WO2015009996A1

WO2015009996A1 - Compositions and methods for treating eosinophilic esophagitis

Info

Publication number: WO2015009996A1
Application number: PCT/US2014/047153
Authority: WO
Inventors: David ARTIS; Mario NOTI; Elia Tait WOJNO
Original assignee: The Trustees Of The University Of Pennsylvania
Priority date: 2013-07-19
Filing date: 2014-07-18
Publication date: 2015-01-22

Abstract

The present invention relates to the treatment of eosinophilic esophagitis (EoE). In one embodiment, the invention relates to a new method of treating EoE by targeting TSLP and/or depleting basophils in the subject in need thereof.

Description

TITLE OF THE INVENTION

COMPOSITIONS AND METHODS FOR TREATING EOSINOPHILIC

ESOPHAGITIS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Serial

No. 61/856,185, filed July 19, 2013, the content of which is incorporated by reference herein in its entirety.

STATEMENT REGARDING FEDERALLYSPONSORED RESEARCH OR

DEVELOPMENT

This invention was made with government support under AI061570, AI087990, AI074878, AI095776, AI102942, AI095466, AI09560, and AI097333, awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Eosinophilic esophagitis (EoE) is a food allergy-associated inflammatory disease that affects children and adults (Liacouras et al, 2011, J.

Allergy Clin. Immunol. 128:3-20; Spergel, 2007, Curr. Opin. Allergy Clin. Immunol. 7:274-278; Abonia and Rothenberg, 2012, Annu. Rev. Med. 63:421-434). In industrialized countries, the incidence of EoE has increased dramatically in the past 30 years, resulting in a considerable public health and economic burden (Liacouras et al, 2011, J. Allergy Clin. Immunol. 128:3-20; Straumann and Simon, 2005, J.

Allergy Clin. Immunol. 115:418^119; Kapel et al, 2008, Gastroenterology 134:1316— 1321). EoE is characterized by esophageal eosinophilia and inflammation and histological changes in the esophagus associated with stricture, dysphagia and food impaction (Spergel, 2007, Curr. Opin. Allergy Clin. Immunol. 7:274-278; Liacouras et al, 2011, J. Allergy Clin. Immunol. 128:3-20; Abonia and Rothenberg, 2012, Annu. Rev. Med. 63 :421-434).

The observations that immune suppression or removal of dietary trigger foods can ameliorate EoE symptoms indicate that EoE is a food antigen- driven disease mediated by aberrant immune responses (Spergel, 2007, Curr. Opin. Allergy Clin. Immunol. 7:274-278; Liacouras et al, 2011, J. Allergy Clin. Immunol. 128:3-20; Markowitz et al, 2003, Am. J. Gastroenterol. 98:777-782). Therefore, targeting the dysregulated immunological pathways that underlie EoE could offer new treatment strategies for this disease. Studies investigating the immunological mechanisms that mediate EoE have shown that various immune cell types, including eosinophils, mast cells, type 2 helper T (TH2) cells that produce interleukin-4 (IL-4), IL-5, and IL-13, and IgE-producing B cells, may contribute to esophageal

inflammation during EoE (Spergel, 2007, Curr. Opin. Allergy Clin. Immunol. 7:274- 278; Liacouras et al, 2011, J. Allergy Clin. Immunol. 128:3-20; Abonia and

Rothenberg, 2012, Annu. Rev. Med. 63:421-434; Mulder and Justinich, 2010, Gut 59:6-7). Further, recent work has shown that there is a strong association between a gain-of- function polymorphism in the gene that encodes the predominantly epithelial cell-derived cytokine TSLP and the development of EoE in children (Rothenberg et al, 2010, Nat. Genet. 42:289-291; Sherrill et al, 2010, J. Allergy Clin. Immunol. 126: 160-165). TSLP is associated with multiple allergic disorders (Rothenberg et al, 2010, Nat. Genet. 42:289-291; Sherrill et al, 2010, J. Allergy Clin. Immunol.

126: 160-165; Ziegler, 2010, Curr. Opin. Immunol. 22:795-799; Ramasamy et al, 2011, J. Allergy Clin. Immunol. 128:996-1005; Liu et al, 2011, PLoS ONE

6:e25099; Hirota et al, 2011, Nat. Genet. 43:893-896; Hunninghake et al, 2010, Allergy 65: 1566-1575) and is thought to promote allergic inflammation by activating dendritic cells, inducing TH2 cell responses, supporting IgE production and eliciting the population expansion of phenotypically and functionally distinct basophils (Ziegler, 2010, Curr. Opin. Immunol. 22:795-799; Siracusa et al, 2011, Nature 477: 229-233; Siracusa et al, 2012, Adv. Immunol. 115: 141-159; Giacomin et al, 2012, J. Immunol. 189:4371-4378; Liu et al., 2007, Annu. Rev. Immunol. 25: 193-219;

Soumelis et al, 2002, Nat. Immunol. 3:673-680). However, whether TSLP directly promotes inflammatory responses associated with EoE and the mechanisms by which polymorphisms in TSLP and increased TSLP expression may contribute to the pathogenesis of EoE in patients has been unknown.

Currently, treatment strategies for EoE are nonspecific and impose a burden on patients. Although swallowed topical steroids can be effective in limiting EoE-associated inflammation, there are concerns regarding the long-term use of steroids, particularly in children (Liacouras et al, 2011, J. Allergy Clin. Immunol. 128:3-20; Straumann and Schoepfer, 2012, Nat. Rev. Gastroenterol. Hepatol. 9:697- 704). Adherence to an elemental diet that eliminates exposure to foods that trigger EoE results in resolution of symptoms in many patients; however, this approach requires disruptive changes in lifestyle and eating habits (Liacouras et al, 201 1, J. Allergy Clin. Immunol. 128:3-20; Straumann and Schoepfer, 2012, Nat. Rev.

Gastroenterol. Hepatol. 9:697-704; Arora et al, 2012, Curr. Gastroenterol. Rep. 14:206-215).

Thus, there is a need in the art for improved compositions and methods to treat EOE. The present invention satisfies this unmet need.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention provides a method for treating a disease or disorder comprising administering to a subject in need thereof an effective amount of an agent that targets thymic stromal lymphopoietin (TSLP).

In one embodiment, the disease or disorder is eosinophilic esophagitis

(EoE).

In one embodiment, targeting TSLP comprises one or more of the level of TSLP and the activity of TSLP.

In one embodiment, the agent that targets TSLP prevents the transcription of the TSLP gene or translation of the TSLP mRNA.

In one embodiment, the agent that targets TSLP interferes with the activity of TSLP.

In one embodiment, the agent that targets TSLP interferes with the interaction between TSLP and its receptor.

In one embodiment, the agent is selected from the group consisting of a small interfering RNA (siRNA), a microRNA, an antisense nucleic acid, a ribozyme, an expression vector encoding a transdominant negative mutant, an antibody, a peptide and a small molecule.

In one embodiment, the method further comprises administering an effective amount of an agent that targets a basophil.

In one embodiment, the agent that targets a basophil inhibits the activity of the basophil.

In one embodiment, the agent that targets a basophil depletes the basophil.

In one embodiment, the agent that targets a basophil is a basophil- depleting antibody. The invention also provides a method for treating a disease or disorder comprising administering to a subject in need thereof an effective amount of an agent that targets a basophil.

In one embodiment, the disease or disorder is eosinophilic esophagitis (EoE).

In one embodiment, the agent that targets a basophil depletes the basophil.

In one embodiment, the agent that targets a basophil is a basophil- depleting antibody.

In one embodiment, the invention further comprises administering an effective amount of agent that targets thymic stromal lymphopoietin (TSLP).

In one embodiment, the agent that targets TSLP prevents the transcription of the TSLP gene or translation of the TSLP mR A.

In one embodiment, the agent that targets TSLP is selected from the group consisting of a small interfering RNA (siRNA), a microRNA, an antisense nucleic acid, a ribozyme, an expression vector encoding a transdominant negative mutant, an antibody, a peptide and a small molecule.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and

instrumentalities of the embodiments shown in the drawings. Figure 1, comprising Figure 1A through Figure 1J, depicts the results of experiments investigating the experimental mouse model of EoE-like disease. (Figure 1A) Schematic of EoE-like disease mouse model in which WT BALB/c mice are epicutaneously sensitized for 14 d with OVA on a developing atopic dermatitis- like skin lesion, challenged intragastrically (i.g.) with OVA on days 14 and 17.5 and sacrificed (sac.) at day 18. (Figure IB) TSLP (ng per mg of ear skin) expression in supernatants of overnight-cultured skin (ears) measured by ELISA. Data are from one experiment (EtOH + OVA, n = 3; MC903, n = 3; MC903 + OVA, n = 4) and are representative of three independent replicates. EtOH, ethanol. (Figure 1C)

Histological sections (H&E staining) from the esophagus. Arrowheads identify tissue- infiltrating eosinophils. Scale bar, 25 μιη. (Figure ID) Number of eosinophils per HPF in the esophagus. (Figure IE) Representative flow cytometry plots showing frequencies of eosinophils in esophageal tissues. Data in Figure lC-Figure IE are from one experiment (EtOH + OVA, n = 3; MC903, n = 3; MC903 + OVA, n = 4) and are representative of three or more independent replicates. (Figure IF)

Frequencies of eosinophils in esophageal tissues, as measured by flow cytometry. Data are from three pooled experiments (EtOH + OVA, n = 7; MC903, n = 8; MC903 + OVA, n = 11). (Figure 1G) Immunofluorescence staining for eosinophils (Siglec-F- specific mAb, red) in esophageal tissues. Counterstaining with DAPI (blue). Scale bar, 25 μιη. Images are representative of two controls and three EoE-like disease samples. (Figure 1H) Representative EM image of an eosinophil in the esophagus of control mice with intact granules with electron dense cores (left) or degranulating eosinophils in MC903 + OVA-treated mice (right), showing loss of electron density in granule cores (red arrow), granule extrusion channels (blue arrow) and loss of granule contents (purple arrow). Scale bar, 2 μιη. (Figure II) mRNA expression of TH2 cytokines (114, 115, 1113), the basophil-specific protease Mcpt8 and Tslp in the esophagus. Data depicted are from one experiment (EtOH + OVA, n = 3; MC903, n = 3; MC903 + OVA, n = 4) and are representative of three independent replicates, y axis shows fold induction compared to controls. (Figure 1 J) Representative images of esophagi, with incidence of impaction. Arrowheads identify impacted food. Data depicted are from two pooled experiments (EtOH + OVA, n = 7, MC903 + OVA, n = 9). All parameters were assessed 12 h post- final oral antigen challenge. Data in Figure 1A-Figure II are from mice challenged twice with OVA, and data in Figure 1 J are from mice challenged six times with OVA. Results are shown as mean ± s.e.m., and a nonparametric, one-way Kruskal-Wallis analysis of variance (ANOVA) with Dunn's post hoc testing was used to determine significance. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2, comprising Figure 2A through Figure 2H, depict the results of experiments demonstrating that TSLP-TSLPR interactions are crucial for the pathogenesis of EoE-like disease. (Figure 2A) Histological sections (H&E staining) from the esophagus of BALB/c Tslpr^+I+ or BALB/c Tslpr ^ mice. Arrowheads identify tissue-infiltrating eosinophils. Scale bar, 25 μιη. (Figure 2B) Number of eosinophils per HPF in the esophagus. (Figure 2C) Representative flow cytometry plots showing frequencies of eosinophils in esophageal tissues. Data in Figure 2A- Figure 2C are from one experiment (Tslpr^+/+ EtOH + OVA, n = 3; Tslpr^+/+ MC903 + OVA, n = 5; Tslpr ' EtOH + OVA, n = 3; Tslpr ^ MC903 + OVA, n = 5) and are representative of three independent replicates. (Figure 2D) Frequencies of eosinophils in esophageal tissues, as measured by flow cytometry. Data are from three pooled experiments ( Tslpr EtOH + OVA, n = 5; Tslpr MC903 + OVA, « = 1 1; Tslpr'^' EtOH + OVA, n = 5; Tslpr ^ MC903 + OVA, n = 12). (Figure 2E) Histological sections (H&E staining) from the esophagus of WT BALB/c mice treated with an isotype control or TSLP-specific mAb (anti-TSLP). Arrowheads identify tissue- infiltrating eosinophils. Scale bar, 50 μιη. (Figure 2F) Number of eosinophils per HPF in the esophagus. (Figure 2G) Representative flow cytometry plots showing frequencies of eosinophils in esophageal tissues. Data in Figure 2E-Figure 2G are from one experiment (EtOH + OVA + IgG, n = 3; MC903 + OVA + IgG, n = 3; MC903 + OVA + anti-TSLP mAb, n = 3) and are representative of three independent replicates. (Figure 2H) Frequencies of eosinophils in esophageal tissues, as measured by flow cytometry. Data are from three pooled experiments (EtOH + OVA +IgG, n = 5; MC903 + OVA + IgG, n = 9; MC903 + OVA + anti-TSLP mAb, n = 10). All parameters were assessed 12 h after final oral antigen challenge. Data are from mice challenged twice with OVA. Results are shown as mean ± s.e.m., and a

