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Title:
COMPOSITIONS COMPRISING GLUTEN
Document Type and Number:
WIPO Patent Application WO/2015/164722
Kind Code:
A1
Abstract:
Provided herein compositions comprising gluten and methods of use thereof. In some aspects, compositions comprising ratios of wheat flour protein, rye flour protein, and barley flour protein are provided and methods of use thereof.

Inventors:
ANDERSON, Robert, P. (152 Spring Street, Shrewsbury, MA, 01545, US)
TYE-DIN, Jason, A. (2/11-17 Gold Street, CollingwoodAustralia, 3066, 3066, AU)
Application Number:
US2015/027489
Publication Date:
October 29, 2015
Filing Date:
April 24, 2015
Export Citation:
Click for automatic bibliography generation   Help
Assignee:
IMMUSANT, INC. (One Kendall Square, Suite B2004Cambridge, MA, 02139, US)
International Classes:
A23J1/00; A21D13/04; A21D13/06; A21D13/08; A23J3/14; A61K38/02; A61P1/00
Domestic Patent References:
WO2003104273A22003-12-18
WO2013016427A12013-01-31
WO2007047303A22007-04-26
WO2003066079A22003-08-14
Foreign References:
EP0905518A11999-03-31
Attorney, Agent or Firm:
MCMAHON, Amy, J. (Wolf, Greenfield & Sacks P.C.,600 Atlantic Avenu, Boston MA, 02210-2206, US)
Download PDF:
Claims:
Claims

What is claimed is:

1. A composition, comprising wheat flour protein, barley flour protein, and rye flour protein in a ratio of 3:2: 1 by weight in grams, respectively.

2. The composition of claim 1, comprising 1.5 grams of wheat flour protein, 1 gram of barley flour protein, and 0.5 grams of rye flour protein.

3. The composition of claim 1 or 2, wherein the composition is a foodstuff.

4. The composition of claim 3, wherein the foodstuff is a baked good.

5. The composition of claim 4, wherein the baked good is a cookie.

6. A kit, comprising the composition of any one of claims 1 to 5.

7. The kit of claim 6, wherein the composition is provided in triplicate.

8. The kit of claim 6, wherein the composition is provided in nonuplicate.

9. A method, comprising:

(a) administering a composition of any one of claims 1 to 5 to a subject suspected of having or having Celiac disease.

10. The method of claim 9, wherein the composition is administered to the subject more than once.

11. The method of claim 10, wherein the composition is administered to the

subject at least once a day for three days.

12. The method of claim 11, wherein the composition is administered to the

subject three times a day for three days.

13. The method of any one of claims 9 to 12, wherein the administration of the composition is oral administration.

14. The method of any one of claims 9 to 13, further comprising:

(b) measuring a T cell response in a sample comprising T cells obtained from the subject after administration to the subject of the composition.

15. The method of claim 14, wherein the measuring of a T cell response in the sample comprises contacting the sample with a second composition comprising at least one gluten peptide and measuring the level of at least one cytokine in the sample.

16. The method of claim 15, wherein the level of the at least one cytokine is measured with an enzyme-linked immunosorbent assay (ELISA) or multiplex bead- based assay.

17. The method of claim 15, wherein the level of the at least one cytokine is measured with an enzyme-linked immunosorbent spot (ELISpot) assay.

18. The method of any one of claims 15 to 17, wherein the at least one cytokine is IFN-γ, IP- 10, or IL-2.

19. The method of any one of claims 15 to 18, wherein the second composition comprises at least one of:

(i) a first peptide comprising the amino acid sequence PFPQPELPY (SEQ

ID NO: 1) and PQPELPYPQ (SEQ ID NO: 2),

(ii) a second peptide comprising the amino acid sequence PFPQPEQPF

(SEQ ID NO: 3) and PQPEQPFPW (SEQ ID NO: 4), or

(iii) a third peptide comprising the amino acid sequence PIPEQPQPY (SEQ ID NO: 5) and the amino acid sequence EQPIPEQPQ (SEQ ID NO: 12).

20. The method of claim 19, wherein the first peptide comprises

LQPFPQPELPYPQPQ (SEQ ID NO: 6); the second peptide comprises

QPFPQPEQPFPWQP (SEQ ID NO: 7); and/or the third peptide comprises

PEQPIPEQPQPYPQQ (SEQ ID NO: 8).

21. The method of claim 19 or 20, wherein the first, second and/or third peptides comprise an N-terminal acetyl group or pyroglutamate group, and/or a C terminal amide group.

22. The method of any one of claims 14 to 21, wherein the sample is obtained from the subject at least one day after administration of the first composition.

