WO2016009253A1 - Recombinant interleukin-15 is a therapeutic agent for the treatment of airway hyperactivity, resistance (obstruction) and fibrosis in chronic asthma - Google Patents

Abstract

The invention method and composition disclosed herein generally related to the treatment and/or management of airway obstruction including resistance, hyperactivity and lung/esophageal fibrosis. We state that rIL-15 or IL-15 overexpressed by its agonist is a novel molecule for the treatment and management of pulmonary functional abnormalities like airway hyperreactivity, obstruction or fibrosis in chronic lung and esophageal inflammation. Chronic asthma is normally associated with the esophageal inflammation, a disease termed as eosinophilic esophagitis (EoE). In particularly invention is identifying the treatment strategy of airway hyperactivity, resistance, Obstruction and fibrosis in experimental asthma including the esophageal fibrosis in eosinophilic esophagitis. Our identified and preclinical tested rIL-15 protein or its super- agonist will be ready to be tested in the clinical trials of chronic asthma and eosinophilic esophagitis patients to treat airway hyperactivity resistance and fibrosis in patients.

Description

INVENTION TITLE

RECOMBINANT INTERLEUKIN-15 IS A THERAPEUTIC AGENT FOR THE TREATMENT OF AIRWAY HYPERACTIVITY, RESISTANCE (OBSTRUCTION) AND FIBROSIS IN CHRONIC ASTHMA

STATEMENT REGARDING THE USE OF NO FEDERAL FUND

[0001 ] The invention was made with "Elite Biosciences, LLC" on private and personal Funding

CROSS REFERERECES TO RELATED APPLICATION

[0002] The present application claims the benefit of priority under 35 U.S.C. ξ 1 19(c) to U.S. Provisional Application number 61/949,369, Recombinant lnterleukin-15 is a therapeutic agent for the treatment of airway obstruction and fibrosis in asthma, filed on March, 07, 2014, which is currently copending herewith and which is incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0003] The invention disclosed herein generally related to the treatment and/or management of airway obstruction including resistance, hyperactivity and lung/esophageal fibrosis. We state that rlL-15 or IL-15 overexpressed by its agonist is a novel molecule for the treatment and management of pulmonary functional abnormalities like airway hyperreactivity, obstruction or fibrosis in chronic lung and esophageal inflammation. Chronic asthma is normally associated with the esophageal inflammation, a disease termed as eosinophilic esophagitis (EoE) [Mishra et al. 2001, J. Clin. Invest. 2001, 103, 83-90].

BACKGROUND [0004] All publications mentioned herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually incorporated by the references. The following description includes information that can be useful in understanding the present subject matter. It is not an admission that any of the information provided herein is prior art of relevant to the presently claimed subject matter, or that any publication specifically or implicitly referenced is prior art.

[0005] Induced IL-15 expression has been observed across a range of pulmonary inflammation, including chronic obstructive airways disease, sarcoidosis and pulmonary tuberculosis, compare to the healthy volunteers or in those with asthma (Muro, et al J Allergy Clin Immunol, 108, 2001, 970-975), Additionally, IL-15 is also found induced in the sputum of steroid-treated patients compared with that of untreated asthmatic patients {Komai-Koma, et al Clin Exp Allergy, 31, 2001, 1441-1448). The IL-15 transgenic expression suppressed development of ovalbumin-induced Tc1 responses in asthma (ishimitsu, et al J Immunol, 166, 200), 1991-2001). Notably, on the basis of current data, IL-15 does not represent a therapeutic target in asthma. In contrast, IL-15 is shown as a pro-inflammatory factor in macrophage-dominant pulmonary conditions. IL-15- mediated T cell and macrophage activation have also been reported; but it has been also shown that IL-15 protects TNFa-induced leukocyte apoptosis (Hiromatsu, et al J Infect Dis, 187 2003, 1442-1451). Furthermore, IL-15 was shown to be an important trigger for IL-17-mediated pulmonary inflammation (Ziolkowska, J Immunol, 164, 2000, 2832-2838) and suggest a direct link between IL-15 and downstream leukocyte effector function and matrix damage.

[0006] The functional role of IL-15 in pancreatic pathology is suggested, as earlier studies identified IL-15 mRNA and protein expression in cytokine-exposed rodent islet cells (Cardozo, et at. Diabetes, 2001, 50: 909-920) suggesting a functional role early in disease onset. IL-15 is induced in the pancreatic islet cells; however, islet cell apoptosis is not protected by IL-15 (Cardozo et al. Diabetologia, 46 2003, 255-266). Together, these studies clearly implicate IL-15 in the inflammatory events leading to the onset of insularities and β islet cell destruction. Whether such pathophysiological elucidation can lead to therapeutic intervention remains unclear.

[0007] Keratinocytes express both IL-15 and IL-15Ra, suggesting a role in autocrine regulation of keratinocytes in dermal inflammatory responses. The dermal induction of IL-15 is associated with cutaneous malignancy

(Mohamadzadeh, J Immunol, 155 1995, 4492-4496 and Dobbeiing, Blood, 92 1998, 252-258). Additionally, local IL-15 expression has been characterized in a variety of inflammatory dermatitis; whereas, its reduced levels are reported in atopic dermatitis associated Th2-mediated inflammation (Rucker, et al. Ear J Immunol, 33, 2003, 3493-3503).