nonparametric, one-way Kruskal-Wallis ANOVA with Dunn's post hoc testing or a nonparametric, two-way ANOVA with Bonferroni's post hoc testing were used to determine significance. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3, comprising Figure 3A through Figure 3F, depicts the results of experiments demonstrating that EoE-like disease development is independent of IgE. (Figure 3 A) OVA-specific serum IgE levels from BALB/c Tslpr^+/+ and Tslpr^~ mice. Data are from one experiment (EtOH + OVA Tslpr^+/+, n = 3; MC903 + OVA Tslpr . n = 4; EtOH + OVA Tslpr^~ , n = 3; MC903 + OVA Tslpr , n = 4) and are representative of three or more independent replicates. (Figure 3B) Histological sections (H&E staining) from the esophagus of BALB/c Igh-7^+l+ and BALB/c Igh-7^~ mice. Arrowheads identify tissue-infiltrating eosinophils. Scale bar, 25 μιη. (Figure 3C) Number of eosinophils per HPF. (Figure 3D) Representative flow cytometry plots showing frequencies of eosinophils in esophageal tissues. Data in Figure 3B-Figure 3D are from one experiment (EtOH + OVA Igh-7^+/+, n = 3; MC903 + OVA Igh-7^+/+, n = 3; EtOH + OVA Igh-T , n = 3; MC903 + OVA Igh-T , n = 4) and are representative of three or more independent replicates. (Figure 3E) Representative EM image of an eosinophil in the esophagus of control Igh-7^+l+ mice with intact granules with electron-dense cores (left) or degranulating eosinophils in MC903 + OVA treated Igh-7^+l+ (middle) or Igh-7^~ (right) mice in various stages of degranulation, with loss of electron density in granule cores (red arrows), formation of granule extrusion channels (blue arrow), complete loss of granule contents (green arrow) and formation of lipid vesicles (yellow arrow). Scale bar, 2 μιη. (Figure 3F) mRNA expression of TH2 cytokines in the esophagus. Data are from one experiment (EtOH + OVA Igh-7^+l+, n = 3; MC903 + OVA Igh-7^+l+, n = 3; EtOH + OVA Igh-T , n = 3; MC903 + OVA Igh-7^~ , n = 3) and are representative of two independent replicates, y axis shows fold induction compared to controls. All parameters were assessed 12 h after final oral antigen challenge. Data are from mice challenged twice with OVA. Results are shown as mean ± s.e.m., and a nonparametric, two-way ANOVA with Bonferroni's post hoc testing was used to determine significance. **P < 0.01; ***P < 0.001.

Figure 4, comprising Figure 4A through Figure 4J, depicts the results of experiments demonstrating that basophils promote EoE-like disease. (Figure 4A) Schematic of in vivo basophil depletion strategy. C57BL/6 (Baso-DTR ) or Baso- DTR⁺ mice were treated with diphtheria toxin (DT) during the course of epicutaneous sensitization. (Figure 4B) Histological sections (H&E staining) from the esophagus. Arrowheads identify tissue-infiltrating eosinophils. Scale bar, 25 μιη. (Figure 4C) Number of eosinophils per HPF in the esophagus. (Figure 4D) Representative flow cytometry plots showing frequencies of eosinophils in esophageal tissues. Data in Figure 4B-Figure 4D are from one experiment (Baso-DTR^~ EtOH + OVA, n = 3; Baso-DTR- MC903 + OVA, n = 3; Baso-DTR+ MC903 + OVA, n = 4) and are representative of three independent replicates. (Figure 4E) Frequencies of eosinophils in esophageal tissues, as measured by flow cytometry. Data depicted are from three pooled experiments (Baso-DTRT EtOH + OVA, n = 7; Baso-DTRT MC903 + OVA, n = 10; Baso-DTR⁺ MC903 + OVA, n = 11). (Figure 4F) Schematic of in vivo basophil depletion strategy using CD200R3 -specific mAb (anti-CD200R3) in WT BALB/c mice. (Figure 4G) Histological sections (H&E staining) from the esophagus.

Arrowheads identify tissue-infiltrating eosinophils. Scale bar, 25 μιη. (Figure 4H) Number of eosinophils per HPF in the esophagus. (Figure 41) Representative flow cytometry plots showing frequencies of eosinophils in esophageal tissues. Data in Figure 4G-Figure 41 are from one experiment (EtOH + OVA + IgG, n = 3; MC903 + OVA + IgG, n = 3; EtOH + OVA + anti-CD200R3 mAb, n = 3; MC903 + OVA + anti-CD200R3 mAb, n = 4) and are representative of three independent replicates. (Figure 4J) Frequencies of eosinophils in esophageal tissues, as measured by flow cytometry. Data are from three pooled experiments (EtOH + OVA + IgG, n = 8; MC903 + OVA + IgG, n = 9; EtOH + OVA + anti-CD200R3 mAb, n = 8; MC903 + OVA + anti-CD200R3 mAb, n = 10). All parameters were assessed 12 h after final oral antigen challenge. Data are from mice challenged twice with OVA. Results are shown as mean ± s.e.m., and a nonparametric, two-tailed Mann- Whitney /-test or a nonparametric, one-way Kruskal-Wallis ANOVA with Dunn's post hoc testing were used to determine significance. * P < 0.05; **P < 0.01 ; ***P < 0.001.

Figure 5, comprising Figure 5A through Figure 51, depicts the results of experiments demonstrating that neutralization of TSLP or depletion of basophils ameliorates established EoE-like disease. (Figure 5A) Schematic of treatment with TSLP-specific mAb in WT BALB/c mice with established EoE-like disease. (Figure 5B) Histological sections (H&E staining) from the esophagus. Arrowheads identify tissue-infiltrating eosinophils. Scale bar, 25 μιη. (Figure 5C) Frequencies of CD45⁺ cells in esophageal tissues, as measured by flow cytometry. (Figure 5D)

Representative flow cytometry plots showing frequencies and total numbers of eosinophils in esophageal tissues. Data in Figure 5B-Figure 5D are from one experiment (MC903 + OVA + IgG, n = 5; MC903 + OVA + anti-TSLP mAb, n = 5) and are representative of three independent replicates. (Figure 5E) Schematic of CD200R3 -specific mAb basophil-depletion treatment in WT BALB/c mice in established EoE-like disease. (Figure 5F) Histological sections (H&E staining) from the esophagus. Arrowheads identify tissue-infiltrating eosinophils. Scale bar, 25 μιη. (Figure 5G) Frequencies of CD45⁺ cells in esophageal tissues, as measured by flow cytometry. (Figure 5H) Representative flow cytometry plots showing frequencies and total numbers of eosinophils in esophageal tissues. Data in Figure 5F-Figure 5H are from one experiment (MC903 + OVA + IgG, n = 4; MC903 + OVA + anti-CD200R3 mAb, n = 5) and are representative of three independent replicates. (Figure 51)

Quantified incidence of food impaction. All parameters were assessed 12 h after final oral antigen challenge. Data are from mice challenged repeatedly with OVA. Results are shown as mean ± s.e.m., and a nonparametric, two-tailed Mann- Whitney ?-test was used to determine significance. *P < 0.05; **P < 0.01.

Figure 6, comprising Figure 6A through Figure 6G, depicts the results of experiments demonstrating that the TSLP -basophil axis is active in human subjects with EoE. (Figure 6A) Number of eosinophils per HPF in esophageal biopsy tissue sections quantified in pediatric control subjects (n = 19) and subjects with active EoE (n = 16) or inactive EoE (n = 15). (Figure 6B) Relative mRNA expression oi TSLP in esophageal biopsies of pediatric control subjects (n = 8) and subjects with active EoE (n = 25) or inactive EoE (n = 10). (Figure 6C) Immunohistochemical staining for TSLP1 in an esophageal biopsy. Data are representative of five subjects with active EoE. Scale bar (main and inset), 100 μιη. (Figure 6D) Basophils were identified by flow cytometry in esophageal biopsies from pediatric subjects with active EoE (plots are from one control subject and one subject with active EoE and are representative of 19 control subjects and 16 subjects with active EoE). (Figure 6E) Frequencies of basophils in the lin compartment in esophageal biopsies from pediatric control subjects (n = 19) and subjects with active EoE (n = 16) or inactive EoE (n = 15). (Figure 6F) Correlation of frequencies of basophils in pediatric esophageal biopsies and the number of eosinophils per HPF observed histologically (n = 50) (Spearman r = 0.6638). (Figure 6G) Frequencies of basophils in the PBMCs of pediatric subjects with EoE who were homozygous (n = 26) or heterozygous for the TSLPrisk polymorphism (n = 26) or who lacked the TSLPrisk polymorphism (n = 9), as identified by flow cytometry. All data are shown as mean ± s.e.m., and a

nonparametric, two-tailed Mann- Whitney ?-test or a nonparametric, one-way Kruskal- Wallis ANOVA with Dunn's post hoc testing were used to determine significance. Correlation analysis was performed using a nonparametric Spearman correlation (sensitivity analyses were performed), and a linear regression of the data is shown. *P < 0.05; **P < 0.01; ***P < 0.001. Figure 7, comprising Figure 7A through Figure 7G, depicts the results of experiments demonstrating epicutaneous sensitization with peanut antigen and antigen-induced immune responses in the GI tract. (Figure 7A) Histological sections (H & E staining) from the esophagus of WT BALB/c mice. Arrows identify tissue- infiltrating eosinophils. Scale bar: 25 μιη. (Figure 7B) Number of eosinophils per hpf in the esophagus. (Figure 7C) Representative flow cytometry plots showing frequencies of eosinophils in esophageal tissues. Data depicted in Figure 7A-Figure 7C are from one experiment (EtOH + CPE, n = 3; MC903, n = 3; MC903 + CPE, n = 4), and are representative of three independent experiments. Representative flow cytometry plots showing frequencies of eosinophils in (Figure 7D) the stomach and (Figure 7E) small intestine of control (EtOH + OVA) and MC903 + OVA treated WT BALB/c mice. TH2 cytokines in cell-free supernatants of antigen re-stimulated (Figure 7F) mesenteric lymph nodes (LN) and (Figure 7G) splenocytes of control (EtOH + OVA) and MC903 + OVA treated mice as measured by ELISA. Data depicted in Figure 7D-Figure 7G are from one experiment (EtOH + OVA, n = 3;

MC903 + OVA, n = 4), and are representative of three independent experiments. All parameters in (a-g) were assessed 12 h post- final oral antigen challenge. All data depicted in Figure 7A-Figure 7G are from mice challenged twice with OVA. Results are shown as mean ± sem, and a non-parametric, two-tailed Mann- Whitney Mest or a non-parametric, two-way ANOVA with Bonferroni post-hoc testing were used to determine significance. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 8, comprising Figure 8A and Figure 8B, depicts the results of experiments demonstrating that OCT analysis reveals epithelial thickening in the esophagus of mice with EoE-like disease. (Figure 8A) Representative OCT images of the esophagus of WT BALB/c mice. Scale bar: 200 μιη. (Figure 8B) Quantification of epithelial thickness of the esophagus as measured by OCT. Data depicted in Figure 8A and Figure 8B are from two pooled experiments (EtOH + OVA, n = 7; MC903 + OVA, n = 9). All parameters were assessed 12 h post-final oral antigen challenge. Data depicted are from mice challenged repeatedly with OVA to induce prolonged inflammation. Results are shown as mean ± sem.

Figure 9, comprising Figure 9A through Figure 9F, depicts the results of experiments demonstrating that epicutaneous sensitization and oral challenge with a model antigen in the context of elevated TSLP levels results in EoE-like disease. (Figure 9A) Schematic of sensitization in the presence of recombinant TSLP (rTSLP). WT BALB/c mice were injected intradermally (i.d.) on the ears with PBS as control or rTSLP in the presence or absence of OVA. (Figure 9B) Representative flow cytometry plots showing frequencies of eosinophils in esophageal tissues. Data depicted are from one experiment (PBS, n = 3; rTSLP, n = 3; rTSLP + OVA, n = 4), and are representative of three independent experiments. (Figure 9C) Schematic of sensitization on tape-stripped skin. WT BALB/c mice were shaved on the back and sensitized with PBS as control or OVA on tape-stripped skin. (Figure 9D) TSLP expression in cell-free supernatants of overnight-cultured skin (ears) as measured by ELISA. (Figure 9E) Representative flow cytometry plots showing frequencies of eosinophils in esophageal tissues. Data depicted in Figure 9D and Figure 9E are from one experiment ( Tslpr PBS + IgG, n = 3; Tslpr OVA + IgG, n = 3; Tslpr PBS + anti-TSLP mAb, n = 3; Tslpr OVA + anti-TSLP mAb, n = 3; Tslpr'- PBS, n = 3; Tslpr'- OVA, n = 3), and are representative of three independent experiments. (Figure 9F) Frequencies of eosinophils in esophageal tissues as measured by flow cytometry. Data depicted are from two pooled experiments (Tslpr^+/+ PBS + IgG, n = 5; Tslpr^+/+ OVA + IgG, n = 6; Tslpr^+/+ PBS + anti-TSLP mAb, n = 5; Tslpr^+/+ OVA + anti-TSLP mAb, n = 7; Tslpr'- PBS, n = 5; Tslpr'- OVA, n = 6). All parameters were assessed 12 h post- final oral antigen challenge. Data depicted are from mice challenged twice with OVA. Results are shown as mean ± sem, and a non-parametric, one-way Kruskal-Wallis ANOVA with Dunn's post-hoc testing or a nonparametric, two-way ANOVA with Bonferroni post-hoc testing were used to determine significance. *, P < 0.05.