4010157

23. The method of claim 22, wherein the first sample is obtained from the subject six days after administration of the composition.

24. The method of any one of claims 9 to 23, wherein the subject is HLA-DQ2.5 positive.

Description:
COMPOSITIONS COMPRISING GLUTEN

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application number 61/983,981, filed April 24, 2014, U.S. provisional application number 62/011,561, filed June 12, 2014, U.S. provisional application number 62/014,676, filed June 19, 2014, U.S. provisional application number 62/057,152, filed September 29, 2014, U.S. provisional application number 62/115,925, filed February 13, 2015, U.S. provisional application number 61/984,028, filed April 24, 2014, U.S. provisional application number 61/984,043, filed April 25, 2014, U.S. provisional application number 62/011,566, filed June 12, 2014, U.S. provisional application number 62/014,681, filed June 19, 2014, U.S.

provisional application number 62/057,163, filed September 29, 2014, U.S. provisional application number 62/115,897, filed February 13, 2015, U.S. provisional application number 61/983,989, filed April 24, 2014, U.S. provisional application number 62/014,666, filed June 19, 2014, U.S. provisional application number 62/009,146, filed June 06, 2014, U.S. provisional application number 62/043,386, filed August 28, 2014, U.S. provisional application number 62/115,963, filed February 13, 2015, U.S. provisional application number 61/983,993, filed April 24, 2014, U.S. provisional application number 62/011,508, filed June 12, 2014, U.S. provisional application number 62/116,052, filed February 13, 2015, U.S. provisional application number 62/043,395, filed August 28, 2014, U.S. provisional application number 62/082,832, filed November 21, 2014, U.S. provisional application number 62/009,090, filed June 6, 2014, U.S. provisional application number 62/014,373, filed June 19, 2014, U.S. provisional application number 62/043,390, filed August 28, 2014, U.S. provisional application number 62/116,002, filed February 13, 2015, U.S. provisional application number 62/011,493, filed June 12, 2014, U.S. provisional application number 62/011,794, filed June 13, 2014, U.S. provisional application number 62/014,401, filed June 19, 2014, U.S. provisional application number 62/116,027, filed February 13, 2015, and U.S. provisional application number 62/011,540, filed June 12, 2014, the contents of each of which are incorporated by reference herein in their entirety. BACKGROUND

Celiac disease (CD) is an autoimmune-like disorder of the small intestine that occurs in people of all ages. CD causes damage to the villi of the small intestine due to an inappropriate immune response to gluten peptides, leading to malabsorption and an increased risk of intestinal cancer. Correctly diagnosing CD is important in order to ensure that those affected by CD receive proper treatment.

SUMMARY

Aspects of the disclosure relate to compositions comprising gluten (e.g., foodstuffs comprising gluten) and methods of use thereof.

In some aspects the disclosure relates to a composition, comprising wheat flour protein, barley flour protein, and rye flour protein in a ratio of 3:2: 1 by weight in grams, respectively. In some aspects the disclosure relates to a composition, comprising 1.5 grams of wheat flour protein, 1 gram of barley flour protein, and 0.5 grams of rye flour protein. In some embodiments of any one of the compositions, the composition is a foodstuff. In some embodiments of any one of the compositions, the foodstuff is a baked good. In some embodiments of any one of the compositions, the baked good is a cookie. Other aspects of the disclosure relate to kits. In some embodiments, the kit comprises a composition, comprising wheat flour protein, barley flour protein, and rye flour protein in a ratio of 3:2: 1 by weight in grams, respectively. In some embodiments, the kit comprises a composition, comprising 1.5 grams of wheat flour protein, 1 gram of barley flour protein, and 0.5 grams of rye flour protein. In some embodiments of any one of the kits, the composition is a foodstuff. In some embodiments, the foodstuff is a baked good. In some embodiments of any one of the kits, the baked good is a cookie. In some embodiments of any one of the kits, the composition is provided in triplicate in the kit. In some embodiments of any one of the kits, the composition is provided in nonuplicate in the kit. In some embodiments of any one of the kits provided, the kit further comprises a gluten peptide composition as described herein. In some embodiments of any one of the kits provided, the kit further comprises one or more binding partners for IP- 10, IFN-γ, and/or IL-2. In some embodiments of any one of the kits provided,

4010157 the binding partners are antibodies or antigen binding fragments thereof.