[0008] Asthma is a chronic, inflammatory pulmonary airway disease characterized by activation of inflammatory cells and mediators, variable obstruction, hyperresponsiveness and remodelling of the airway (Bousquet et al Am J. Res. Crit. Care Med 2000, 161:1720-1745; Postma and Kerstjens, Am J. Res. Crit. Care Med. 198, 158:S187-192; Skloot et al. 1985, 96:2393-2403). In the United States, nearly 25 million of the population, 8% adult and 9% children are suffering from asthma, a chronic inflammatory pulmonary disorder responsible for over 5000 deaths/year (http://www.aafa.org). Antigen presenting cells (APCs) interact with and activate T cell subsets (e.g. Th2) and initiate a series of immunological responses. A number of investigations provide evidence that asthma is a Th2-type malady that is induced in response to food and environmental antigens (Hogan et al. Nature immunology 2001, 2:353-360; Mishra et al. The Journal of clinical investigation 2001, 107:83-90; Rayapudi et al. Journal of leukocyte biology 2011, 88:337-346). [0009] The airway inflammatory response in asthma is characterized by induced expression of multiple genes encoding cytokines, chemokines, and adhesion molecules, which are associated with recruitment of eosinophils and Th2 lymphocytes (Cohn, et al. Annual Review of Immunology 2004, 22:789-815). In addition to the characteristic Th2-mediated eosinophilic inflammatory response found in the airway during acute asthma episodes, chronic asthma is characterized by structural changes that are termed airway remodeling (Cohn, et al. Annual Review of Immunology 2004, 22:789-815; Davies et al. J. Allergy Clin. Immunol. 2003, 111:215-225).

[0010] Remodeling-associated changes in the airway include peribronchial fibrosis with increased deposition of collagen (types I, III, and V), smooth muscle hypertrophy/hyperplasia, and mucus secretion (Cohn, et al. Annual Review of Immunology 2004, 22:789-815; Davies et al. J. Allergy Clin. Immunol. 2003, 111:215-225). Repeated cycles of inflammation and repair in the airway in chronic asthma are considered to be the driving force for airway remodeling.

[001 1 ] Recent studies in mice (Cho et a. Journal of clinical investigation 2004, 113:551-560; Humbles et al. Science 2004, 305:1776-1779) and humans have supported an important role for eosinophilic inflammation in allergen-induced airway remodeling. (Flood-Page, et al. The Journal of clinical investigation 2003, 112:1029-1036). Notably, eosinophils are not the only cells that promote remodelling, airway remodeling is also induced by Th2 and Th3 cytokines that also contributes to the development of airway hyper-reactivity and irreversible loss of lung function (Al Heialy et al. Respirology 2011, 16:589-597; Al-Muhsen et al. Journal of Allergy and Clinical Immunology 2011, 128:451-462). Fibrosis is important as it can occur early in the pathogenesis of asthma, and be associated with severity and resistance to therapy (Durrani et al. The Journal of allergy and clinical immunology 12011, 28:439-448). [0012] Fibrosis is not directly targeted or effectively treated by current asthma drugs including corticosteroids, characterization of airway fibrosis and how it is regulated will be essential for the development of novel therapies for asthma and other lung diseases (Becky Kelly, et al. American Journal of Respiratory and Critical Care Medicine 162:883-890). Airway and parenchymal fibrosis can also be caused by other chronic inflammatory processes, infections, and exposure to a number of environmental agents like asbestos, silica, ionizing radiation and adverse side effects of certain medications (du Bois, Journal of the European Respiratory Society 2011, 20:132-133; Jeffery, American journal of respiratory and critical care medicine 2001; Naik, et al. American journal of physiology.2012, 164.S28-38; Phillips et al. Pulmonary pharmacology & therapeutics 2012, 25:281-285. Wells and Keiieher American journal of respiratory and critical care medicine 2013, 187:677-679; Zolak and de Andrade, Immunology and allergy clinics of North America 2012, 32:473-485).

[0013] Despite the worldwide severity of fibrosis in asthma and other pulmonary disorders, no affective therapy for patients has materialized (Raghu et al. American Journal of Respiratory and Critical Care Medicine 2006, 174:810-816; Rodriguez et al. Thorax 2011, 66:1043-1050). Furthermore, airway hypersensitivity (AHR) induced by Th2 cytokines like IL-13 is implicated in mucus hypersecretion, eosinophil recruitment and goblet cell hyperplasia (Bala, et al. Med Wieku Rozwoj 2004, 8:33-42; Brombacher, et al. BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology 2000, 22:646-656; Bias and Lee, American Journal of Respiratory and Critical Care Medicine 2011, 183:957-958; Bias, et al. Chest 2003, 123.339S-345S).

[0014] Chronic asthma is a pulmonary disease characterized by inflammation and narrowing of the lung's small and medium airways, which eventuates in bronchial hyperactivity/obstruction and fibrosis (Kerstjens, Am J. Res. Crit. Care Med. 198, 158.S187-192; Skloot et al. 1985, 96:2393-2403). Bronchial obstruction and fibrosis is developed not only in chronic asthma; but also subsequent to cigarette smoking, certain viral infections, exposure to number of environmental pollutants, as adverse effects of certain medicines, and autoimmune diseases, (du Bois, Journal of the European Respiratory Society 2011, 20:132-133; Jeffery, American Journal of Respiratory and Critical Care Medicine 2001; Naik, et al. American Journal of Physiology.2012, 164:S28-38; Phillips et al. Pulmonary Pharmacology &Ttherapeutics 2012, 25:281-285. Wells and Keiieher American Journal of Respiratory and Critical Care Medicine 2013, 187:677-679; Zolak and de Andrade, Immunology and Allergy Clinics of North America 2012, 32:473-485).