Figure 10, comprising Figure 10A and Figure 10B, depicts the results of experiments demonstrating the reduced epithelial thickening of the esophagus and absence of food impaction in Tslpr^~'^~ mice. (Figure 10A) Quantification of epithelial thickness of the esophagus of BALB/c Tslpr^+/+ and BALB/c Tslpr^~'^~ mice as measured by OCT. Data depicted are from one experiment (EtOH + OVA Tslpr^+/+, n = 6; MC903 + OVA Tslpr^+/+, n = 9; EtOH + OVA Γ ^Α, n = 4; MC903 + OVA Tslpr^, n = 6). (Figure 10B) Table summarizing the incidence of food impaction in the esophagus. Data depicted are from two pooled experiments (EtOH + OVA Tslpr . n = l; MC903 + OVA Tslpr . n = 14; EtOH + OVA Tslpr'-, n = 6 ;

MC903 + OVA T lpr'-, n = 10). All parameters were assessed 12 h post-final oral antigen challenge. Data depicted are from mice challenged repeatedly with OVA to induce extended inflammation. Results are shown as mean ± sem. Figure 1 1, comprising Figure 1 1A through Figure 1 ID, depicts the results of experiments demonstrating peripheral basophil responses. (Figure 1 1 A) Representative flow cytometry plots showing frequencies of basophils in the periphery of BALB/c Tslpr^+/+ and BALB/c Tslpr^-1' mice. Data depicted are from one experiment (EtOH + OVA Tslpr . n = 3; MC903 + OVA Tslpr . n = 3; EtOH + OVA Tslpr-/-, n = 3; MC903 + OVA Tslpr^~'^~, n = 4), and are representative of three or more independent experiments. For statistical analysis, MC903 + OVA Tslpr^+/+ and MC903 + OVA Tslpr^~'^~ are compared. (Figure 1 IB) Representative flow cytometry plots showing frequencies of basophils in the periphery of BALB/c Igh- 7^+/+ and BALB/c Igh-7^~'^~ mice. Data depicted are from one experiment (EtOH+OVA Igh-7^+/+, n=3; MC903+OVA Igh-7^+/+, n=3; EtOH+OVA Igh-7^-, n = 3; MC903 + OVA Igh-7^~'^~, n = 4), and are representative of three independent experiments. For statistical analysis, MC903 + OVA Igh-7^+/+ and MC903 + OVA Igh-7^- are compared. (Figure 11C) Representative flow cytometry plots showing frequencies of basophils in the periphery of C57BL/6 Baso-DTR- and C57BL/6 Baso-DTR⁺ mice. Data depicted are from one experiment (EtOH + OVA Baso-DTR , n = 3; MC903 + OVA Baso-DTR- , n = 3; MC903 + OVA Baso-DTR⁺, n = 4), and are representative of three independent experiments. For statistical analysis, MC903 + OVA Baso-DTR- and MC903 + OVA Baso-DTR⁺ are compared. (Figure 1 ID) Representative flow cytometry plots showing frequencies of basophils in the periphery of control antibody or anti-CD200R3 mAb treated WT BALB/c mice. Data depicted are from one experiment (EtOH + OVA+ IgG, n = 3; MC903 + OVA + IgG, n = 3; EtOH + OVA + anti-CD200R3 mAb, n = 3; MC903 + OVA + anti-CD200R3 mAb, n = 4), and are representative of three independent experiments. For statistical analysis, MC903 + OVA + IgG or MC903 + OVA + anti-CD200R3 mAb are compared. All parameters were assessed 12 h post-final oral antigen challenge. All data depicted are from mice challenged twice with OVA. Results are shown as mean ± sem, and a non-parametric, two-way ANOVA with Bonferroni post-hoc testing was used to determine significance. *, P < 0.05; **, P < 0.01; ***, P < 0.001 .

Figure 12, comprising Figure 12A through Figure 12C, depicts the results of experiments demonstrating the reduced inflammatory responses in the esophagus of mice with EoE-like disease depleted of basophils. mRNA expression levels of (Figure 12A) TH2 cytokines (114, 1113), (Figure 12B) Cclll, and (Figure 12C) the basophil-specific protease Mcpt8 in the esophagus of C57BL/6 Baso-DTR- and C57BL/6 Baso-DTR⁺ mice. Data depicted in Figure 12A-Figure 12C are from one experiment (EtOH + OVA Baso-DTRr, n = 3; MC903 + OVA Baso-DTRr, n = 4; MC903 + OVA Baso-DTR⁺, n = 4), and are representative of two independent experiments. All parameters were assessed 12 h post-final oral antigen challenge. Data depicted are from mice challenged twice with OVA. Results are shown as mean ± sem, and a nonparametric, one-way Kruskal-Wallis ANOVA with Dunn's post-hoc testing was used to determine significance. *, P < 0.05; ***, P < 0.001.

Figure 13, comprising Figure 13 A through Figure 13C, depicts the results of experiments demonstrating elevated basophil responses in adult subjects with EoE positively correlate with esophageal eosinophil counts. (Figure 13 A) Number of eosinophils per hpf in adult esophageal biopsy tissue sections were quantified for control subjects (n = 6), subjects with active EoE (n = 9), and subjects with inactive EoE (n = 3). (Figure 13B) Frequencies of basophils in the lin compartment in esophageal biopsies from control subjects (n = 6), active subjects with EoE (n = 9), and inactive subjects with EoE (n = 3). (Figure 13C) Correlation of frequencies of basophils in the lin compartment in adult esophageal biopsies and the number of eosinophils per hpf observed histologically (n = 18) (Spearman r = 0.5282). Data are shown as mean ± sem. Correlation analysis was performed using a non-parametric Spearman correlation (sensitivity analyses were performed), and a linear regression of the data is displayed. *, P < 0.05.

Figure 14 depicts a proposed model of the relationship between a gain- of-function TSLP polymorphism (TSLPrisk), peripheral basophil responses, and the development of EoE in humans. In humans that do not carry the TSLPrisk polymorphism, exposure to antigens at epithelial barriers may induce TSLP expression which can result in local (1) and systemic (2) TSLP-elicited basophil responses. Encounter with the antigen in the esophagus may promote additional TSLP expression and mobilization of TSLP-elicited basophil populations from the blood to esophageal tissue (3). The studies presented here suggest that TSLP-elicited basophils and their products contribute to esophageal inflammation, including the accumulation of eosinophils, and other immune cells, such as T cells, B cells, and mast cells (4). In humans that carry the TSLPrisk polymorphism, exposure to antigens at epithelial barriers is more likely to result in enhanced TSLP expression (5) associated with TSLP-elicited peripheral basophilia (6), which together increase the likelihood of, but are not required for, developing EoE in the context of antigen-induced TSLP over- expression in the esophagus (7). It is currently unknown whether the TSLPrisk polymorphism and peripheral basophilia also promote increased accumulation of basophils and eosinophils in the esophagus in the context of EoE.

DETAILED DESCRIPTION

The present invention is directed to methods and compositions for treatment, inhibition, prevention, or reducing eosinophilic esophagitis (EoE) and related diseases in a subject. In one embodiment, the invention is related to compositions and methods for targeting one or more of thymic stromal lymphopoietin (TSLP) and basophils in order to ameliorate EoE and related diseases in a subject. Examples of related diseases include but are not limited to Barrets esophagus and other inflammatory diseases characterized by TSLP production, esosinophilia and basophils.

In one embodiment, the invention provides compositions and methods for affecting one or more of the level, production, and activity of TSLP as a therapy against EoE and related diseases. In another embodiment, the invention provides compositions and methods for affecting one or more of the level, production, and activity of basophils as a therapy against EoE and related diseases. Accordingly, the invention provides compositions and methods for targeting the TSLP -basophil axis for the clinical management of EoE and related diseased in a subject.

An aspect of the present invention comprises a method for interfering with the activity of a TSLP comprising administering to a subject an effective amount of a composition comprising an inhibitor of TSLP. In an embodiment of the present invention, the composition prevents the transcription of the TSLP gene or translation of TSLP mRNA. In another embodiment of the present invention, the composition interferes with one or more of the activity of TSLP and the interaction between TSLP and its receptor (e.g., TSLPR). In one embodiment, the composition that interferes with one or more of the activity of TSLP and the interaction between TSLP and TSLPR can comprise an antibody or a fragment thereof that binds to at least a portion of TSLP, an antibody or a fragment thereof that binds to at least a portion of TSLPR, a peptide, a nucleic acid, or small molecule.

An aspect of the present invention comprises a method for depleting basophils in a subject in order to ameliorate EoE and related diseases in a subject comprising administering to the subject an effective amount of a composition comprising a basophil depleting agent. In one embodiment, a basophil depleting agent includes but is not limited to a toxin, an antibody or fragment thereof, and the like.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice of and/or for the testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used according to how it is defined, where a definition is provided.

It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

"About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or in some instances ±10%, or in some instances ±5%, or in some instances ±1%, or in some instances ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

The term "abnormal" when used in the context of organisms, tissues, cells or components thereof, refers to those organisms, tissues, cells or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from those organisms, tissues, cells or components thereof that display the "normal" (expected) respective characteristic. Characteristics which are normal or expected for one cell or tissue type, might be abnormal for a different cell or tissue type.

The term "antibody," as used herein, refers to an immunoglobulin molecule which specifically binds with an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. The an antibody in the present invention may exist in a variety of forms where the antigen binding portion of the antibody is expressed as part of a contiguous polypeptide chain including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv) and a humanized antibody (Harlow et al, 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al, 1989, In: Antibodies: A

Laboratory Manual, Cold Spring Harbor, New York; Houston et al, 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

The term "antibody fragment" refers to at least one portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, sdAb (either VL or VH), camelid VHH domains, scFv antibodies, and multi-specific antibodies formed from antibody fragments. The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it was derived. Unless specified, as used herein an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

An "antibody heavy chain," as used herein, refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and which normally determines the class to which the antibody belongs.

An "antibody light chain," as used herein, refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappy (κ) and lambda (λ) light chains refer to the two major antibody light chain isotypes.

By the term "synthetic antibody" as used herein, is meant an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.

The term "antigen" or "Ag" as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an "antigen" as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.

A "disease" is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.

In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.

A disease or disorder is "alleviated" if the severity of a sign or symptom of the disease or disorder, the frequency with which such a sign or symptom is experienced by a patient, or both, is reduced.

The term "eosinophil protein" refers to, but is not limited to, eosinophil surface proteins, eosinophil granule proteins, and secretory products of eosinophils. "Effective amount" or "therapeutically effective amount" are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result. Such results may include, but are not limited to, the inhibition of virus infection as determined by any means suitable in the art.

As used herein "endogenous" refers to any material from or produced inside an organism, cell, tissue or system.

As used herein, the term "exogenous" refers to any material introduced from or produced outside an organism, cell, tissue or system.

The term "inhibit," as used herein, means to suppress or block an activity or function by at least about ten percent relative to a control value. Preferably, the activity is suppressed or blocked by 50% compared to a control value, more preferably by 75%, and even more preferably by 95%.

As used herein, an "instructional material" includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the compositions and methods of the invention. The instructional material of the kit of the invention may, for example, be affixed to a container which contains the nucleic acid, peptide, and/or composition of the invention or be shipped together with a container which contains the nucleic acid, peptide, and/or composition. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the compound be used cooperatively by the recipient.

"Isolated" means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

The term "operably linked" refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

"Parenteral" administration of an immunogenic composition includes, e.g., subcutaneous (s.c), intravenous (i.v.), intramuscular (i.m.), or intrasternal inj ection, or infus ion techniques .

The term "polynucleotide" as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric "nucleotides." The monomeric nucleotides can be hydro lyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR™, and the like, and by synthetic means.

As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

"Sample" or "biological sample" as used herein means a biological material from a subject, including but is not limited to organ, tissue, exosome, blood, plasma, saliva, urine and other body fluid. A sample can be any source of material obtained from a subject.

The terms "subject," "patient," "individual," and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human.

The term "therapeutic" as used herein means a treatment and/or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.

A "vector" is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, poly lysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.

Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

Description

The present invention is based on the identification of a role for TSLP and basophils in EoE and related diseases and that targeting the TSLP -basophil axis offers therapeutic outcome for subjects having EoE and related diseases. The results presented herein demonstrate that TSLP and basophils, but not IgE, are required for the development of experimental EoE-like disease in an animal model and that antibody-mediated neutralization of TSLP or depletion of basophils is effective in preventing the development of experimental EoE-like disease. Accordingly, the invention provides compositions and methods for targeting one or more of TSLP and basophils as a novel therapy for treating EoE and related diseases in a subject. Compositions

In various embodiments, the present invention includes compositions for inhibiting the level or activity of TSLP in a subject, a tissue, or an organ in need thereof. In various embodiments, the present invention includes compositions for depleting basophils in a subject, a tissue, or an organ in need thereof.