Other aspects of the disclosure relate to a method, comprising administering a composition comprising wheat flour protein, barley flour protein, and rye flour protein in a ratio of 3:2: 1 by weight in grams, respectively, to a subject suspected of having or having 5 Celiac disease. In some embodiments, the method comprises administering a composition comprising 1.5 grams of wheat flour protein, 1 gram of barley flour protein, and 0.5 grams of rye flour protein to a subject suspected of having or having Celiac disease. In some embodiments of any one of the methods, the composition is a foodstuff. In some

embodiments of any one of the methods, the foodstuff is a baked good. In some

o embodiments of any one of the methods, the baked good is a cookie. In some embodiments of any one of the methods, the composition is administered to the subject more than once. In some embodiments of any one of the methods, the composition is administered to the subject at least once a day for three days. In some embodiments of any one of the methods, the composition is administered to the subject three times a day for three days. In some

5 embodiments, the administration of the composition is oral administration.

In some embodiments of any one of the methods described above, the method further comprises measuring a T cell response in a sample comprising T cells obtained from the subject after administration to the subject of the composition. In some embodiments, the measuring of a T cell response in the sample comprises contacting the sample with a second o composition comprising at least one gluten peptide and measuring the level of at least one cytokine in the sample. In some embodiments, the level of the at least one cytokine is measured with an enzyme-linked immunosorbent assay (ELISA) or multiplex bead-based assay. In some embodiments, the level of the at least one cytokine is measured with an enzyme-linked immunosorbent spot (ELISpot) assay. In some embodiments, the at least one5 cytokine is IFN-γ, IP-10, or IL-2. In some embodiments, the second composition comprises at least one of:

(i) a first peptide comprising the amino acid sequence PFPQPELPY (SEQ ID NO: 1) and PQPELPYPQ (SEQ ID NO: 2),

(ii) a second peptide comprising the amino acid sequence PFPQPEQPF (SEQ ID

4010157 NO: 3) and PQPEQPFPW (SEQ ID NO: 4), or

(iii) a third peptide comprising the amino acid sequence PIPEQPQPY (SEQ ID NO: 5). In some embodiments, the second composition comprises at least one of:

(i) a first peptide comprising the amino acid sequence PFPQPELPY (SEQ ID 5 NO: 1) and PQPELPYPQ (SEQ ID NO: 2),

(ii) a second peptide comprising the amino acid sequence PFPQPEQPF (SEQ ID NO: 3) and PQPEQPFPW (SEQ ID NO: 4), or

(iii) a third peptide comprising the amino acid sequence PIPEQPQPY (SEQ ID NO: 5) and the amino acid sequence EQPIPEQPQ (SEQ ID NO: 12). In some embodiments, o the first peptide comprises LQPFPQPELPYPQPQ (SEQ ID NO: 6); the second peptide

comprises QPFPQPEQPFPWQP (SEQ ID NO: 7); and/or the third peptide comprises PEQPIPEQPQPYPQQ (SEQ ID NO: 8). In some embodiments, the first, second and/or third peptides comprise an N-terminal acetyl group or pyroglutamate group, and/or a C terminal amide group. In some embodiments, the sample is obtained from the subject at least one day5 after administration of the first composition. In some embodiments, the first sample is

obtained from the subject six days after administration of the composition. In some embodiments, the subject is HLA-DQ2.5 positive.

BRIEF DESCRIPTION OF THE DRAWINGS

o The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1 is a series of four graphs showing the amount of interferon-gamma (IFNy) and 5 IP- 10 in whole blood samples collected from multiple subjects one day before beginning a three-day oral gluten challenge (day 0) and six days after beginning the oral gluten challenge (day 6). The whole blood samples were contacted with medium or peptide pool 1 and the level of IFNy or IP- 10 was measured by MAGPIX® assay (Luminex). The subjects are indicated as follows: subject 1: filled-in circle, subject 2: filled-in square, subject 3: filled- in

4010157 point-up triangle, subject 4: filled-in point-down triangle, subject 5: filled- in diamond, subject 6: unfilled circle, subject 7: unfilled square, subject 8: unfilled point-up triangle, subject 9: unfilled point-down triangle, and subject 10: unfilled diamond.

FIG. 2A is a graph showing the amount of IFNy as measured by ELISA in blood

5 samples. The amount is shown as the amount in the blood sample contacted with peptide pool 1 minus the amount in the blood sample contacted with medium (pool 1 - medium). Individual points indicate blood drawn from each of 10 subjects.

FIG. 2B is a graph showing the amount of IFNy as measured by MAGPIX® in blood samples. The amount is shown as the amount in the blood sample contacted with peptide o pool 1 minus the amount in the blood sample contacted with medium (pool 1 - medium).

Individual points indicate blood drawn from each of 10 subjects.

FIG. 2C is a graph showing the amount of IP- 10 as measured by MAGPIX® in blood samples. The amount is shown as the amount in a blood sample contacted with peptide pool 1 minus the amount in a blood sample contacted with medium (pool 1 - medium). Individual5 points indicate blood drawn from each of 10 subjects.