[0015] About 140,000 Americans have been diagnosed with different types of pulmonary asthma including fibrosis. Apart from bronchial obstruction, airway hyperactivity and remodeling in chronic asthma, other pulmonary functional abnormalities like idiopathic pulmonary fibrosis (IPF) affects between 132,000- 200,000 people (Johnson and Raghu. European Respiratory Journal 2005, 26:755-758; Raghu et al. American Journal of Respiratory and Critical Care Medicine 2006, 174:810-816).

[0016] Approximately 50,000 new cases are diagnosed each year and as many as 40,000 Americans die from IPF each year (Johnson and Raghu. European Respiratory Journal 2005, 26:755-758; Raghu et al. American Journal of Respiratory and Critical Care Medicine 2006, 174:810-816) Therefore, a great need to continue with innovative fundamental studies to uncover new possibilities for the therapeutic interventions for airway hyperactivity/obstruction and fibrosis. Currently, anti-inflammatory corticosteroid inhalers and other medications are available to treat airway inflammation and obstruction. These treatments show a significant reduction in inflammation; but fail to restrict or reverse the progression of the bronchial airway obstruction and fibrosis. Our proposal provides a nonsteroidal treatment for airway obstruction and remodeling in chronic asthma.

[0017] IL-15 is a yC cytokine and a member of the four a-helix bundle cytokine family. IL-15 is produced by a number of inflammatory cells including dendritic cells and epithelial cells (Under et al. The Journal of Investigative Dermatology 1998, 110:457-458; Morelli, et al. Blood 2001, 98:1512-1523). IL-15 is a requisite for the generation or maintenance of specific hematopoietic lineages. In the absence of IL-15 or IL-15 receptor (IL-15R)a, defects are observed in naive and memory CD8 T cells, intestinal intraepithelial lymphocytes (IEL), and natural killer (NK) and NKT lineages (Kennedy and Park, Journal of Clinical Immunology 1996, 16:134-143; Lodolce, Immunity 1998, 9:669-676).

[0018] Depending on the lymphoid lineage or stage of differentiation, IL-15 can have various roles in development and homeostasis. IL-15 can act to increase survival, induce proliferation, and/or drive differentiation of hematopoietic cells. The receptor for IL-15 is composed of an IL-15Ra chain, capable of binding IL-15 with high affinity in the absence of other receptor subunits, the IL-15/IL-2 receptor β chain, and the yC chain (Anderson, et al. Journal of Biological Chemistry 1995, 270:29862-29869; Giri, et al. Journal of Leukocyte Biology 1995, 57:763-766).

[0019] Soluble IL-15 can bind the IL-15R complex and induce signals in a manner similar to other cytokines and cytokine receptors (Giri, et al. Journal of leukocyte biology 1995, 57:763-766; Grabstein, et al. Science 1994, 264:965- 968). In allergic inflammation models IL-15 has been shown to increase cytokine production (Mori, et al. Journal of immunology 1996, 156:2400-2405), stimulate antibody production (Ruckert et al. Journal of Immunology 1998, 174:5507- 5515), and inhibit apoptosis of T cells, B cells and epithelial cells (Bulfone-Paus et al. Nature Medicine 1997, 3:1124-1128; Under et al. The Journal of Investigative Dermatology 1998, 110:457-458). IL-15 has been implicated in the inflammatory responses of various infectious and autoimmune diseases including IBD, sarcoidosis, and chronic active hepatitis (Agostini et al. Journal of Immunology 1996, 157:910-918; Kakumu et al. Clin Exp Immunol 1997, 109:458- 463; Kirman and Nielsen, Am J Gastroenterol 1996, 91:1789-1794). [0020] Blocking of endogenous IL-15 in vivo by a soluble IL-15Ra (slL-15Ra) antagonist has previously been shown to be a very efficient treatment in inflammatory, collagen-induced arthritis and OVA-induced pulmonary inflammation {Ohteki, et al. Current Molecular Medicine 2002, 2:371-380 Ohteki et al. Nature Immunology 2001, 2:1138-1143; Ruckert et al. European Journal of Immunology 2005, 28:3312-3320). Although, it is clear that IL-15 is important for the development of the hematopoietic lineages and has a role in allergic inflammation, the mechanism of IL-15-mediated actions is not well defined. Therefore, it would be of interest to know the role and mechanism of IL-15-inuced allergic esophageal eosinophilic inflammation.

SUMMARY

[0021 ] Last decade, the health of the western world is threatened by immune- based diseases and currently is rising in prevalence (ISAAC. 1998. Lancet 351:1225-1232Broide, D.H. 2001. J Allergy Clin Immunol 108: S65-71). Experimentation in the asthma and eosinophilic esophagitis (EoE) field has largely focused on analysis of the cellular and molecular events induced by allergen exposure in sensitized animals and humans. Th2 and Th3 cytokines are thought to induce asthma including airway restriction (airway hyperactivity) and EoE pathogenesis through the secretion of an array of cytokines that activate inflammatory and residential effector pathway {Ray, A., and Cohn, L. J Clin Invest 1999, 104:985-993; Wills-Karp, M. Annu Rev Immunol 1999, 17:255-281; Rothenberg, et al. J Allergy Clin Immunol 108: 891-894; Mishra, A. Immunology and Allergy Clinic of North America, 2009; 20; 29-40).

[0022] More recently, attention has focused on the pathogenesis of lung, airway and esophageal remodeling in the setting of chronic lung and esophageal inflammation. In particular, it has been appreciated that the allergic lung and esophagus is characterized by thickening of the basement membrane, collagen deposition, and mucus metaplasia and these process occur in the development of lung or esophageal fibrosis in asthma and EoE. A number of reports indicate that IL-15 level is increased in the patients suffering from chronic asthma and EoE {Wills-Karp, M. et al. 1998, Science 282:2258-2261; Bochner, B.S. et al. Annu Rev Immnol 1994, 12:295-335; Wills-Karp, M. et al. J Allergy Clin Immunol 2001, 107:9-18; Lai, S. Yet al. Embo Journal 1996, 15:4506-4514; Zhu X. et al. Gastroenterology; 2010, 139:182-93).