In various embodiments, the compositions of the invention decrease the amount of polypeptide of TSLP, the amount of mRNA of TSLP, the amount of activity of TSLP, or a combination thereof. In various embodiments, the compositions of the invention decrease the amount of basophils in a subject, the amount of activity of basophils in a subject, or a combination thereof.

It will be understood by one skilled in the art, based upon the disclosure provided herein, that a decrease in the level of TSLP encompasses the decrease in the expression, including transcription, translation, or both. The skilled artisan will also appreciate, once armed with the teachings of the present invention, that a decrease in the level of TSLP includes a decrease in the activity of TSLP. Thus, decrease in the level or activity of TSLP includes, but is not limited to, decreasing the amount of polypeptide of TSLP, and decreasing transcription, translation, or both, of a nucleic acid encoding TSLP; and it also includes decreasing any activity of TSLP as well. In one embodiment, the invention provides a generic concept for inhibiting one or more of TSLP and basophils as a therapy against EoE and related diseases. In one embodiment, the composition of the invention comprises an inhibitor of one or more of TSLP and basophils. In one embodiment, the inhibitor is selected from the group consisting of a small interfering RNA (siRNA), a microRNA, an antisense nucleic acid, a ribozyme, an expression vector encoding a transdominant negative mutant, an intracellular antibody, a peptide and a small molecule.

One skilled in the art will appreciate, based on the disclosure provided herein, that one way to decrease the mRNA and/or protein levels of TSLP in a cell is by reducing or inhibiting expression of the nucleic acid encoding TSLP. Thus, the protein level of TSLP in a cell can also be decreased using a molecule or compound that inhibits or reduces gene expression such as, for example, siRNA, an antisense molecule or a ribozyme. siRNA

In one embodiment, siRNA is used to decrease the level of TSLP. RNA interference (RNAi) is a phenomenon in which the introduction of double- stranded RNA (dsRNA) into a diverse range of organisms and cell types causes degradation of the complementary mRNA. In the cell, long dsRNAs are cleaved into short 21-25 nucleotide small interfering RNAs, or siRNAs, by a ribonuclease known as Dicer. The siRNAs subsequently assemble with protein components into an RNA- induced silencing complex (RISC), unwinding in the process. Activated RISC then binds to complementary transcript by base pairing interactions between the siRNA antisense strand and the mRNA. The bound mRNA is cleaved and sequence specific degradation of mRNA results in gene silencing. See, for example, U.S. Patent No. 6,506,559; Fire et al, 1998, Nature 391(19):306-31 1; Timmons et al, 1998, Nature 395:854; Montgomery et al, 1998, TIG 14 (7):255-258; David R. Engelke, Ed., RNA Interference (RNAi) Nuts & Bolts of RNAi Technology, DNA Press, Eagleville, PA (2003); and Gregory J. Hannon, Ed., RNAi A Guide to Gene Silencing, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2003). Soutschek et al. (2004, Nature 432: 173-178) describe a chemical modification to siRNAs that aids in intravenous systemic delivery. Optimizing siRNAs involves consideration of overall G/C content, C/T content at the termini, Tm and the nucleotide content of the 3' overhang. See, for instance, Schwartz et al, 2003, Cell, 115: 199-208 and Khvorova et al., 2003, Cell 1 15:209-216. Therefore, the present invention also includes methods of decreasing levels of TSLP at the protein level using RNAi technology.

In other related aspects, the invention includes an isolated nucleic acid encoding an inhibitor, wherein an inhibitor such as an siRNA or antisense molecule, inhibits TSLP, a derivative thereof, a regulator thereof, or a downstream effector, operably linked to a nucleic acid comprising a promoter/regulatory sequence such that the nucleic acid is preferably capable of directing expression of the protein encoded by the nucleic acid. Thus, the invention encompasses expression vectors and methods for the introduction of exogenous DNA into cells with concomitant expression of the exogenous DNA in the cells such as those described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York) and as described elsewhere herein. In another aspect of the invention, TSLP or a regulator thereof, can be inhibited by way of inactivating and/or sequestering TSLP, or a regulator thereof. As such, inhibiting the effects of TSLP can be accomplished by using a transdominant negative mutant.

In another aspect, the invention includes a vector comprising an siRNA or antisense polynucleotide. Preferably, the siRNA or antisense polynucleotide is capable of inhibiting the expression of TSLP. The incorporation of a desired polynucleotide into a vector and the choice of vectors is well-known in the art as described in, for example, Sambrook et al, supra, and Ausubel et al, supra, and elsewhere herein.

The siRNA or antisense polynucleotide can be cloned into a number of types of vectors as described elsewhere herein. For expression of the siRNA or antisense polynucleotide, at least one module in each promoter functions to position the start site for RNA synthesis.

In order to assess the expression of the siRNA or antisense polynucleotide, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers are known in the art and include, for example, antibiotic -resistance genes, such as neomycin resistance and the like.

Antisense nucleic acids

In one embodiment of the invention, an antisense nucleic acid sequence which is expressed by a plasmid vector is used to inhibit TSLP. The antisense expressing vector is used to transfect a mammalian cell or the mammal itself, thereby causing reduced endogenous expression of TSLP.

Antisense molecules and their use for inhibiting gene expression are well known in the art (see, e.g., Cohen, 1989, In: Oligodeoxyribonucleotides,

Antisense Inhibitors of Gene Expression, CRC Press). Antisense nucleic acids are DNA or RNA molecules that are complementary, as that term is defined elsewhere herein, to at least a portion of a specific mRNA molecule (Weintraub, 1990, Scientific American 262:40). In the cell, antisense nucleic acids hybridize to the corresponding mRNA, forming a double-stranded molecule thereby inhibiting the translation of genes.

The use of antisense methods to inhibit the translation of genes is known in the art, and is described, for example, in Marcus-Sakura (1988, Anal.

Biochem. 172:289). Such antisense molecules may be provided to the cell via genetic expression using DNA encoding the antisense molecule as taught by Inoue, 1993, U.S. Patent No. 5, 190,931.

Alternatively, antisense molecules of the invention may be made synthetically and then provided to the cell. Antisense oligomers of between about 10 to about 30, and more preferably about 15 nucleotides, are preferred, since they are easily synthesized and introduced into a target cell. Synthetic antisense molecules contemplated by the invention include oligonucleotide derivatives known in the art which have improved biological activity compared to unmodified oligonucleotides (see U.S. Patent No. 5,023,243).

Compositions and methods for the synthesis and expression of antisense nucleic acids are as described elsewhere herein.

Ribozymes

Ribozymes and their use for inhibiting gene expression are also well known in the art (see, e.g., Cech et al, 1992, J. Biol. Chem. 267: 17479-17482; Hampel et al, 1989, Biochemistry 28:4929-4933; Eckstein et al, International Publication No. WO 92/07065; Altman et al, U.S. Patent No. 5, 168,053). Ribozymes are RNA molecules possessing the ability to specifically cleave other single-stranded RNA in a manner analogous to DNA restriction endonucleases. Through the modification of nucleotide sequences encoding these RNAs, molecules can be engineered to recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech, 1988, J. Amer. Med. Assn. 260:3030). A major advantage of this approach is the fact that ribozymes are sequence-specific.

There are two basic types of ribozymes, namely, tetrahymena-type (Hasselhoff, 1988, Nature 334:585) and hammerhead-type. Tetrahymena-type ribozymes recognize sequences which are four bases in length, while hammerhead- type ribozymes recognize base sequences 11-18 bases in length. The longer the sequence, the greater the likelihood that the sequence will occur exclusively in the target mRNA species. Consequently, hammerhead-type ribozymes are preferable to tetrahymena-type ribozymes for inactivating specific mRNA species, and 18-base recognition sequences are preferable to shorter recognition sequences which may occur randomly within various unrelated mRNA molecules.

In one embodiment of the invention, a ribozyme is used to inhibit TSLP. Ribozymes useful for inhibiting the expression of a target molecule may be designed by incorporating target sequences into the basic ribozyme structure which are complementary, for example, to the mRNA sequence of TSLP of the present invention. Ribozymes targeting TSLP may be synthesized using commercially available reagents (Applied Biosystems, Inc., Foster City, CA) or they may be genetically expressed from DNA encoding them.

Small Molecules

When the inhibitor of the invention is a small molecule, a small molecule agonist may be obtained using standard methods known to the skilled artisan. Such methods include chemical organic synthesis or biological means.

Biological means include purification from a biological source, recombinant synthesis and in vitro translation systems, using methods well known in the art.

Combinatorial libraries of molecularly diverse chemical compounds potentially useful in treating a variety of diseases and conditions are well known in the art as are method of making the libraries. The method may use a variety of techniques well-known to the skilled artisan including solid phase synthesis, solution methods, parallel synthesis of single compounds, synthesis of chemical mixtures, rigid core structures, flexible linear sequences, deconvolution strategies, tagging techniques, and generating unbiased molecular landscapes for lead discovery vs. biased structures for lead development.

In a general method for small library synthesis, an activated core molecule is condensed with a number of building blocks, resulting in a combinatorial library of covalently linked, core-building block ensembles. The shape and rigidity of the core determines the orientation of the building blocks in shape space. The libraries can be biased by changing the core, linkage, or building blocks to target a

characterized biological structure ("focused libraries") or synthesized with less structural bias using flexible cores.

Antagonist

In another aspect of the invention, TSLP can be inhibited by way of inactivating and/or sequestering TSLP. As such, inhibiting the effects of TSLP can be accomplished by using a transdominant negative mutant. Alternatively an antibody specific for TSLP, otherwise known as an antagonist to TSLP may be used. In one embodiment, the antagonist is a protein and/or compound having the desirable property of interacting with a binding partner of TSLP (e.g., TSLP receptor; TSLPR) and thereby competing with the corresponding protein. In another embodiment, the antagonist is a protein and/or compound having the desirable property of interacting with TSLP and thereby sequestering TSLP. In another embodiment, the antagonist is a protein and/or compound having the desirable property of neutralizing TSLP.

As will be understood by one skilled in the art, any antibody that can recognize and bind to an antigen of interest is useful in the present invention. Methods of making and using antibodies are well known in the art. For example, polyclonal antibodies useful in the present invention are generated by immunizing rabbits according to standard immunological techniques well-known in the art (see, e.g., Harlow et al, 1988, In: Antibodies, A Laboratory Manual, Cold Spring Harbor, NY). Such techniques include immunizing an animal with a chimeric protein comprising a portion of another protein such as a maltose binding protein or glutathione (GSH) tag polypeptide portion, and/or a moiety such that the antigenic protein of interest is rendered immunogenic (e.g., an antigen of interest conjugated with keyhole limpet hemocyanin, KLH) and a portion comprising the respective antigenic protein amino acid residues. The chimeric proteins are produced by cloning the appropriate nucleic acids encoding the marker protein into a plasmid vector suitable for this purpose, such as but not limited to, pMAL-2 or pCMX.

However, the invention should not be construed as being limited solely to methods and compositions including these antibodies or to these portions of the antigens. Rather, the invention should be construed to include other antibodies, as that term is defined elsewhere herein, to antigens, or portions thereof. Further, the present invention should be construed to encompass antibodies, inter alia, bind to the specific antigens of interest, and they are able to bind the antigen present on Western blots, in solution in enzyme linked immunoassays, in fluorescence activated cells sorting (FACS) assays, in magenetic-actived cell sorting (MACS) assays, and in

immunofluorescence microscopy of a cell transiently transfected with a nucleic acid encoding at least a portion of the antigenic protein, for example.

One skilled in the art would appreciate, based upon the disclosure provided herein, that the antibody can specifically bind with any portion of the antigen and the full-length protein can be used to generate antibodies specific therefor. However, the present invention is not limited to using the full-length protein as an immunogen. Rather, the present invention includes using an immunogenic portion of the protein to produce an antibody that specifically binds with a specific antigen. That is, the invention includes immunizing an animal using an immunogenic portion, or antigenic determinant, of the antigen.

Once armed with the sequence of a specific antigen of interest and the detailed analysis localizing the various conserved and non-conserved domains of the protein, the skilled artisan would understand, based upon the disclosure provided herein, how to obtain antibodies specific for the various portions of the antigen using methods well-known in the art or to be developed.

The skilled artisan would appreciate, based upon the disclosure provided herein, that that present invention includes use of a single antibody recognizing a single antigenic epitope but that the invention is not limited to use of a single antibody. Instead, the invention encompasses use of at least one antibody where the antibodies can be directed to the same or different antigenic protein epitopes.

The generation of polyclonal antibodies is accomplished by inoculating the desired animal with the antigen and isolating antibodies which specifically bind the antigen therefrom using standard antibody production methods such as those described in, for example, Harlow et al. (1988, In: Antibodies, A Laboratory Manual, Cold Spring Harbor, NY).