DETAILED DESCRIPTION

Celiac disease (CD, also sometimes referred to as Coeliac disease, C(o)eliac sprue, non-tropical sprue, endemic sprue, gluten enteropathy, etc.) is defined by the presence of o small intestinal inflammation that improves or normalizes with exclusion of dietary gluten derived from foods including wheat, barley and rye. Celiac disease is one of a cluster of diseases associated with autoantibody production (IgA specific for transglutaminase-2) and T-cell mediated organ-specific immunopathology that are strongly associated with HLA- DR3-DQ2 and DR4-DQ8 haplotypes. In celiac disease peptides derived from an exogenous 5 antigen, dietary gluten, are recognized by pathogenic T cells, such as CD4 + T cells. Celiac disease occurs in people of all ages after gluten has been included in the diet, e.g., middle infancy onward. Celiac disease affects approximately 1 % of people in Europe and North America. In many of those affected, Celiac disease is unrecognized, but this clinical oversight is now being rectified with greater clinical awareness.

4010157 Celiac disease occurs in genetically susceptible individuals who possess either HLA- DQ2 encoded by HLA-DQAl *05 and HLA-DQBl *02 (accounting for about 90% of individuals), variants of HLA-DQ2, or HLA-DQ8. Without wishing to be bound by theory, it is believed that these individuals mount an inappropriate HLA-DQ2-and/or DQ8-restricted CD4 + T cell-mediated immune response to peptides derived from the aqueous-insoluble proteins of wheat flour, gluten, and related proteins in rye and barley.

As disclosed herein, testing was performed to determine whether CD4 + T cells specific for immunodominant gluten peptides derived from wheat, barley and rye prolamins (plant storage proteins with a characteristically high proline content, which include gliadin, hordein, and secalin) could be detected six days after a gluten challenge in subjects with

Celiac disease. The gluten challenge involved ingestion of 3 cookies per day for 3 days, each cookie containing a ratio of 3:2: 1 of wheat flour protein, barley flour protein, and rye flour protein by weight in grams, respectively: 1.5 grams of wheat flour protein, 1 gram of barley flour protein, and 0.5 grams of rye flour protein each. Ex vivo whole blood assays and peripheral blood mononuclear cell ELISpot assays confirmed that ingestion of 3 cookies per day for 3 days was sufficient to induce release of interferon-γ (IFN-γ) and interferon-γ- induced protein 10 (IP- 10) in the whole blood assay and the ELISpot assay. Thus, this composition is useful for challenge methods, e.g., in subjects with Celiac disease.

Accordingly, aspects of the disclosure relate to compositions comprising gluten, e.g., a ratio of 3:2: 1 of wheat flour protein, barley flour protein, and rye flour protein by weight in grams, respectively, such as 1.5 grams of wheat flour protein, 1 gram of barley flour protein, and 0.5 grams of rye flour protein, and methods of use thereof.

Compositions comprising gluten

Aspects of the disclosure relate to a composition comprising a ratio of 3:2: 1 of wheat flour protein, barley flour protein, and rye flour protein by weight in grams, respectively, such as 1.5 grams of wheat flour protein, 1 gram of barley flour protein, and 0.5 grams of rye flour protein. In some embodiments, the wheat flour protein is contained within wheat flour,

4010157 the barley flour protein is contained within barley flour, and the rye flour protein is contained within rye flour. In some embodiments, the composition is a foodstuff. In some

embodiments, the foodstuff is a baked good (e.g., breads, cookies, muffins, cakes, etc.). In some embodiments, baked good is a cookie. Multiple compositions of wheat flour protein, barley flour protein, and rye flour protein may be administered to a subject such that the subject receives 4.5 grams of wheat flour protein, 3 grams of barley flour protein, and 1.5 grams of rye flour protein per day. Preferably, such compositions test positive with an R5- ELISA.

In some embodiments of any one of the compositions provided herein, the

composition comprises additional components such as, e.g., fillers, sweetening agents, flavoring agents, coloring agents, thickening agents, and preserving agents.

In some embodiments of any one of the compositions provided herein, the

composition comprises 20% flour protein (e.g., 20% gluten), 12% sugar, and 12% glucose, such as by weight (e.g., dry weight). In some embodiments of any one of the compositions provided herein, the composition comprises 20% flour protein (e.g., in a 3:2: 1 ratio of wheat flour protein, barley flour protein, and rye flour protein), 12% sugar, and 12% glucose, such as by weight (e.g., dry weight).