[0023] The lung and esophagus are the primary sites of entry for noxious environmental or food allergens that can trigger airway or esophagus remodeling, which contributes significantly to chronic asthma and eosinophil esophagitis (EoE). The Important pathologic components associated to the tissue remodeling include fibrosis and abnormal innate and adaptive immune responses. The asthma and EOE patients are associated with structural changes within the lung and esophagus including sub-epithelial fibrosis as well as chronic eosinophil's infiltration.

[0024] Significant bodies of data have suggested that the fibroblast proliferation and collagen accumulation in lung and esophagus promotes tissue fibrosis. Here, we show that allergen challenge wild type mice develop esophageal and lung fibrosis. The anti-MBP immunostaining showed that Aspergillus fumigatus challenged mice significantly increased eosinophil's infiltration in the lung and esophagus and a pharmacological delivery of IL-15 significantly reduced proinflammatory cytokine IL-4 and IL-5 in the BALF and in lung homogenates. IL- 15 treated allergen challenged mice show a significantly reduced fibroblast and collagen accumulation in lungs and in esophagus.

[0025] Mechanistically, our in vitro studies indicate that IL-15 promotes apoptosis or death of primary lung fibroblast. In conclusion we first time report that recombinant (r) IL-15 treatment can be used as therapeutic agent for the treatment of lung and esophageal fibrosis as well as airway obstruction (hyperactivity) in bronchial asthma. BRIEF INVENTION DESCRIPTION

[00261 Recombinant IL-15 is a future therapeutic agent to treat the lung and esophageal fibrosis in asthma and eosinophilic esophagitis (EoE), not limited to only theses two diseases but may be applied to other tissue fibrosis. Most importantly, our data first time provide a therapeutic molecule (rlL-15) for the treatment of airway hyperreactivity and resistance in bronchial chronic asthma.

[0027] Currently no perfect therapy is available to treat this life threatening lung functional abnormalities. rlL-15 down regulates IL-13 and restricts goblet cell hyperplasia responsible for airway hyperreactivity and kills the fibroblasts that are responsible for collagen production, tissue remodeling and fibrosis. Fibroblast is the potent inducer of tissue fibrosis.

[0028] We would like to protect our invention by filling this international patent, so that our invention is protected from other investigators and no one can copy and perform these experiments in their facility and claim these inventions as their own. Here below we provide the experimental data that support our claim that rlL-15 or its related agonist treatment is a novel therapeutic strategy to treat thee life threatening functional lung abnormalities. Interestingly, tissue fibrosis is also one of the major characteristics tissue abnormality is in a number of cancers. Therefore, rlL-15 therapy may also be beneficial for the treatment of cancers, in particular to the pancreatic cancer, in which tissue fibrosis occurs.

DETAILED DISCRETIONS OF DRAWINGS and INVENTION

[0029] Figure 1. rlL-15 treatment reduces fibrosis-associated proteins like a- SMA and collagen-1 and TGF-β in the lung following the induction of experimental asthma in mice. The Western Blot immunobloting analysis of fibrosis associated proteins in the lung homogenates in response to aspergillus, saline and rlL-15 (15 microgram/2x/week) intratracheal ly treated aspergillus challenge mice is shown in the figure below. The aspergillus challenge (group 1 ) induces all the three proteins level compare to saline (group 2), but rlL-15 treated and aspergllus challenged (group 3) mice show reduced expression compare to the aspergillus treated (group 1 ) alone. GAPDH (housekeeping gene) expression shows that equal level of proteins is analyzed for a-SMA, TGF-β and collagen-1 protein levels in the lungs of each mouse. These data indicates that rlL-15 treatment protects fibrosis by down regulating fibrosis-inducing genes in the lung.

[0030] Figure: 2. rlL-15 pharmacological treatment protects mice from the induction of asthma and fibrosis associated Th2 (IL-5 and IL-13) and Th3 (TGF-beta) cytokines in Aspergillus-induced experimental asthma.

Pharmacological delivery of the rlL-15 (15 microgram/2x/week) intratracheally in the lungs of Aspergillus fumigatus challenged Balb/C mice reduces Th-2 (IL-4, IL- 5, and IL-13) and Th-3 (TGF-β) cytokines in the lung (A-C). Data shown is mean+/- SD, =1 1 mice/group. The cytokines levels in the lung homogenates were examined by performing ELISA analysis using the manufacturer protocol (B&D Biosciences). These data indicates that rlL-15 treatment protects fibrosis, airway hyperreativity and resistance by down regulating inflammation, fibrosis and airway hyperreactivity-inducing cytokines in the lung.

[0031] Figure 3. rlL-15 pharmacological treatment protects mice from the lung accumulation of fibroblast in Aspergillus-induced experimental asthma. The fibroblast accumulation in the lungs of saline, aspergillus and rlL- 15 (15microgram/2x/week) intratracheally delivered aspergillus challenged mice was detected by performing anti-FSP1 antibody. A representative photomicrograph of the anti-FSP-1 (fibroblast specific protein-1 ) immunostained lung sections in response to saline (A, (Original magnification 200x). The aspergillus challenged mice lung show high accumulation of fibroblast accumulation compare to saline challenged mice (B, original magnification 200x) and a reduced number of fibroblast accumulation in the lungs following rlL-15 treatment is shown (C). The Quantitative analysis of percent anti-FSP1 positive cells in saline, aspergillus and IL-15 treated aspergillus challenged groups of mice are shown (D). Data is expressed as mean ±SD, n=12/group. These data

indicates that rlL-15 treatment protects fibrosis by reducing the accumulation of fibroblast in the lung. Note Fibroblast are responsible for promoting fibrosis.