Monoclonal antibodies directed against full length or peptide fragments of a protein or peptide may be prepared using any well-known monoclonal antibody preparation procedures, such as those described, for example, in Harlow et al. (1988, In: Antibodies, A Laboratory Manual, Cold Spring Harbor, NY) and in Tuszynski et al. (1988, Blood, 72: 109-1 15). Quantities of the desired peptide may also be synthesized using chemical synthesis technology. Alternatively, DNA encoding the desired peptide may be cloned and expressed from an appropriate promoter sequence in cells suitable for the generation of large quantities of peptide. Monoclonal antibodies directed against the peptide are generated from mice immunized with the peptide using standard procedures as referenced herein.

Nucleic acid encoding the monoclonal antibody obtained using the procedures described herein may be cloned and sequenced using technology which is available in the art, and is described, for example, in Wright et al. (1992, Critical Rev. Immunol. 12: 125-168), and the references cited therein. Further, the antibody of the invention may be "humanized" using the technology described in, for example, Wright et al, and in the references cited therein, and in Gu et al. (1997, Thrombosis and Hematocyst 77:755-759), and other methods of humanizing antibodies well- known in the art or to be developed.

The present invention also includes the use of humanized antibodies specifically reactive with epitopes of an antigen of interest. The humanized antibodies of the invention have a human framework and have one or more complementarity determining regions (CDRs) from an antibody, typically a mouse antibody, specifically reactive with an antigen of interest. When the antibody used in the invention is humanized, the antibody may be generated as described in Queen, et al. (U.S. Patent No. 6, 180,370), Wright et al, (supra) and in the references cited therein, or in Gu et al. (1997, Thrombosis and Hematocyst 77(4):755-759). The method disclosed in Queen et al. is directed in part toward designing humanized

immunoglobulins that are produced by expressing recombinant DNA segments encoding the heavy and light chain complementarity determining regions (CDRs) from a donor immunoglobulin capable of binding to a desired antigen, such as an epitope on an antigen of interest, attached to DNA segments encoding acceptor human framework regions. Generally speaking, the invention in the Queen patent has applicability toward the design of substantially any humanized immunoglobulin. Queen explains that the DNA segments will typically include an expression control DNA sequence operably linked to the humanized immunoglobulin coding sequences, including naturally-associated or heterologous promoter regions. The expression control sequences can be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells or the expression control sequences can be prokaryotic promoter systems in vectors capable of transforming or transfecting prokaryotic host cells. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the introduced nucleotide sequences and as desired the collection and purification of the humanized light chains, heavy chains, light/heavy chain dimers or intact antibodies, binding fragments or other immunoglobulin forms may follow (Beychok, Cells of Immunoglobulin Synthesis, Academic Press, New York, (1979), which is incorporated herein by reference).

The invention also includes functional equivalents of the antibodies described herein. Functional equivalents have binding characteristics comparable to those of the antibodies, and include, for example, hybridized and single chain antibodies, as well as fragments thereof. Methods of producing such functional equivalents are disclosed in PCT Application WO 93/21319 and PCT Application WO 89/09622.

Functional equivalents include polypeptides with amino acid sequences substantially the same as the amino acid sequence of the variable or hypervariable regions of the antibodies. "Substantially the same" amino acid sequence is defined herein as a sequence with at least 70%, preferably at least about 80%, more preferably at least about 90%, even more preferably at least about 95%, and most preferably at least 99% homology to another amino acid sequence (or any integer in between 70 and 99), as determined by the FASTA search method in accordance with Pearson and Lipman, 1988 Proc. Nat'l. Acad. Sci. USA 85: 2444-2448. Chimeric or other hybrid antibodies have constant regions derived substantially or exclusively from human antibody constant regions and variable regions derived substantially or exclusively from the sequence of the variable region of a monoclonal antibody from each stable hybridoma. Single chain antibodies (scFv) or Fv fragments are polypeptides that consist of the variable region of the heavy chain of the antibody linked to the variable region of the light chain, with or without an interconnecting linker. Thus, the Fv comprises an antibody combining site.

Functional equivalents of the antibodies of the invention further include fragments of antibodies that have the same, or substantially the same, binding characteristics to those of the whole antibody. Such fragments may contain one or both Fab fragments or the F(ab')2 fragment. The antibody fragments contain all six complement determining regions of the whole antibody, although fragments containing fewer than all of such regions, such as three, four or five complement determining regions, are also functional. The functional equivalents are members of the IgG immunoglobulin class and subclasses thereof, but may be or may combine with any one of the following immunoglobulin classes: IgM, IgA, IgD, or IgE, and subclasses thereof. Heavy chains of various subclasses, such as the IgG subclasses, are responsible for different effector functions and thus, by choosing the desired heavy chain constant region, hybrid antibodies with desired effector function are produced. Exemplary constant regions are gamma 1 (IgGl), gamma 2 (IgG2), gamma 3 (IgG3), and gamma 4 (IgG4). The light chain constant region can be of the kappa or lambda type.

The immunoglobulins of the present invention can be monovalent, divalent or polyvalent. Monovalent immunoglobulins are dimers (HL) formed of a hybrid heavy chain associated through disulfide bridges with a hybrid light chain. Divalent immunoglobulins are tetramers (H2L2) formed of two dimers associated through at least one disulfide bridge.

In one embodiment, the invention includes a TSLP-specific mAb that neutralizes TSLP. In another embodiment, the invention includes an antibody that can deplete basophils. In one embodiment, the antibody that can deplete basophils is the basophil-depleting CD200R3 -specific mAb. Modulators of Basophils

Included in the invention are compositions that act to modulate an activity of basophils. This can be done, for example, by modulating basophils.

"Activity," as used in context of basophils, refers to any function or process of a composition disclosed herein and includes, for example, transcription, translation, post-translational modification, translocation, homophilic or heterophils binding, secretion, endocytosis, or degradation. Disclosed therefore are compositions that inhibit one or more activities of basophils as provided herein. These compositions are referred to herein as basophil inhibitors. Inhibition or a form of inhibition, such as inhibit or inhibiting, as used herein means to restrain or limit. Reduce or a form of reduce, such as reducing or reduces, as used herein, means to diminish, for example in size or amount. It is understood that wherever one of inhibit or reduce is used, unless explicitly indicated otherwise, the other can also be used. For example, if something is referred to as "inhibited," it is also considered referred to as "reduced."

Therapeutics

The invention provides methods of treating EoE and related diseases comprising targeting one or more of TSLP and basophils. One aspect of the invention provides a method of treating EoE and related diseases in a subject in need thereof, the method comprising administering to the subject an effective amount of an inhibitor of TSLP. Another aspect of the invention provides a method of treating EoE and related diseases in a subject in need thereof, the method comprising administering to the subject an effective amount of a basophil-depleting agent.

Administration of the therapeutic agent in accordance with the present invention may be continuous or intermittent, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. The administration of the agents of the invention may be essentially continuous over a preselected period of time or may be in a series of spaced doses. Both local and systemic administration is contemplated. The amount administered will vary depending on various factors including, but not limited to, the composition chosen, the particular disease, the weight, the physical condition, and the age of the mammal, and whether prevention or treatment is to be achieved. Such factors can be readily determined by the clinician employing animal models or other test systems which are well known to the art

One or more suitable unit dosage forms having the therapeutic agent(s) of the invention, which, as discussed below, may optionally be formulated for sustained release (for example using microencapsulation, see WO 94/07529, and U.S. Pat. No. 4,962,091 the disclosures of which are incorporated by reference herein), can be administered by a variety of routes including parenteral, including by intravenous and intramuscular routes, as well as by direct injection into the diseased tissue. For example, the therapeutic agent or modified cell may be directly injected into the tumor. The formulations may, where appropriate, be conveniently presented in discrete unit dosage forms and may be prepared by any of the methods well known to pharmacy. Such methods may include the step of bringing into association the therapeutic agent with liquid carriers, solid matrices, semi-solid carriers, finely divided solid carriers or combinations thereof, and then, if necessary, introducing or shaping the product into the desired delivery system.

When the therapeutic agents of the invention are prepared for administration, they are preferably combined with a pharmaceutically acceptable carrier, diluent or excipient to form a pharmaceutical formulation, or unit dosage form. The total active ingredients in such formulations include from 0.1 to 99.9% by weight of the formulation. A "pharmaceutically acceptable" is a carrier, diluent, excipient, and/or salt that is compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof. The active ingredient for administration may be present as a powder or as granules; as a solution, a suspension or an emulsion.

Pharmaceutical formulations containing the therapeutic agents of the invention can be prepared by procedures known in the art using well known and readily available ingredients. The therapeutic agents of the invention can also be formulated as solutions appropriate for parenteral administration, for instance by intramuscular, subcutaneous or intravenous routes.

The pharmaceutical formulations of the therapeutic agents of the invention can also take the form of an aqueous or anhydrous solution or dispersion, or alternatively the form of an emulsion or suspension.

Thus, the therapeutic agent may be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and may be presented in unit dose form in ampules, pre-filled syringes, small volume infusion containers or in multi-dose containers with an added preservative. The active ingredients may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredients may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use. It will be appreciated that the unit content of active ingredient or ingredients contained in an individual aerosol dose of each dosage form need not in itself constitute an effective amount for treating the particular indication or disease since the necessary effective amount can be reached by administration of a plurality of dosage units. Moreover, the effective amount may be achieved using less than the dose in the dosage form, either individually, or in a series of administrations.

The pharmaceutical formulations of the present invention may include, as optional ingredients, pharmaceutically acceptable carriers, diluents, solubilizing or emulsifying agents, and salts of the type that are well-known in the art. Specific non- limiting examples of the carriers and/or diluents that are useful in the pharmaceutical formulations of the present invention include water and physiologically acceptable buffered saline solutions, such as phosphate buffered saline solutions pH 7.0-8.0.

The agents of this invention can be formulated and administered to treat a variety of disease states by any means that produces contact of the active ingredient with the agent's site of action in the body of the organism. They can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic active ingredients or in a

combination of therapeutic active ingredients. They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.

In general, water, suitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions. Solutions for parenteral administration contain the active ingredient, suitable stabilizing agents and, if necessary, buffer substances. Antioxidizing agents such as sodium bisulfate, sodium sulfite or ascorbic acid, either alone or combined, are suitable stabilizing agents. Also used are citric acid and its salts and sodium Ethylenediaminetetraacetic acid (EDTA). In addition, parenteral solutions can contain preservatives such as benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, a standard reference text in this field.

The active ingredients of the invention may be formulated to be suspended in a pharmaceutically acceptable composition suitable for use in mammals and in particular, in humans. Such formulations include the use of adjuvants such as muramyl dipeptide derivatives (MDP) or analogs that are described in U.S. Patent Nos. 4,082,735; 4,082,736; 4, 101,536; 4, 185,089; 4,235,771 ; and 4,406,890. Other adjuvants, which are useful, include alum (Pierce Chemical Co.), lipid A, trehalose dimycolate and dimethyldioctadecylammonium bromide (DDA), Freund's adjuvant, and IL-12. Other components may include a polyoxypropylene-polyoxyethylene block polymer (Pluronic®), a non-ionic surfactant, and a metabolizable oil such as squalene (U.S. Patent No. 4,606,918).

Additionally, standard pharmaceutical methods can be employed to control the duration of action. These are well known in the art and include control release preparations and can include appropriate macromolecules, for example polymers, polyesters, polyamino acids, polyvinyl, pyrolidone, ethylenevinylacetate, methyl cellulose, carboxymethyl cellulose or protamine sulfate. The concentration of macromolecules as well as the methods of incorporation can be adjusted in order to control release. Additionally, the agent can be incorporated into particles of polymeric materials such as polyesters, polyamino acids, hydrogels, poly (lactic acid) or ethylenevinylacetate copolymers. In addition to being incorporated, these agents can also be used to trap the compound in microcapsules.

Accordingly, the pharmaceutical composition of the present invention may be delivered via various routes and to various sites in an mammal body to achieve a particular effect (see, e.g., Rosenfeld et al, 1991 ; Rosenfeld et al., 1991a; Jaffe et al., supra; Berkner, supra). One skilled in the art will recognize that although more than one route can be used for administration, a particular route can provide a more immediate and more effective reaction than another route. Local or systemic delivery can be accomplished by administration comprising application or instillation of the formulation into body cavities, inhalation or insufflation of an aerosol, or by parenteral introduction, comprising intramuscular, intravenous, peritoneal, subcutaneous, intradermal, as well as topical administration.

The active ingredients of the present invention can be provided in unit dosage form wherein each dosage unit, e.g., a teaspoonful, tablet, solution, or suppository, contains a predetermined amount of the composition, alone or in appropriate combination with other active agents. The term "unit dosage form" as used herein refers to physically discrete units suitable as unitary dosages for human and mammal subjects, each unit containing a predetermined quantity of the compositions of the present invention, alone or in combination with other active agents, calculated in an amount sufficient to produce the desired effect, in association with a pharmaceutically acceptable diluent, carrier, or vehicle, where appropriate. The specifications for the unit dosage forms of the present invention depend on the particular effect to be achieved and the particular pharmacodynamics associated with the pharmaceutical composition in the particular host.