In some embodiments of any one of the compositions provided herein, the weight in grams of each flour protein is dry weight. The amount of wheat flour protein, barley flour protein, and rye flour protein can be determined using any method known in the art (see, e.g., Moore et al. Total Protein Methods and Their Potential Utility to Reduce the Risk of Food Protein Adulteration. Comprehensive Reviews in Food Science and Food Safety. Vol. 9, Issue 4 (2010) and AOAC International. 2005. Official methods of analysis. 17th ed.

Gaithersburg , Md.:AOAC International). In some embodiments, the amount of wheat flour protein, barley flour protein, and rye flour protein are determined by measuring the total nitrogen content in each of the wheat, barley and rye flours. Total nitrogen content can be measured using any method known in the art, e.g., using the Kjeldahl method or the Dumas (combustion) method. In some embodiments, the amount of flour protein is a flour protein amount in a commercially- available wheat flour, barely, flour or rye flour and the amount of

4010157 flour protein (e.g., in grams) is the amount determined by the manufacturer, which is provided on a food contents label or a nutritional information label for the commercially- available flour.

Gluten Challenge

In some embodiments, methods provided herein comprise a gluten challenge and/or measuring a T cell response in one or more samples obtained from a subject before, during, or after a gluten challenge.

In some embodiments, a composition comprising gluten (e.g., comprising a ratio of 3:2: 1 of wheat flour protein, barley flour protein, and rye flour protein by weight in grams, respectively, such as 1.5 grams of wheat flour protein, 1 gram of barley flour protein, and 0.5 grams of rye flour protein) is administered to the subject for a defined period of time in order to activate gluten-reactive CD4 + T cells and/or mobilize such CD4 + T cells in the subject. The composition comprising gluten may be administered using the methods of gluten challenge known in the art. Currently, the standard gluten challenge lasts for several weeks (e.g., 4 weeks or more) and involves high doses of orally administered gluten peptides (e.g., 5-30 grams of gluten daily usually in the form of one or more slices of wheat bread or other baked goods that include gluten). Some studies suggest that a shorter challenge, e.g., through use of as little as 3 days of oral gluten challenge, is sufficient to activate and/or mobilize gluten-reactive CD4 + T cells (Anderson R, van Heel D, Tye-Din J, Barnardo M, Salio M, Jewell D, and Hill A. T cells in peripheral blood after gluten challenge in coeliac disease. Gut 2005; 54;1217-1223; and In vivo antigen challenge in Celiac disease identifies a single transglutaminase-modified peptide as the dominant A-gliadin T cell epitope. Anderson R, Degano P, Godkin A, Jewell D, and Hill A. Nature Medicine. 2000;6(3):337-342.).

In some embodiments, the challenge comprises administering a composition comprising a ratio of 3:2: 1 of wheat flour protein, barley flour protein, and rye flour protein by weight in grams, respectively, such as 1.5 grams of wheat flour protein, 1 gram of barley flour protein, and 0.5 grams of rye flour protein. In some embodiments, the wheat flour protein is comprised within wheat flour, the barely flour protein is comprised within barley flour, and the rye flour protein is comprised within rye flour. In some embodiments, the

4010157 challenge comprises administering the composition to the subject prior to determining a T cell response as described herein.

The administration may occur more than once. In some embodiments, administration is daily for 3 days. In some embodiments, administration is at least once daily for 3 days. In 5 some embodiments, administration is 3 times a day for 3 days, e.g., such that the subject receives 4.5 grams of wheat flour protein, 3 grams of barley flour protein, and 1.5 grams of rye flour protein per day. In some embodiments, the composition comprising gluten is administered to the subject three times a day for three days.

Administration of the composition comprising gluten may be self-administration by o the subject or administration by a qualified individual, e.g., a medical practitioner such as a doctor or nurse. Such administration may be through any method known in the art.

Compositions suitable for each administration route are well known in the art (see, e.g., Remington: The Science and Practice of Pharmacy, 21st Ed. Lippincott Williams & Wilkins, 2005). In some embodiments, administration of the composition comprising gluten is oral5 administration.

Suitable forms of oral administration include foodstuffs (e.g., baked goods such as breads, cookies, muffins, cakes, etc.), tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs.

Compositions intended for oral use may be prepared according to methods known to the art o for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents such as sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations.