[0032]. Figure: 4. Histopathological representation of reduced collagen (indicator of tissue fibrosis) accumulation in the lung and esophagus of rlL- 15 treatment in the aspergillus-induced mouse model of asthma and EoE. The collagen accumulation in the lung and esophagus of mice following saline (1 A), aspergillus (1 B) challenged and Aspergillus challenged treated with recombinant rlL-15 treatment (15 microgram/2x/week) intratracheally is shown in the lung (1 C) (Original magnification 125x). The collagen accumulation following saline (2D), Aspergillus (2E) and rlL-15 treated Aspergillus challenged (2F) mouse esophagus is shown. Collagen accumulation is detected by trichrome staining, the saline treated mice show a base line collagen accumulation in the lung and esophagus that is significantly induced following Aspergillus treatment and reduced to baseline following rlL-15 treatment. Induced collagen accumulation is a characteristics feature of tissue remodeling and fibrosis.

[0033] Figure: 5. rlL-15 promotes concentration dependent fibroblast ( cells that promote fibrosis) apoptosis. A. Flowcytometric analysis of apoptosis cells and dead cells treated with or without rlL-15 for 24h. Annexin-V positive cells are the apoptotic cells, live dead marker are the dead cells (a) control cells without rlL-15, treated with medium alone (b). Fibroblasts treated with 10 pg/ml of rlL-15 (c). Fibroblasts treated with 100 pg/ml of rlL-15 (d). Fibroblasts treated with 10 ng/ml and (e) 100ng/ml rlL-15. In [B], we detect cleaved caspase detection a marker of apoptosis, following in vitro IL-15 treatment in cultured cells by immunoflorescence staining. These data clearly shows that recombinant rlL- 15 kills the fibroblast that is the major cells in promoting tissue fibrosis. The data is accordance with the other data shown in figure 3 that indicates rlL-15 treatment reduces the fibroblast in vivo accumulation in the lung following the induction of experimental asthma.

[0034]. Figure: 6. Pharmacological delivery of rlL-15 reduces goblet cell hyperplasia (cells responsible for mucus production and airway resistance) in the lungs of mice following the induction of Aspergillus-induced experimental asthma. A representative light microscopic photomicrograph of lungs sections of saline, aspergillus and aspergillus challenged IL-15 treated (intratracheally, 15 microgram/2x/week) mice following Periodic Acid Schiff (PAS) staining show mucus producing cells (A to C) (Original magnification 200x). PAS positive cells are rarely observed in the saline treated mice (A). An increase number of PAS positive cells were observed in aspergillus challenged mice compared to saline challenged mice (B) that is significantly decreases in aspergillus challenged IL-15 treated mice (C). Quantitative analysis of percent (%) PAS positive cells in saline, aspergillus and rlL-15 treated aspergillus challenged groups of mice are shown (D). Data is expressed as mean ±SD, n=12/group. These data indicates that rlL-15 treatment reduces the number of mucus producing cells. Induced mucus secretion induces airway hyperreactivity and resistance in asthma. [0035] Figure: 7. rlL-15 improves airway hyperreactivity (PENH response) as measured by methyl choline response in aeroallergen-induced experimental asthma. The allergen challenged mice treated with IL-15 show improved airway hyperreactivty (hyperactivity) (green line graph) compared to the mice challenged with Aspergillus allergen, (pink line graph). It is interesting to note that, we found that airway hyperactivity (measured by increase PENH response values after methacholine [MCH] challenge) is protected in in allergen challenged and rlL-15 (15 microgram, intranasal, 2x/week) delivered mice compare to the wild-type Aspergillus challenged mice (Figure 7). Further work is in progress to define the mechanism by which IL-15 protects airway hyperactivity in experimental asthma. Herein, we show that rlL-15 treatment protect allergen- induced airway hyperreactivity in aspergillus-induced experimental asthma in mice.

[0036] Figure: 8. rlL-15 is a novel therapeutic molecule for the treatment of airway obstruction in experimental asthma. The protective role of rlL-15 on lung function was assessed further by determined by examining the airway resistance and compliance in the experimental asthmatic mice. Herein, we present an additional data of improved airway resistance in rlL-15 (intranasal, 15 g, 2 times/week) treated mice compare to non-treated mice following the induction of Aspergillus-induced experimental asthma. A baseline airway resistance using tracheal intubation was shown in saline treated and saline challenged mice; whereas, we show a significantly increased of the airway resistance at highest dose of methacholine (25mg/ml) concomitant with decreased compliance (Figure A, B) in saline treated Aspergillus challenged mice. The rlL-15 treated and Aspergillus challenged mice show a significantly (p<0.02) decrease of resistance compare to saline-treated Aspergillus challenged mice (Figure A). These data support that rlL-15 is a novel therapeutic agent for the treatment of allergen-induced airway obstruction in Aspergillus-induced experimental asthma in mice. These presented data indicates that rlL-15 treatment or IL-15 overexpression by other means for example in the form of IL- 15 superagonist is a novel therapy for airway obstruction in chronic asthma.