These methods described herein are by no means all-inclusive, and further methods to suit the specific application will be apparent to the ordinary skilled artisan. Moreover, the effective amount of the compositions can be further approximated through analogy to compounds known to exert the desired effect.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore, specifically point out the preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure. Example 1 : Thymic stromal lymphopoietin-elicited basophil responses promote eosinophilic esophagitis

Described herein is the development and use of a new mouse model of EoE-like disease that developed independently of IgE, but was dependent on TSLP and basophils as targeting TSLP or basophils during the sensitization phase limited disease. Notably, therapeutic TSLP neutralization or basophil depletion also ameliorated established EoE-like disease. In human subjects with EoE, elevated TSLP expression and exaggerated basophil responses in esophageal biopsies was observed. Further, it was found that a gain-of-function TSLP polymorphism was associated with increased basophil responses in patients with EoE. Together, the data presented herein demonstrate that the TSLP -basophil axis contributes to the pathogenesis of EoE and could be therapeutically targeted to treat the disease.

The materials and methods employed in these experiments are now described.

Material and Methods:

Mice

Male and female BALB/c and C57BL/6 mice were purchased from the Jackson Laboratories. BALB/c Tslpr^+I+ and BALB/c Tslpr ^ mice were provided by Amgen, through Charles River Laboratories. BALB/c Igh-7^~ mice and C57BL/6 Baso -DTR mice were bred. All mice were used at 8-12 weeks of age, and all experiments employed age-, gender- and genetic strain-matched controls to account for any variations in data sets compared across experiments. Mice were bred and housed in specific pathogen-free conditions. Mice requiring medical attention were provided with appropriate veterinary care by a licensed veterinarian and were excluded from the experiments described. No other exclusion criteria existed.

Reagents and treatments

Mice were treated daily with 2 nmol MC903 (calcipotriol, Tocris Bioscience) in 20 μΐ of 100% EtOH applied to the ears in the presence of 100 μg

OVA for 14 d. As a vehicle control, the same volume of EtOH and OVA was applied. For tape-stripping, mice were shaved on the back, tape-stripped six times and sensitized with 100 μg OVA or saline as control daily for 14 d. For TSLP injections, mice were subcutaneously injected with 5 μg rTSLP in the presence of 100 μg OVA on days 0, 3, 6, 9 and 12. For controls, mice were injected subcutaneously with PBS or rTSLP alone. For CPE sensitization, CPE was made from whole roasted peanuts (Sainsbury's Ltd.) sterilized by gamma irradiation (Lillico Biotech) that were ground in an airflow cabinet using a pestle and mortar. The resulting paste was solubilized in pH 7.4 PBS (Gibco) and sonicated for two 20-min periods, with mixing in between. The solution was then filtered through a 75-μιη tissue filter (BD Biosciences) to remove large particles of debris. Lipopolysaccharide content was tested (Lonza) and reported less than 0.006 ng mL ¹. Mice were treated daily with 2 nmol MC903 in 20 μΐ of 100% EtOH on ears in the presence of 100 μg CPE for 14 d. As a vehicle control, the same volume of EtOH and CPE was applied. Mice were challenged i.g. with 50 mg OVA or 10 mg CPE on days 14 and 17.5 and killed on day 18. Upon first i.g. OVA or CPE challenge, mice were continuously fed water containing 1.5 g L ¹ OVA or given continuous access to whole roasted peanut. Mice subjected to repeated challenge with OVA to induce prolonged inflammation in the esophagus were challenged i.g. with 50 mg OVA on days 14, 17.5, 18, 20, 22, 24 and 26 and killed on day 27. For depletion with TSLP-specific mAbl7, mice were injected with 500 μg of control IgG or TSLP-specific mAb commercially produced by Amgen

intraperitoneally every 3 d during the course of the experiment starting at day -1 or every other day starting at day 18. For basophil depletion by diphtheria toxin treatment, Baso-DTR⁺ or Baso-DTR^" littermate control mice were treated with 500 ng diphtheria toxin (Sigma) intraperitoneally on days -1, 3, 7 and 12. For depletion with CD200R3 -specific mAb (Bal03) (Obata et al, 2007, Blood 1 10:913-920), mice were injected with 100 μg of control IgG or CD200R3 -specific mAb intravenously every 4 d during the course of the experiment starting at day -1 or every other day starting at day 18. To assess food impaction in the esophagus, mice exposed to prolonged esophageal inflammation were fasted for at least 30 min and up to 2 h. Mice were then killed, and their esophagi were examined for the presence of impacted food.

Cohort of human subjects with eosinophilic esophagitis Pediatric participants from a cohort of control subjects or subjects with

EoE were analyzed. Adult participants from a cohort of control subjects or subjects with EoE being treated were also assessed. Written consent was obtained from all participants or their parents or legal guardians, and for pediatric participants, verbal assent from the child was additionally obtained. Subjects defined as having EoE had no other chronic condition except asthma, allergic rhinitis, food allergy, urticaria or atopic dermatitis. Control subjects presented with epigastric abdominal pain but had normal endoscopic and microscopic results. Pediatric subjects with EoE were on proton pump inhibitor therapy, but subjects on systemic corticosteroid treatment or antibiotics were excluded. Subjects with active EoE had an esophageal eosinophil count of >15 per HPF after 8 weeks of treatment with a proton pump inhibitor.

Subjects with inactive EoE had previously been diagnosed with active EoE but had an esophageal eosinophil count of < 15 per HPF at the time of sample collection. During routine endoscopy, three esophageal biopsies were collected for histological analysis of esophageal eosinophil counts. During the same procedure, two esophageal tissue biopsies were collected for research purposes, for either real-time PCR,

immunohistochemistry or flow cytometry. For flow cytometry, single-cell suspensions were made by filtering the mechanically disrupted tissue through a 70-μιη filter (BD Biosciences) for flow cytometry. Peripheral blood from pediatric subjects from a cohort of control subjects or subjects with active or inactive EoE that were genotyped for a gain-of-function TSLP polymorphism was analyzed. Written consent was obtained from all participants or their parents or legal guardians, and for pediatric participants, verbal assent from the child was additionally obtained. Peripheral blood was collected by venipuncture, and serum was isolated. PBMCs were isolated by Ficoll gradient as previously described (Siracusa et al, 201 1, Nature 477: 229-233), and cells were analyzed by flow cytometry. For genotyping of pediatric subjects with EoE, all samples were genotyped on either the Illumina HumanHap 550 or 610 BeadChips according to the manufacturer's protocols. Data normalization and canonical genotype clustering were carried out using the Illumina Genome Studio package. Samples with call rate <98% were excluded from further analysis.

Human real-time PCR and immunohistochemistry

For real-time PCR analysis of gene expression in human esophageal biopsies, human subject biopsy samples were collected and placed in KNAlater (Ambion). RNA was isolated using the mirVana miRNA Isolation Kit according to the manufacturer's recommendations (Ambion) and reverse transcribed using a high- capacity cDNA reverse transcriptase kit (Applied Biosystems). Quantitative real-time PCR was performed using the Taqman Fast Universal PCR Master Mix kit and pre- formulated TaqMan Gene Expression Assays for TSLP (Applied Biosystems).

Reactions were performed in triplicate using 96-well optical plates on a StepOnePlus Real-Time PCR System (Applied Biosystems). GAPDH was used as an endogenous control to normalize the samples using the CT method of relative quantification, where CT is the threshold cycle. For immunohistochemical staining for human TSLP, human esophageal biopsies were embedded in paraffin and sectioned. Sections were deparaffinized and stained with a primary human TSLP-specific mAb or an isotype control antibody (commercially produced by Merck Research Laboratories), and positive staining was visualized using the DAB substrate kit (Vector Laboratories).

Flow cytometry For mouse studies, esophageal tissues of two or three mice were pooled within each replicate experiment, opened longitudinally, digested in 1 mg mL^~ ¹ collagenase/DNase (Roche) for 30 min, and mashed through 70-μιη nylon mesh filters. Single-cell suspensions were incubated with Aqua Live/Dead Fixable Dye (Invitrogen) for dead cell exclusion and stained with fluorochrome-conjugated mAbs purchased from eBioscience specific for CD3e (145-2C11, 1 :300), CD4 (GK1.5, 1 :300), CD 8 (53-6.7, 1 :300), NK1.1 (PK136, 1 :300), CD19 (eBiolD3, 1 :300), FceRl (MAR-1, 1 :200), IgE (23G3, 1 :200), CD45 (30-F 11, 1 :200), CD49b (DX5, 1 :200,), CD1 17 (c-kit, 1 :200), fluorochrome-conjugated mAbs purchased from Biolegend specific for CD l lc (N418, 1 :200), CD5 (53-7.3, 1 :300), B220 (RA3-6B2, 1 :300) and Siglec-F (E50-2440, 1 :200, BD Biosciences, or fluorochrome-conjugated mAbs purchased from eBioscience specific for human CD19 (HIB 19, 1 :200), CD45 (HI30, 1 : 100), CD49b (ebioY418, 1 :200), FceRl (AER-37, 1 :50), CD123 (6H6, 1 : 100) and c-kit (104D2, 1 :30) or fluorochrome-conjugated mAbs purchased from BD

Bioscience specific for human CD56 (B159, 1 :200), CD1 lc (B-ly6, 1 :200) and

TCRa (IP26, 1 :200). For intracellular staining, surface-stained cells were washed, fixed in 2% paraformaldehyde, permeabilized using eBioscience Permeabilization Buffer (eBioscience) according to manufacturer instructions, stained intracellularly with human 2D7-specific mAb (2D7, 1 :25) (eBioscience), washed and resuspended in flow cytometry buffer. All cells were run on a four-laser 14-color LSR II (BD

Biosciences), and Flow Jo 8.7.1 (Tree Star) was used to analyze data. Mouse eosinophils were identified as live, lin (CD3,CD5,CD19,CD l lc,NKl . l),

CD45⁺Siglec-F⁺ side-scatter (SSC)-high cells. Mouse basophils were identified as live, l T (CD3,CD5,CD19,CD1 lc,NKl . l), c-kirCD49b⁺IgE⁺ cells (or as FceRT cells in Igh-7^~ mice). Human basophils in the esophageal biopsy were identified as live, lhT (CD19,CD56,CD1 lc,TCRa ), CD49b⁺FceRI⁺c-kir2D7⁺ cells. Human basophils in the PBMCs were identified as live, lin (CD19,CD56,CD1 lc,TCRa ),

CD123⁺FceRI⁺ cells. Optical coherence tomography

An OCT system operating at 1.3-μιη center wavelength at 47 kHz axial scan rate (-30 frames per s) was developed and used for obtaining volumetric images of freshly excised mouse esophagus. The axial and transverse resolutions were 6 μιη and 10 μιη in tissue, respectively, and the imaging depth was approximately 2 mm, sufficient to image through the entire thickness of the mouse esophagus. Prior to OCT imaging, the esophagus was removed from the mouse, and a plastic tube with 0.75- mm outer diameter was inserted, allowing for the lumenal surface to be clearly differentiated in cross-sectional images. The esophagus was immersed in saline solution to remove light reflection from the surface. Subsequently, three-dimensional OCT images were obtained from multiple locations along the esophagus, with each data set covering 3 x 1.5 x 1.5 mm3. The thickness values of the squamous epithelial layer were measured from cross-sectional OCT images every 200 μιη along the esophagus within each data set. Average squamous epithelial thickness values from the middle of the esophagus were calculated from each mouse by an investigator blinded to group allocations and were used for comparison between different groups.

Mouse cell cultures. ELISA. real-time PCR. histology and electron microscopy

To measure spontaneous release of TSLP, whole ears were incubated for 12 h in complete culture medium (DMEM, 10% FBS), and cell-free supernatants were stored for a TSLP ELISA using a commercially available kit (eBioscience). For antigen re-stimulation, splenocytes or mesenteric lymph node cells were isolated, and single-cell suspensions were stimulated with 200 μg OVA for 72 h. Cell-free supernatants were used for standard sandwich ELISA. Antigen-specific IgE responses were measured as described previously (Zhang et al, 2009, Proc. Natl. Acad. Sci. USA 106: 1536-1541). For histological analysis, at necroscopy, the esophagus was fixed in 4% paraformaldehyde and embedded in paraffin, and 5-μιη sections were cut and stained with hematoxylin and eosin (H&E). For immunofluorescence, sections were deparrafinized and stained with biotinylated SiglecF-specific mAb from R&D Systems (BAF 1706, 1 :200), followed by secondary staining with Cy3 -conjugated streptavidin (Jackson Laboratory) and counterstaining with DAPI (Molecular Probes). For EM, esophageal tissues were fixed with 2.5% glutaraldehyde, 2.0%

paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, overnight at 4 °C. After buffer washes, the samples were post-fixed in 2.0% osmium tetroxide for 1 h at room temperature and rinsed in dEkO before en bloc staining with 2% uranyl acetate. After dehydration through a graded ethanol series, the tissue was infiltrated and embedded in Embed-812 (Electron Microscopy Sciences). Thin sections were stained with uranyl acetate and lead citrate and examined with a JEOL 1010 electron microscope fitted with a Hamamatsu digital camera and AMT Advantage image capture software. For real-time PCR analysis, R A was isolated from esophageal tissue using an RNeasy mini kit (Qiagen) or the mirVana miRNA isolation kit (Ambion) according to the manufacturer's instructions. cDNA was generated using a Superscriptll reverse transcription kit (Invitrogen). Real-time quantitative PCR was performed on cDNA using SYBR green master mix (Applied Biosystems) and commercially available primer sets from Qiagen (Quantitect primer assays). Samples were run on a real-time PCR system (ABI 7500; Applied Biosystems), normalized to β-actin and displayed as fold induction over controls.