T Cell Responses and Measurement Thereof

5 Aspects of the disclosure relate to a determination or measurement of a T cell

response in a sample comprising T cells from a subject, such as a subject having or suspected of having Celiac disease. In some embodiments, a composition comprising gluten (e.g., comprising a ratio of 3:2: 1 of wheat flour protein, barley flour protein, and rye flour protein by weight in grams, respectively, such as 1.5 grams of wheat flour protein, 1 gram of barley

4010157 flour protein, and 0.5 grams of rye flour protein) is administered to a subject and is capable of activating a CD4 + T cell in a subject, e.g., a subject with Celiac disease. The term "activate" or "activating" or "activation" in relation to a CD4 + T cell refers to the presentation by an MHC molecule of an epitope on one cell to an appropriate T cell receptor on a second CD4 + T cell, together with binding of a co-stimulatory molecule by the CD4 + T cell, thereby eliciting a "T cell response", in this example, a CD4 + T cell response. Such a T cell response can be measured ex vivo, e.g., by measuring a T cell response in a sample comprising T cells from the subject.

As described herein, an elevated T cell response, such as an elevated CD4 + T cell response, from a sample comprising T cells from a subject after administration of a composition comprising gluten to the subject compared to a control T cell response can correlate with the presence or absence of Celiac disease in the subject. Accordingly, aspects of the disclosure relate to methods that comprise determining or measuring a T cell response in a sample comprising T cells from a subject, e.g., having or suspected of having Celiac disease.

In some embodiments, measuring a T cell response in a sample comprising T cells from a subject comprises contacting the sample with a composition comprising at least one gluten peptide.

For example, whole blood or PBMCs obtained from a subject who has been exposed to a gluten peptide (e.g., by administration of a first composition comprising a gluten peptide) may be contacted with the composition in order to stimulate T cells in the whole blood sample.

Measuring a T cell response can be accomplished using any assay known in the art (see, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001, Current

Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Microarray technology is described in Microarray Methods and Protocols, R. Matson, CRC Press, 2009, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York). In some embodiments, measuring a T cell response

4010157 comprises an MHC Class II tetramer assay, such as flow cytometry with MHC Class II tetramer staining (see, e.g., Raki M, Fallang LE, Brottveit M, Bergseng E, Quarsten H, Lundin KE, Sollid LM: Tetramer visualization of gut-homing gluten- specific T cells in the peripheral blood of Celiac disease patients. Proceedings of the National Academy of Sciences of the United States of America 2007; Anderson RP, van Heel DA, Tye-Din JA, Barnardo M, Salio M, Jewell DP, Hill AV: T cells in peripheral blood after gluten challenge in coeliac disease. Gut 2005, 54(9): 1217-1223; Brottveit M, Raki M, Bergseng E, Fallang LE,

Simonsen B, Lovik A, Larsen S, Loberg EM, Jahnsen FL, Sollid LM et al: Assessing possible Celiac disease by an HLA-DQ2-gliadin Tetramer Test. The American journal of

gastroenterology 2011, 106(7): 1318-1324; and Anderson RP, Degano P, Godkin AJ, Jewell DP, Hill AV: In vivo antigen challenge in Celiac disease identifies a single transglutaminase- modified peptide as the dominant A-gliadin T cell epitope. Nature Medicine 2000, 6(3):337- 342).

In some embodiments, measuring a T cell response in a sample comprising T cells from a subject comprises measuring a level of at least one cytokine in the sample. In some embodiments, measuring a T cell response in a sample comprising T cells from a subject comprises contacting the sample with a composition comprising at least one gluten peptide as described herein and measuring a level of at least one cytokine in the sample. In some embodiments, the at least one cytokine is IFN-γ, IP- 10, or IL-2. Exemplary IFN-γ, IP- 10 (also known as CXCL10), and IL-2 sequences are provided below.

>gi I 323 422 857 I ref I M_0 0 1565 . 3 I Homo sapiens chemokine ( C-X-C mot i f ) l igand 1 0 ( CXCL 1 0 ) , mRNA

CTTTGCAGATAAATATGGCACACTAGCCCCACGTTTTCTGAGACATTCCTCAATTGCTTA GACATATTCT GAGCCTACAGCAGAGGAACCTCCAGTCTCAGCACCATGAATCAAACTGCCATTCTGATTT GCTGCCTTAT

CTTTCTGACTCTAAGTGGCATTCAAGGAGTACCTCTCTCTAGAACTGTACGCTGTAC CTGCATCAGCATT AGTAATCAACCTGTTAATCCAAGGTCTTTAGAAAAACTTGAAATTATTCCTGCAAGCCAA TTTTGTCCAC GTGTTGAGATCATTGCTACAATGAAAAAGAAGGGTGAGAAGAGATGTCTGAATCCAGAAT CGAAGGCCAT CAAGAATTTACTGAAAGCAGTTAGCAAGGAAAGGTCTAAAAGATCTCCTTAAAACCAGAG GGGAGCAAAA TCGATGCAGTGCTTCCAAGGATGGACCACACAGAGGCTGCCTCTCCCATCACTTCCCTAC ATGGAGTATA