[0037] Figure: 9. Recombinant (r) IL-15 pharmacological delivery show comparable eosinophilic lung and esophageal inflammation in Aspergillus challenge and 11-15 treated Aspergillus challenged mice following the induction of experimental EoE and asthma in mice. Pharmacological delivery of the rlL-15 (15 microgram/2xweek) in the lungs of Aspergillus fumigatus

I. Histological evidence of eosinophilic esophageal inflammation in mice following saline, Aspergillus and IL-15 treated Aspergillus challenged mice.

II. Histological evidence of eosinophilic lung inflammation in saline, Aspergillus and rlL-15 treated Aspergillus challenged mice challenged wild type Balb/C mice increased eosinophils accumulation in the esophagus and rlL-15 treatment not able to protect tissue eosinophilia in the esophagus. A representative photomicrograph of the anti-MBP immunostaining of esophageal tissue sections is shown (I, A-C). Saline challenge (A), Aspergillus challenge (B) and rlL-15 treated Aspergillus challenge (C). The experiments was conducted three times with 4 mice each group. These data is in accordance with the earlier published data that IL-15 promote EoE (Zhu X. et al. Gastroenterology; 2010, 139:182-93)

Pharmacological delivery of the rlL-15 (15 microgram/2xweek) in the lungs of Aspergillus fumigatus challenged wild type Balb/C mice increased eosinophils accumulation in the lung and rlL-15 treatment not able to protect tissue eosinophilia in lung. A representative photomicrograph of the anti-MBP immunostaining of lung tissue sections is shown (II, A-C.) Saline challenge (A), Aspergillus challenge (B) and rlL-15 treated Aspergillus challenge (C). The experiments was conducted three times with 4 mice each group.

[0038] These presented data and drawings in this international patent application is a supportive evidence for the future treatment strategies for asthma pathogenesis. Data and drawings presented here strongly support that rlL-15 treatment is novel therapy for chronic asthma airway resistance, hyperactivity, obstruction and fibrosis. Although delivery of a protein to treat airway disease is expensive; therefore, alternatively IL-15 super-agonist will be developed to replace rlL-15 for therapeutic use.

[0039] TREATMENT compound (rlL-15) or agonist used as therapeutic agents BASED ON INVENTIONS as described above can be ADMIN ISTRATERED through oral or parenteral delivery routes (subcutaneous or Intravenous or intradermal, intratracheally) or via Inhaler. Such therapeutics can be administered by any pharmaceutically acceptable carrier, including, for example, any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substance is known in the art. Supplementary active compounds can also be incorporated into the compositions. A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Routes of administration include for example, but are not limited to intravenous, intramuscular and oral, and the like.

[0040] Solutions or suspensions used for appropriate routes of administration, including, for example but not limited to parenteral, intradermal, or subcutaneous application, and the like, can include, for example, the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidant such as ascorbic acid or solution bisulfate; chelating agents such as ethylenediaminetetraaccetic acid; buffers such as acetates, citrates, or phosphates and agents for the adjustment of tonicity such as sodium chloride o dextrose, and the like. The pH can be adjusted with acids or bases, such as for example, hydrochloric acid or sodium hydroxide, and the like. The parenteral preparations can be enclosed in, for examples, disposable syringes, or multiple dose vials made of glass or plastic, and the like.

[0041 ] Pharmaceutical compositions suitable for injectable use include, for example, sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparations of sterile injectable solutions or dispersion, and the like. For intravenous administration, suitable carriers include; for example, physiological saline, bacteriostatic water. Cremophor ELTM (BASF, Parsippany, NJ), Prolonged absorption of the injectable composition such as, for example aluminum monoestcarate and gelatin, and the like, phosphate buffered saline (PBS), and the like. In all cases, the composition should be fluid to the extent that easy syringability exists.

[0042] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powder for preparation of sterile injectable solutions, the perfect method of preparation are vacuum drying and freeze-dying which yields a powder of a active ingredients plus any additional desired ingredients from a previously sterile-filtered solution thereof.

[0043] Oral composition generally includes an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tables, for example, oral administration, the agent can be contained in enteric forms to survive the stomach or further coated or mixed to be released in a particular region of the gastrointestinal tract. Similarly, inhaled compound included an inert diluent that agent can be contained in the respective organ or airway and survive for longer time. [00444] It is specifically advantageous to formulate oral or parenteral compositions in dosage unit form for case of administration and uniformity of dosage. "Dosage unit form" as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated, each unit containing a pre-determined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Those of skill in the art know such details.

[0045] Certain embodiments of the invention include using quantification data from a gene-expression (mRNA) analysis and/or form a tissue protein analysis, from a sample of blood, urine, saliva, lung or esophageal tissue or the like. Embodiments of the invention include not only methods of conducting and interpreting such tests but also include reagents, kits, assay and the like, the conducting the analysis.

[0046] The results disclosed and concluded herein, for the induction of esophageal and lung fibrosis and their treatment strategy including airway hyperreactivity, restriction and compliance following rlL-15 treatment, provide a basis for proposing a novel molecule for the treatment of chronic asthma or esophageal fibrosis in EoE. For example the reduction in the tissue accumulation of collagen, profibrotic cytokines like TGF-beta and IL-13. The IL- 13 cytokine is also critical for inducing airway hyperreactivity in chronic asthma. Thus even in situation in which a given cytokine or inflammatory cells like fibroblast or eosinophils moderately or weakly affected, the combination with one or more others, provide an enhanced clarity on the affect of the treatment molecule, rlL-15. Accordingly, the methods and materials of the invention are expressly contemplated to be used both alone and in combination with others.