Statistical analysis

Results are shown as mean ± s.e.m. To determine group sizes necessary for adequate statistical power, power analysis was performed using preliminary data sets for all analyses presented. Mice of were assigned at random to treatment groups for all mouse studies. Mouse studies were not performed in a blinded fashion, except where indicated. Analyses of basophil responses in esophageal biopsy samples and peripheral blood were conducted in such a manner that the investigator was blinded to the disease state (number of eosinophils per HPF in the biopsy) and TSLP genotype until after flow cytometric analyses were completed. Analysis of TSLP expression levels in the biopsies of control subjects and those with EoE were not performed in a blinded fashion. All inclusion and exclusion criteria for mouse and human studies were pre-established. For mouse studies, statistical significance was determined using a nonparametric, two-tailed Mann- Whitney ?-test, a nonparametric, one-way Kruskal-Wallis ANOVA test followed by Dunn's post hoc testing or a non-parametric, two-way ANOVA followed by

Bonferroni's post hoc testing. For human studies, a nonparametric, two-tailed Mann- Whitney ?-test or a nonparametric, one-way Kruskal-Wallis ANOVA followed by Dunn's post hoc testing were used. Correlation analysis was performed using a nonparametric Spearman correlation (sensitivity analyses were performed), and a linear regression of the data is displayed. All data meet the assumptions of the statistical tests used. Within each group there is an estimate of variation, and the variance between groups is similar. For each statistical analysis, appropriate tests were selected based on whether the data was normally distributed and whether multiple comparisons were made. Results were considered significant at < 0.05. Statistical analyses were performed using Prism version 5.0a (GraphPad Software).

The results of the experiments are now described.

A new mouse model of experimental EoE-like disease

To investigate whether TSLP directly promotes EoE disease pathogenesis, a new mouse model of EoE-like disease that is associated with exaggerated TSLP production was developed. Multiple studies in mouse models and humans suggest that sensitization to food allergens may occur at sites where the skin barrier is disrupted, such as atopic dermatitis lesions (Hsieh et al, 2003, Clin. Exp. Allergy 33: 1067-1075; Lack, 2012, J. Allergy Clin. Immunol. 129: 1 187-1 197; van den Oord and Sheikh, 2009, Br. Med. J. 339:b2433). Thus, a model was employed in which mice were epicutaneously sensitized to a food antigen, ovalbumin (OVA) on a developing atopic dermatitis-like skin lesion induced by topical treatment with the vitamin D analog MC903 (Siracusa et al, 201 1, Nature 477: 229-233; Li et al, 2006, Proc. Natl. Acad. Sci. USA 103: 1 1736-1 1741 ; Li et al, 2009, J. Invest. Dermatol. 129:498-502; Leyva-Castillo et al, 2013, J. Invest. Dermatol. 133 : 154-163) (Figure 1A). Consistent with previous reports (Siracusa et al, 2011, Nature 477: 229-233; Li et al, 2006, Proc. Natl. Acad. Sci. USA 103 : 1 1736-1 1741 ; Li et al, 2009, J. Invest. Dermatol. 129:498-502; Leyva-Castillo et al, 2013, J. Invest. Dermatol. 133: 154- 163), wild-type (WT) BALB/c mice treated epicutaneously with the vitamin D analog MC903 showed increased TSLP expression in the skin compared to ethanol vehicle- treated mice (Figure IB). Epicutaneous sensitization to and subsequent oral challenge with OVA resulted in the development of experimental EoE-like disease that was characterized by inflammation, edema and eosinophilia in the esophagus, as measured histologically and quantified by enumeration of eosinophils per high-power field (HPF) (Figure 1C and Figure ID). Flow cytometric analysis (Figure IE and Figure IF) and immunofluorescence staining (Figure 1G) also demonstrated that there was an accumulation of eosinophils in esophageal tissues of mice with EoE-like disease, and electron microscopic (EM) analysis revealed the presence of degranulated eosinophils in these tissues (Figure 1H). Significantly higher expression of genes that encode TH2 cytokines and the basophil-specific protease Mcpt8 was also observed. Further, a trend toward increased Tslp expression in esophageal tissues of mice with EoE-like disease was observed compared to control mice (Figure II). Further, a similar pattern of EoE-like disease in mice that were epicutaneously sensitized to crude peanut extract (CPE) on an atopic dermatitis-like skin lesion was observed (Figure 7A through Figure 7C), confirming that sensitization to a natural food allergen in the presence of elevated amounts of TSLP results in experimental EoE-like disease. Eosinophil accumulation in this model was not restricted to the esophagus, as mice with EoE-like disease also showed eosinophilia in the gastrointestinal tract after epicutaneous sensitization and oral challenge with OVA (Figure 7D and Figure 7E) associated with antigen-specific TH2 cytokine responses in the mesenteric lymph node and spleen (Figure 7F and Figure 7G).

In addition to the immunological parameters that define human EoE, mouse EoE-like disease in this model is also associated with physiological changes in esophageal tissue and signs of esophageal dysfunction, including food impaction, which occurs in approximately 40% of patients with EoE (Spergel, 2007, Curr. Opin. Allergy Clin. Immunol. 7:274-278; Liacouras et al, 2011, J. Allergy Clin. Immunol. 128:3-20; Abonia and Rothenberg, 2012, Annu. Rev. Med. 63:421^134; DeBrosse et al, 201 1, J. Allergy Clin. Immunol. 128: 132-138). To assess whether clinical manifestations of EoE were present in the experimental mouse model of EoE-like disease, mice that had existing EoE-like disease were challenged repeatedly with OVA to induce prolonged esophageal inflammation. Although analysis using optical coherence tomography (OCT), which allows for high-resolution imaging of live biological tissues based on optical scattering (Huang et al, 1991, Science 254: 1178- 1181 ; Zhou et al, 2012, Gastrointest. Endosc. 76:32-40), revealed that EoE-like disease was characterized by minimal changes in the thickness of the esophageal epithelium, (Figure 8A and Figure 8B), prolonged esophageal inflammation was also associated with food impaction in the esophagus. Approximately 30% of fasted mice with EoE-like disease exhibited food impaction at the time of killing, but food impaction in the esophagus of control (ethanol)-treated mice was never observed (Figure 1J). Collectively, these data indicate that this new model of EoE-like disease is characterized by a number of immunological and pathophysiological changes in esophageal tissues and signs of esophageal dysfunction similar to those observed in humans with EoE (Bhattacharya et al, 2007, Hum. Pathol. 38: 1744-1753; Blanchard et al, 201 1, J. Allergy Clin. Immunol. 127:208-217; Hsu Blatman et al, 2011, J. Allergy Clin. Immunol. 127: 1307-1308; Justinich et al, 1997, J. Pediatr. Gastroenterol. Nutr. 25: 194-198; Spergel, 2007, Curr. Opin. Allergy Clin. Immunol. 7:274-278; Liacouras et al, 201 1, J. Allergy Clin. Immunol. 128:3-20; Abonia and Rothenberg, 2012, Annu. Rev. Med. 63 :421-434). EoE-like disease is dependent on TSLP but independent of IgE

To determine whether TSLP directly promotes the pathogenesis of experimental EoE-like disease in mice, WT BALB/c (Tslpr^+I+) mice or mice deficient in the TSLP receptor (TSLPR) (Tslpr^~ ) were epicutaneously sensitized to OVA followed by oral antigen challenge (see Figure 1A). Whereas sensitized and challenged Tslpr^+I+ mice showed esophageal eosinophilia and associated

inflammation, Tslpr^~ mice did not develop esophageal eosinophilia (Figure 2A though Figure 2D). Using an alternative approach to abrogate TSLP signaling, it was found that multiple systemic treatments with a monoclonal antibody (mAb) that neutralizes TSLP during epicutaneous sensitization with OVA in WT BALB/c mice also limited eosinophil infiltration in the esophagus after oral challenge (Figure 2E through Figure 2H).

To test whether TSLP was sufficient for the development of EoE-like disease during epicutaneous sensitization, mice were intradermally injected with exogenous recombinant TSLP (rTSLP) in the presence or absence of OVA and were challenged orally (Figure 9A). Mice sensitized to OVA in the presence of rTSLP also showed esophageal eosinophilia after oral challenge compared to mice treated with OVA alone or rTSLP alone (Figure 9B). In a complementary approach, Tslpr^+I+ mice treated with control antibody or a TSLP-specific mAb, and Tslpr^~ mice were sensitized with OVA on tape-stripped skin (Figure 9C). Tape-stripping was associated with elevated local TSLP production following physical perturbation of the skin barrier (Oyoshi et al, 2010, J. Allergy Clin. Immunol. 126:976-984) (Figure 9D). Whereas Tslpr^+I+ mice treated with control antibody that were sensitized to OVA on tape-stripped skin showed esophageal eosinophilia after oral antigen challenge, Tslpr^+I+ mice treated with a TSLP-specific mAb and Tslpr^~ mice did not develop esophageal eosinophilia (Figure 9E and Figure 9F). Finally, the contribution of TSLP to the development of clinical signs of EoE-like disease was assessed. Repeated challenge with OVA following sensitization in the presence of MC903 was not associated with changes in the thickness of the esophageal epithelium. However, prolonged esophageal inflammation was also associated with an increased incidence of food impaction in the esophagus in Tslpr^+/+ but not Tslpr^~ mice (Figure 10A and Figure 10B). Collectively, these data indicate that TSLP-TSLPR interactions are necessary and sufficient for the development of experimental EoE-like disease in mice.

TSLP-TSLPR interactions are known to promote the production of IgE

(Yoo et al., 2005, J. Exp. Med. 202:541-549; Jessup et al, 2008, J. Immunol.

181 :431 1-4319), a key mediator of allergic inflammation (Finkelman, 2007, J.

Allergy Clin. Immunol. 120:506-515), and class-switched B cells have been observed in the esophagus of patients with EoE (Lucendo et al, 2007, Am. J. Surg. Pathol. 31 : 598-606; Vicario et al, 2010, Gut 59: 12-20; Mulder and Justinich, 2010, Gut 59:6- 7). In addition, MC903 -induced TSLP expression was associated with high systemic OVA-specific IgE amounts (Figure 3A), suggesting that TSLP-dependent EoE-like disease in mice might be IgE dependent. To directly test this, IgE-sufficient WT BALB/c (Igh-7^+l+) mice and IgE-deficient (Igh-7^~ ) mice were epicutaneously sensitized to OVA in the presence of MC903. Following oral challenge with antigen, both Igh-7^+l+ and Igh-7^~ mice showed equivalent EoE-like disease, characterized by esophageal inflammation, elevated tissue eosinophilia (Figure 3B through Figure 3D), the presence of degranulated eosinophils in the esophagus (Figure 3E) and significant increases in gene expression of TH2 cytokines in esophageal tissues (Figure 3F). These data demonstrate that EoE-like disease can occur in an IgE-independent manner and are consistent with recent findings from clinical studies suggesting that treatment with a IgE-specific mAb does not ameliorate EoE in most patients (Rocha et al, 2001 Eur. J. Pediatr. 170: 1471-1474; Foroughi et al, 2007 J. Allergy Clin. Immunol. 120: 594-601 ; Stone et al, 2008 Clin. Exp. Immunol. 38: 1858-1865; Sampson et al, 201 1 J. Allergy Clin. Immunol. 127: 1309-1310. Together, these data indicate that manipulation of the IgE pathway may not be an effective therapeutic approach for the treatment of EoE.

EoE-like disease depends on basophils

In addition to its role in promoting B cell and IgE responses, TSLP expression is associated with the selective expansion of a distinct population of basophils (Siracusa et al, 201 1, Nature 477: 229-233; Siracusa et al, 2012, Adv. Immunol. 1 15: 141-159). Consistent with this hypothesis, MC903-induced expression of TSLP in the skin was associated with TSLP-dependent, IgE-dependent systemic basophil responses (Figure 11 A and Figure 1 IB). To assess whether basophils contribute to the development of experimental EoE-like disease, an established genetic approach was employed to deplete basophils in vivo. C57BL/6 mice in which the diphtheria toxin receptor (DTR) is exclusively expressed by basophils (Baso- DTR⁺ mice)(Siracusa et al, 2011, Nature 477: 229-233; Giacomin et al, 2012, J. Immunol. 189:4371-4378; Sawaguchi et al, 2012, J. Immunol. 188: 1809-1818) and DTR-negative littermate controls (Baso-DTR- mice) were epicutaneously sensitized and orally challenged with OVA while being treated with diphtheria toxin (Figure 4A). Consistent with results observed in BALB/c mice (Figure IB), increased local and systemic TSLP production was observed in C57BL/6 Baso-DTR^" and Baso-

DTR⁺ mice sensitized to OVA in the context of MC903 treatment. Notably, whereas Baso-DTR^" mice that were epicutaneously sensitized and orally challenged with OVA showed high frequencies of eosinophils in the esophagus, depletion of basophils in Baso-DTR⁺ mice (Figure 11C) led to a reduction in esophageal eosinophilia (Figure 4B through Figure 4E) and a reduction in expression of genes related to TH2 cytokine responses (Figure 12A through Figure 12C).

