay (ELISA), immunohistochemistry (IHC), immunofluorescence, flow cytometry, or Western blotting.

15. The pharmaceutical composition according to claim 3, wherein the mRNA expression level is measured in a biological sample obtained from the subject.

16. The pharmaceutical composition according to claim 15, wherein the biological sample is selected from respiratory epithelial biopsy, bronchial swab, bronchoalveolar lavage fluid (BALF), sputum, serum, plasma, or nasal mucosal lining fluid.

17. The pharmaceutical composition according to claim 1 or 2, wherein the IL-33 antagonist inhibits the activity of IL-33ox, thereby increasing the protective function of club cells in the airway epithelium.

18. The pharmaceutical composition according to claim 17, wherein increasing the protective function of club cells in the airway epithelium comprises increasing the activity of one or more proteins selected from CCSP, SCGB3A1, WFDC2, beta-microseminoprotein, lactotransferrin, SPLUNC1, SLPI, C3, HLA-DRA, CXCL1, CD74, CXCL17, MDK, TGM2, HLA-DRB1, CXCL8, CXCL2, HLA-DRB5, CX3CL1, and HLA-DPA1.

19. The pharmaceutical composition according to claim 18, wherein increasing the protective function of club cells in the airway epithelium comprises increasing the activity of CCSP and / or SPLUNC1.

20. The pharmaceutical composition according to claim 1 or 2, wherein the use reduces the annual exacerbation rate in the subject, and / or the use reduces the frequency of acute exacerbations of COPD (AECOPD) in the subject.

21. The pharmaceutical composition according to claim 1 or 2, wherein the IL-33 antagonist is an IL-33ox antagonist.

22. The pharmaceutical composition according to claim 1 or 2, wherein the IL-33 antagonist is an antibody or an antigen-binding fragment thereof.

23. The pharmaceutical composition according to claim 22, wherein the antibody or antigen-binding fragment specifically binds to reduced IL-33 (IL-33red).

24. The pharmaceutical composition according to claim 1 or 2, wherein the IL-33 antagonist is an anti-IL-33 antibody comprising a VH domain having HCDR1 having the sequence described in SEQ ID NO: 1, HCDR2 having the sequence described in SEQ ID NO: 2, and HCDR3 having the sequence described in SEQ ID NO: 3, and a VL domain having LCDR1 having the sequence described in SEQ ID NO: 5, LCDR2 having the sequence described in SEQ ID NO: 6, and LCDR3 having the sequence described in SEQ ID NO:

7.

25. The pharmaceutical composition according to claim 1 or 2, wherein the IL-33 antagonist is an anti-IL-33 antibody comprising a VH domain having the sequence described in Sequence ID No. 4 and a VL domain having the sequence described in Sequence ID No.

8.

26. The pharmaceutical composition according to claim 1 or 2, wherein reducing respiratory tract infections means reducing the frequency of respiratory tract infections in the subject.

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