EXAMPLES [0054] The following non-limiting examples are provided to further illustrate embodiments of the inventions disclosed herein. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches that have been found to function well in the practice of he invasion, and thus can be considered to constitute examples of mode for its practice. However, those of skill in the at should, in light of the present disclosure, appreciate that may changes can e made in the specific embodiments that are delivered and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1

IL-15 is induced in experimental and human asthma

Earlier microarray analysis indicated that IL-15 is increased in the murine lung following the induction of experimental asthma (Zimmermann N. et al. Journal of clinical investigation 2003, 111(12): 1863-1874). In addition, earlier it has been also shown that the IL-15 gene is induced in human asthma (Muro, et al. Journal of allergy and clinical immunology 2001 , 108(6): 970-975). Therefore, to further confirm these data, we subsequently performed IL-15 protein levels in the BALF of saline/allergen challenged mice (experimental asthma) and normal individuals and asthma patients by performing ELISA. Our analysis demonstrated a ~ 6-7- fold induction in the levels of IL-15 in experimental and human asthma compared to the respective controls (Figure A, B). Data is shown mean +/-SD, n=5/group.

EXAMPLE 2

Pharmacological delivery of rlL-15 down regulates pro-fibrotic cytokines like IL-13, TGFp in experimental asthma.

Allergen challenged mice developed experimental asthma and lung fibrosis that mimics the human disease. Therefore, rlL-15 was pharmacologically delivered directly to the lungs of allergen challenged mice. The analysis indicated that rlL- 15 lung delivery down regulated cytokines such as IL-13 and TGFp levels to the baseline in the lungs compare to the Aspergillus challenged mice. Both cytokines were analyzed by performing ELISA in the mice lung homogenates following the induction of experimental asthma and data is shown in (Figure 2 A and B). Data is expressed as mean ± SD, n=10 mice per group.

EXAMPLE 3

Pharmacological delivery of rlL-15 reduces allergen-induced goblet cell hyperplasia in mouse model of experiential asthma.

The mucus production in asthma is related to the airway obstruction or hyperactivity. Therefore, the goblet cell hyperplasia was examined following the rlL-15 treated and allergen challenged mice. We show the data in figure 6 that rlL-15 treated mice have significantly reduced goblet cell hyperplasia compare to non-treated aspergillus challenged mice. Data is expressed as mean ± SD, n=10 mice per group.

EXAMPLE 4

Pharmacological delivery of rlL-15 reduces allergen-induced airway hyperreactivity (PENH response) in mice.

Based on our findings that the levels of IL-13 is reduced by rlL-15 pharmacological delivery, we next tested the hypothesis that allergen-induced airway hyperreactivity is protected in response to rlL-15 delivery in mice. The mice we challenged with the aspergillus with or without rlL-15 treatment and saline were tested for PENH values in response to methacholine by whole body plethysmography. rlL-15 treatment significantly diminishes airway hyperreactivity in murine aspergillus experimental model of asthma. rlL-15 treated aspergillus- challenged mice display a significantly reduced (p<0.01 ) PENH values compared to aspergillus-challenged (p<0.001 ) mice from the saline control mice. Data is expressed as mean ± SD n=12 mice/group and shown in figure 7. EXAMPLE 5

rlL-15 is a novel therapeutic molecule for the treatment of airway obstruction in experimental asthma.

The protective role of rlL-15 on lung function was assessed further by determined by examining the airway resistance and compliance in the experimental asthmatic mice. Herein, we demonstrated improved airway resistance in rlL-15 (15pg, 2 times/week) treated mice compare to non-treated mice following the induction of Aspergillus-induced experimental asthma. A baseline airway resistance using tracheal intubation was shown in saline treated and saline challenged mice; whereas, we show a significantly increased of the airway resistance at highest dose of methacholine (25mg/ml) concomitant with decreased compliance (Data is shown in Figure 8 A, B) in saline treated Aspergillus challenged mice.

EXAMPLE 6

Lung and esophageal fibrosis develops in human and experimental asthma and eosinophilic esophagitis.

Airway fibrosis is evident in human chronic asthma (Minshall, et al. American journal of respiratory cell and molecular biology 1997, 17(3): 326-333 and Roche et al. Lancet 1989, 1(8637): 520-524) and eosinophilic esophagitis (Mishra et al. 2008, Gastrentrology. 134, 204-214); therefore, we next established that similar airway fibrosis occurs in the murine model of allergen challenged asthma and we will employ for our studies. The experimental model of asthma involves intranasal challenge to the mice with saline or aeroallergens (Aspergillus fumigatus extract). Our experimentation indicated that aeroallergen challenged mice develop eosinophilic inflammation and airway fibrosis in experimental asthma and eosinophilic esophagitis that mimics human disease. Fibrosis was assessed in the animal model by staining tissue sections with Masson Trichrome staining. Thick perivascular and peribronchial collagen accumulation in the lung and lamina propria collagen in the esophagus was observed in the allergen challenged mice compared to the saline challenged mice. We show these data in Figure 3. Interestingly mice challenged with rlL-15 show decreased collagen in the esophagus and lung following allergen challenge.

EXAMPLE 7

Pharmacological delivery of rlL-15 reduces collagen accumulation in the lungs of experimental asthma model.

The total collagen accumulation in the lungs of aspergillus challenged rlL-15 treated and saline vehicle control treated murine lungs by Sircol reagent (Biodyne Science, UK). The total collagen analysis was performed in the whole lung homogenates following the manufacturers' protocol and normalized with the total protein of each lung. The analysis indicated that Aspergillus-induced total collagen was significantly reduced in the rlL-15 treated mice lung compare to only Aspergillus challenged mice, as shown in the figure. Data is expressed as mean ± SD, n=10 mice per group.