TGTCAAGCCATAATTGTTCTTAGTTTGCAGTTACACTAAAAGGTGACCAATGATGGT CACCAAATCAGCT GCTACTACTCCTGTAGGAAGGTTAATGTTCATCATCCTAAGCTATTCAGTAATAACTCTA CCCTGGCACT ATAATGTAAGCTCTACTGAGGTGCTATGTTCTTAGTGGATGTTCTGACCCTGCTTCAAAT ATTTCCCTCA CCTTTCCCATCTTCCAAGGGTACTAAGGAATCTTTCTGCTTTGGGGTTTATCAGAATTCT CAGAATCTCA AATAACTAAAAGGTATGCAATCAAATCTGCTTTTTAAAGAATGCTCTTTACTTCATGGAC TTCCACTGCC

4010157 ATCCTCCCAAGGGGCCCAAATTCTTTCAGTGGCTACCTACATACAATTCCAAACACATAC AGGAAGGTAG AAATATCTGAAAATGTATGTGTAAGTATTCTTATTTAATGAAAGACTGTACAAAGTAGAA GTCTTAGATG TATATATTTCCTATATTGTTTTCAGTGTACATGGAATAACATGTAATTAAGTACTATGTA TCAATGAGTA ACAGGAAAATTTTAAAAATACAGATAGATATATGCTCTGCATGTTACATAAGATAAATGT GCTGAATGGT TTTCAAAATAAAAATGAGGTACTCTCCTGGAAATATTAAGAAAGACTATCTAAATGTTGA AAGATCAAAA GGTTAATAAAGTAATTATAACTAAGAAAAAAAAAAAA (SEQ ID NO: 13 )

>gi I 149999382 I ref I P_001556 . 2 I C-X-C motif chemokine 10 precursor [Homo sapiens ]

MNQTAILICCLIFLTLSGIQGVPLSRTVRCTCI S I SNQPVNPRSLEKLEI IPASQFCPRVEI IATMKKKG EKRCLNPESKAIKNLLKAVSKERSKRSP (SEQ ID NO: 14 )

>gi I 149999382 : 22 -98 C-X-C motif chemokine 10 mature protein [Homo sapiens] VPLSRTVRCTCI SI SNQPVNPRSLEKLEI IPASQFCPRVEI IATMKKKGEKRCLNPESKAIKNLLKAVSK ERSKRSP (SEQ ID NO: 15 )

>gi I 56786137 I ref I NM_000619 . 2 I Homo sapiens interferon, gamma ( IFNG ) , mRNA CACATTGTTCTGATCATCTGAAGATCAGCTATTAGAAGAGAAAGATCAGTTAAGTCCTTT GGACCTGATC AGCTTGATACAAGAACTACTGATTTCAACTTCTTTGGCTTAATTCTCTCGGAAACGATGA AATATACAAG TTATATCTTGGCTTTTCAGCTCTGCATCGTTTTGGGTTCTCTTGGCTGTTACTGCCAGGA CCCATATGTA AAAGAAGCAGAAAACCTTAAGAAATATTTTAATGCAGGTCATTCAGATGTAGCGGATAAT GGAACTCTTT TCTTAGGCATTTTGAAGAATTGGAAAGAGGAGAGTGACAGAAAAATAATGCAGAGCCAAA TTGTCTCCTT TTACTTCAAACTTTTTAAAAACTTTAAAGATGACCAGAGCATCCAAAAGAGTGTGGAGAC CATCAAGGAA GACATGAATGTCAAGTTTTTCAATAGCAACAAAAAGAAACGAGATGACTTCGAAAAGCTG ACTAATTATT CGGTAACTGACTTGAATGTCCAACGCAAAGCAATACATGAACTCATCCAAGTGATGGCTG AACTGTCGCC

AGCAGCTAAAACAGGGAAGCGAAAAAGGAGTCAGATGCTGTTTCGAGGTCGAAGAGC ATCCCAGTAATGG TTGTCCTGCCTGCAATATTTGAATTTTAAATCTAAATCTATTTATTAATATTTAACATTA TTTATATGGG GAATATATTTTTAGACTCATCAATCAAATAAGTATTTATAATAGCAACTTTTGTGTAATG AAAATGAATA TCTATTAATATATGTATTATTTATAATTCCTATATCCTGTGACTGTCTCACTTAATCCTT TGTTTTCTGA CTAATTAGGCAAGGCTATGTGATTACAAGGCTTTATCTCAGGGGCCAACTAGGCAGCCAA CCTAAGCAAG