Using an alternative approach, epicutaneously sensitized and orally challenged WT BALB/c mice were treated with a mAb specific for CD200R3 (Bal03) to deplete basophils (Obata et al, Blood 110; 913-920) (Figure 4F). Mice in which basophils were depleted during sensitization (Figure 1 ID) showed a reduced accumulation of eosinophils in the esophagus compared to control mAb-treated mice after oral challenge with OVA (Figure 4G through Figure 4J). Collectively, these results indicate that basophils are major contributors to the pathogenesis of experimental EoE-like disease in mice and represent a new therapeutic target to treat this disease in patients.

TSLP or basophils can be targeted to treat EoE-like disease

As TSLP and basophils were required during sensitization for the development of EoE-like disease in mice, it was next tested whether the TSLP- basophil pathway could be therapeutically targeted to treat established EoE-like disease. First, mice were sensitized and challenged with OVA to establish EoE-like disease and were then treated systemically with either an isotype control or a neutralizing TSLP-specific mAb during repeated antigen challenge (Figure 5A). Whereas mice with established EoE-like disease treated with a control antibody showed esophageal eosinophilia, mice that were treated with a TSLP-specific mAb had decreased esophageal eosinophilia, as measured histologically (Figure 5B). Flow cytometric analysis also revealed that the total immune cell infiltrate and esophageal eosinophilia were significantly reduced in mice treated with a TSLP-specific mAb compared to mice treated with a control mAb (Figure 5C and Figure 5D).

To test whether basophils contributed to the maintenance of EoE-like disease, mice with established EoE-like disease were treated with an isotype control or basophil-depleting CD200R3 -specific mAb during repeated OVA challenge (Figure 5E). Similar to the results observed after neutralization of TSLP, specific depletion of basophils resulted in decreased esophageal eosinophilia, as measured histologically (Figure 5F), and flow cytometric analysis also showed a reduction in total immune cell infiltrate and eosinophil numbers in the esophagus (Figure 5G and Figure 5H). To test whether neutralization of TSLP or depletion of basophils was also associated with a resolution of signs of esophageal dysfunction, mice with established EoE-like disease were treated with a control antibody, TSLP-specific mAb, or CD200R -specific mAb and assessed them for the incidence of food impaction. Whereas food impaction was observed in about 30% of mice treated with a control antibody, impaction was not observed in mice in which TSLP or basophil responses were blocked (Figure 51). Taken together, these data demonstrate that TSLP neutralization or basophil depletion can be used to ameliorate inflammation and clinical symptoms of established experimental EoE-like disease in mice.

The TSLP -basophil axis is associated with EoE in humans

The roles of TSLP and basophils in experimental EoE-like disease in mice (Figure 2 and Figure 4) and the established association between gain-of-function polymorphisms in TSLP and EoE in human pediatric subjects (Rothenberg et al, 2010, Nat. Genet. 42:289-291; Sherrill et al, 2010, J. Allergy Clin. Immunol.

126: 160-165) prompted an investigation into whether the TSLP -basophil pathway contributes to the pathogenesis of EoE in humans. To assess whether the TSLP- basophil axis is active in human subjects with EoE, TSLP expression and basophil responses in esophageal biopsies from a cohort of pediatric subjects was examined. This patient population was stratified on the basis of the number of eosinophils counted in histologic sections from esophageal biopsies from the following groups: (i) control subjects without EoE, (ii) subjects with active EoE (>15 eosinophils per HPF) and (iii) subjects with inactive EoE (<15 eosinophils per HPF and a prior clinical history of active EoE) (Figure 6A). In agreement with previous studies (Sherrill et al, 2010, J. Allergy Clin. Immunol. 126: 160-165; Rothenberg et al, 2010, Nat. Genet. 42:289-291), TSLP expression in esophageal biopsies was higher in subjects with active EoE compared to control subjects or subjects with inactive EoE (Figure 6B). Immunohistochemical staining revealed that stratified squamous epithelial cells showed positive staining for TSLP in esophageal biopsies from subjects with active EoE (Figure 6C). Flow cytometric analysis was then used to identify and quantify the inflammatory cell infiltrate in biopsies. Notably, higher frequencies of cells with a phenotype consistent with that of basophils (Hn^~CD49b⁺FceRI⁺c-ki 2D7⁺) was observed in esophageal biopsies from subjects with active EoE compared to those from control subjects or subjects with inactive EoE (Figure 6D and Figure 6E).

Further, the frequency of basophils positively correlated (Spearman r = 0.6638) with the number of eosinophils counted per HPF in histological sections of esophageal biopsies (Figure 6F). Additionally, a cohort of adult subjects was able to be stratified on the basis of the number of eosinophils counted in histologic sections (Figure 13A). Consistent with results observed in pediatric subjects (Figure 6D through Figure 6F), adult subjects with active EoE had a higher (although not statistically significant) frequency of basophils in the esophageal biopsy, as measured using flow cytometry, that positively correlated (Spearman r = 0.5282) with the number of eosinophils counted per HPF in histological sections (Figure 13B and Figure 13C). Collectively, these data indicate that the TSLP -basophil axis is associated with active EoE in pediatric and adult subjects.

These findings, coupled with the association between the development of EoE and a previously identified gain-of-function polymorphism in TSLP associated with TSLP overexpression (TSLPrisk) (Rothenberg et al, 2010, Nat. Genet. 42:289- 291), suggested that there may be an association between the TSLPrisk polymorphism and enhanced basophil responses in human subjects with EoE. To directly test this, a separate cohort of pediatric subjects with active or inactive EoE genotyped for the presence of the TSLPrisk polymorphism was assessed for basophil frequencies among peripheral blood mononuclear cells (PBMCs). Subjects who were homozygous or heterozygous for the TSLPnsk polymorphism had significantly higher basophil frequencies in their PBMCs than subjects with EoE who did not carry the TSLPrisk polymorphism (Figure 6G), which suggests a genetic link between a gain-of-function TSLP polymorphism, increased peripheral basophil responses and EoE. This data suggest a model in which patients that carry the TSLPrisk polymorphism have a predisposition toward TSLP overexpression and associated peripheral basophilia that may increase the likelihood of developing EoE after encounter with trigger antigens (Figure 14).

Targeting of the TSLP -basophil axis

Described herein is a new mouse model in which epicutaneous sensitization to a model food antigen followed by oral antigen challenge results in EoE-like disease. It is demonstrated that TSLP and basophils, but not IgE, are required for the development of experimental EoE-like disease in mice and that antibody-mediated neutralization of TSLP or depletion of basophils is effective in preventing the development of experimental EoE-like disease. Targeting TSLP or basophils was also effective in treating established EoE-like disease in mice. In addition, it is identified herein that the presence of enhanced basophil responses in the esophageal biopsy tissue of human subjects with EoE and a genetic link between a gain-of-function polymorphism in TSLP and increased peripheral basophil responses.

The model of EoE-like disease reported herein is associated with several characteristics of EoE in humans, including esophageal eosinophilia and associated esophageal dysfunction. In addition, this model is also characterized by gastrointestinal eosinophilia and systemic TH2 cytokine responses. EoE in humans is defined as a disease associated with eosinophilia in the esophagus. However, patients with EoE often suffer from coexisting allergic disorders such as atopic dermatitis, allergic rhinitis, asthma or intestinal food allergy (Liacouras et al, 201 1, J. Allergy Clin. Immunol. 128:3-20; Arora et al, 2012, Curr. Gastroenterol. Rep. 14:206-215; Brown-Whitehorn and Spergel, 2010, Expert Rev. Clin. Immunol. 6: 101-109). These observations suggest that a subset of individuals with EoE with coexisting allergic diseases may present with manifestations of allergic disease at tissue sites outside of the esophagus (Straumann et al, 2005, Inflamm. Bowel Dis. 1 1 :720-726). Thus, the mouse model of EoE-like disease described herein may recapitulate a pan-allergic disease state present in some humans who have EoE and suffer from additional allergic diseases.

Previous studies in mouse models and humans have identified various immunological factors that are associated with EoE (Mishra et al, 2002, J. Immunol. 168: 2464-2469; Liacouras et al, 2011, J. Allergy Clin. Immunol. 128:3-20; Abonia and Rothenberg, 2012, Annu. Rev. Med. 63:421-434; Bhattacharya et al, 2007, Hum. Pathol. 38: 1744-1753; Blanchard et al, 2011, J. Allergy Clin. Immunol. 127:208- 217; Hsu Blatman et al, 2011, J. Allergy Clin. Immunol. 127: 1307-1308; Justinich et al, 1997, J. Pediatr. Gastroenterol. Nutr. 25: 194-198; Akei et al, 2005,

Gastroenterology 129:985-994; Mavi et al, 2012, Am. J. Physiol. Gastrointest. Liver Physiol. 302:G1347-G1355; Rajavelu et al, 2012, Am. J. Physiol. Gastrointest. Liver Physiol. 302:G645-G654; Mishra et al, 2007, J. Leukoc. Biol. 81 :916-924; Mishra and Rothenberg, 2003, Gastroenterology 125: 1419-1427; Spergel, 2007, Curr. Opin. Allergy Clin. Immunol. 7:274-278). However, recent clinical trials that have targeted some of these factors, including IgE and IL-5, have failed to ameliorate symptoms of disease (Liacouras et al, 2011, J. Allergy Clin. Immunol. 128:3-20; Rocha et al, 2011, Eur. J. Pediatr. 170: 1471-1474; Foroughi et al, 2007, J. Allergy Clin.

Immunol. 120:594-601; Sampson et al, 2011, J. Allergy Clin. Immunol. 127: 1309- 1310; Spergel et al, 2012, J. Allergy Clin. Immunol. 129:456-463; Castro et al, 2011, Am. J. Respir. Crit. Care Med. 184: 1125-1132), suggesting that these factors may not be essential for the pathogenesis of EoE. The demonstration that EoE-like disease in mice can develop independently of IgE but is dependent on TSLP and basophils may explain why previous clinical trials employing other candidate biologic therapies have not been successful. The identification of a role for TSLP and basophils in experimental EoE-like disease in mice, coupled with the association between TSLP and basophil responses and EoE in humans, indicate that targeting the TSLP -basophil axis offers new opportunities for the clinical management of EoE in patients.

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMS What is claimed is:

1. A method for treating a disease or disorder comprising administering to a subject in need thereof an effective amount of an agent that targets thymic stromal lymphopoietin (TSLP).

2. The method of claim 1, wherein the disease or disorder is eosinophilic esophagitis (EoE).

3. The method of claim 1, wherein targeting TSLP comprises one or more of the level of TSLP and the activity of TSLP.

4. The method of claim 1, wherein the agent that targets TSLP prevents the transcription of the TSLP gene or translation of the TSLP mRNA.

5. The method of claim 1, wherein the agent that targets TSLP interferes with the activity of TSLP.

6. The method of claim 1, wherein the agent that targets TSLP interferes with the interaction between TSLP and its receptor.

7. The method of claim 1, wherein the agent is selected from the group consisting of a small interfering RNA (siRNA), a microRNA, an antisense nucleic acid, a ribozyme, an expression vector encoding a transdominant negative mutant, an antibody, a peptide and a small molecule.

8. The method of claim 1, further comprising administering an effective amount of an agent that targets a basophil.

9. The method of claim 8, wherein the agent that targets a basophil inhibits the activity of the basophil.

10. The method of claim 8, wherein the agent that targets a basophil depletes the basophil.

11. The method of claim 8, wherein the agent that targets a basophil is a basophil-depleting antibody.

12. A method for treating a disease or disorder comprising administering to a subject in need thereof an effective amount of an agent that targets a basophil.

13. The method of claim 12, wherein the disease or disorder is eosinophilic esophagitis (EoE).

14. The method of claim 12, wherein the agent that targets a basophil inhibits the activity of the basophil.

15. The method of claim 12, wherein the agent that targets a basophil depletes the basophil.

16. The method of claim 12, wherein the agent that targets a basophil is a basophil-depleting antibody.

17. The method of claim 12, further comprising administering an effective amount of agent that targets thymic stromal lymphopoietin (TSLP).

18. The method of claim 17, wherein targeting TSLP comprises one or more of the level of TSLP and the activity of TSLP.

19. The method of claim 17, wherein the agent that targets TSLP prevents the transcription of the TSLP gene or translation of the TSLP mRNA.

20. The method of claim 17, wherein the agent that targets TSLP interferes with the activity of TSLP.

21. The method of claim 17, wherein the agent that targets TSLP interferes with the interaction between TSLP and its receptor.

22. The method of claim 17, wherein the agent is selected from the group consisting of a small interfering RNA (siRNA), a microRNA, an antisense nucleic acid, a ribozyme, an expression vector encoding a transdominant negative mutant, an antibody, a peptide and a small molecule.