EXAMPLE 8

Pharmacological delivery of rlL-15 reduces lung expression of fibrosis- associated proteins in experimental asthma.

The pharmacological delivery of rlL-15 to the lung during aspergillus challenge reduces fibrosis-associated proteins such as, a-SMA and collagen-1. The protein expression was analyzed by performing a western blot analysis for both these proteins in the lung homogenates of aspergillus-challenged mice, aspergillus challenged treated with rlL-15 or saline challenged control mice. The Western immune-blotting analysis showed enhanced levels of a-SMA and collagen-1 proteins following aspergillus challenged compare to saline challenge. Importantly, rlL-15 treatment blocked the increase in both of these proteins to the baseline levels observed in saline treated mice. Data is shown in Figure 1 D. This data demonstrates rlL-15's anti-fibrotic effect in an experimental asthma mouse model. EXAMPLE 9

rlL-15 treatment promotes fibroblast apoptosis/death.

The lung primary fibroblast was exposed in vitro to the various concentration of rlL-15 for 24 hours. The fibroblast apoptosis/death was examined by flowcytometric analysis using AnnexinV and Live dead marker (eBioscience). Fibroblasts treated with various concentration of rlL-15 indicated a concentration dependent fibroblast apoptosis/death. Data is shown in the figure 4. The fibroblast apoptosis was also examined by caspase-3 cleavage in rlL-15 exposed primary lung fibroblasts. Fibroblasts treated with rlL-15 in vitro demonstrated greater Caspase-3 cleavage compared to fibroblasts not treated with rlL-15. DAPI mounting material is used to show the cell nucleus in blue and FITC stain for caspase-3 cleavage in green in the figures 4.

EXAMPLE 10

rlL-15 pharmacological delivery protects mice from fibroblast accumulation in the lung following the induction of experimental asthma.

The fibroblast accumulation in the lungs was detected by anti-FSP-1 (fibroblast specific protein-1 ) immunostaining in the tissue sections following aspergilus and rlL-15 treatment in mice. The allergen challenged mice accumulated a number of anti-fibroblast (FSP-1 ) antibody stained cells compare to saline challenged mice that accumulation of fibroblast is reduced in the lungs following recombinant IL- 15 treatment. Data is shown in the figure 2E.

[0053] Herein all these examples and drawing showed in this patent application, establishes a treatment strategy for allergen induced airway hyperreactivity, airway obstruction, and lung and esophageal fibrosis by using fundamental murine models of asthma and eosinophilic esophagitis. The details provide strong evidence that rlL-15 is a novel therapeutic protein to be used for the treatment for allergen-induced airway hyperreactivity, obstruction, resistance and fibrosis.

Claims

CLAIM What is claimed is:

1. A method treating patients having chronic asthma for airway obstruction, resistance and hyperactivity with airway fibrosis.

2. A method treating esophageal fibrosis develops in eosinophilic esophagitis patients.

The rlL-15 treatment in the mouse model of chronic asthma, airway hyperactivity and obstruction and resistance is improved.

The rlL-15 treatment in the mouse model of chronic asthma and eosinophilic esophagitis fibrosis is improved.

3. Methods used for claim 1 and 2 is experimental asthma and eosinophilic esophagitis in mice was induced by challenging the mice with intranasal Aspergillus fumigatus extract as per the protocol reported earlier (Mishra. et al 2001 , J. Clin Invest. 107, 83-90).

4. Methods used for claim 1 and 2 for rlL-15 recombinant protein was purchased from PeproTech, Rocky Hill, NJ, USA

5. Methods used claim 1 and 2 to determine the lung or esophageal fibrosis by using the Masson trichrome stain reported earlier (Mishra et al. 2008, Gastroenterology. 134, 204-214).

6. Method used claim 1 and 2 to detect mucus cells using the Periodic Acid Schiff (PAS) staining reported earlier (Carson, et al. American Society for Clinical Pathology Press, pp. 137-139. ISBN 978-0-89189-581 -7. Periodic acid-Schiff- -diastase (PAS-D, PAS diastase) stain is periodic acid-Schiff (PAS) stain used in combination with diastase, an enzyme that breaks down glycogen.

7. Method used claim 1 and 2 to detect profibrotic cytokines using the commercial ELISA kit of life technology by the method described earlier (Mishra et al. 2008, Gastroenterology. 134, 204-214).

8. Method used claim 1 and 2 to treat the mice with rlL-15 protein was earlier reported (Madhavi et al. Clinical & Translational Immunology (2014) 3, e9; doi: 10.1038/cti.2013.13; published online 10 Jan 2014).

9. Methods used for claim 1 and 2 to examine airway hyperactivity, restriction and compliance was earlier reported (Irvin and Bates, Respiratory Research, 2003, 4:4 and Takeda t al. J. Exp. Med. 1997, 188, 449-454)

10. A method used for claim 1 and 2 to detect fibroblast apoptosis using flowcytometry is earlier reported (Zhu et al. 2009, AJP-Gastroenterology, 297, 550-558).

1 1. Methods used for claim 1 and 2 to detect tissue fibroblast were detected commercially available anti-FSP1 antibody and eosinophil by anti-MBP antibody (purchase from Mayo clinic, Arizona from Dr J. Lee) by using the immunostaining protocol earlier reported (Mishra. et al 2001 , J. Clin Invest. 107, 83-90)

12. Methods used in claim 1 and 2 for any analysis kits or reagents that are not mentioned and used in any analysis and reported in this patient application was procured from the commercial vendors, such as life sciences, USA, eBiosciences, USA, R&D Biesciences, USA. Polly Scientific, USA, Fisher, USA. etc.