ATCCCATGGGTTGTGTGTTTATTTCACTTGATGATACAATGAACACTTATAAGTGAA GTGATACTATCCA GTTACTGCCGGTTTGAAAATATGCCTGCAATCTGAGCCAGTGCTTTAATGGCATGTCAGA CAGAACTTGA ATGTGTCAGGTGACCCTGATGAAAACATAGCATCTCAGGAGATTTCATGCCTGGTGCTTC CAAATATTGT TGACAACTGTGACTGTACCCAAATGGAAAGTAACTCATTTGTTAAAATTATCAATATCTA ATATATATGA ATAAAGTGTAAGTTCACAACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 16 )

>gi I 56786138 I ref I NP_000610 . 2 I interferon gamma precursor [Homo sapiens] MKYTSYILAFQLCIVLGSLGCYCQDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWK EESDRKIMQS QIVSFYFKLFKNFKDDQS IQKSVETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVM AELSPAAKTGKRKRSQMLFRGRRASQ (SEQ ID NO: 17 )

>gi I 56786138 : 24- 166 interferon gamma mature protein [Homo sapiens]

QDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLF KNFKDDQS IQKSV ETIKEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAELSPAAKTGKRK RSQMLFRGRR ASQ (SEQ ID NO: 18 )

4010157 >gi I 125661059 I ref I M_000586.3 I Homo sapiens interleukin 2 (IL2), mRNA AGTTCCCTATCACTCTCTTTAATCACTACTCACAGTAACCTCAACTCCTGCCACAATGTA CAGGATGCAA CTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACAAACAGTGCACCTACTTCAAGT TCTACAAAGA AAACACAGCTACAACTGGAGCATTTACTGCTGGATTTACAGATGATTTTGAATGGAATTA ATAATTACAA GAATCCCAAACTCACCAGGATGCTCACATTTAAGTTTTACATGCCCAAGAAGGCCACAGA ACTGAAACAT CTTCAGTGTCTAGAAGAAGAACTCAAACCTCTGGAGGAAGTGCTAAATTTAGCTCAAAGC AAAAACTTTC ACTTAAGACCCAGGGACTTAATCAGCAATATCAACGTAATAGTTCTGGAACTAAAGGGAT CTGAAACAAC ATTCATGTGTGAATATGCTGATGAGACAGCAACCATTGTAGAATTTCTGAACAGATGGAT TACCTTTTGT CAAAGCATCATCTCAACACTGACTTGATAATTAAGTGCTTCCCACTTAAAACATATCAGG CCTTCTATTT ATTTAAATATTTAAATTTTATATTTATTGTTGAATGTATGGTTTGCTACCTATTGTAACT ATTATTCTTA ATCTTAAAACTATAAATATGGATCTTTTATGATTCTTTTTGTAAGCCCTAGGGGCTCTAA AATGGTTTCA CTTATTTATCCCAAAATATTTATTATTATGTTGAATGTTAAATATAGTATCTATGTAGAT TGGTTAGTAA AACTATTTAATAAATTTGATAAATATAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 19)

>gi I 28178861 I ref I NP_000577.2 I interleukin-2 precursor [Homo sapiens] MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML TFKFYMPKKA TELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLI SNINVIVLELKGSETTFMCEYADETATIVEFLNR WITFCQSIISTLT (SEQ ID NO: 20)

>gi I 28178861 I ref I NP_000577.2 I interleukin-2 mature protein [Homo sapiens] APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLE EELKPLEEVLNLAQS KNFHLRPRDLI SNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQS I I STLT (SEQ ID NO: 21)

In some embodiments, measuring a T cell response comprises measuring a level of at least one cytokine. Levels of at least one cytokine include levels of cytokine RNA, e.g., mRNA, and/or levels of cytokine protein. In a preferred embodiment, levels of the at least one cytokine are protein levels.

In some embodiments of any one of the methods provided herein, the method further comprises recording the level(s) or the result(s) of the assessing and/or measuring.

Assays for detecting cytokine RNA include, but are not limited to, Northern blot analysis, RT-PCR, sequencing technology, RNA in situ hybridization (using e.g., DNA or RNA probes to hybridize RNA molecules present in the sample), in situ RT-PCR (e.g., as described in Nuovo GJ, et al. Am J Surg Pathol. 1993, 17: 683-90; Komminoth P, et al. Pathol Res Pract. 1994, 190: 1017-25), and oligonucleotide microarray (e.g., by hybridization of polynucleotide sequences derived from a sample to oligonucleotides attached to a solid surface (e.g., a glass wafer with addressable location, such as Affymetrix microarray

(Affymetrix®, Santa Clara, CA)). Designing nucleic acid binding partners, such as probes, is

4010157 well known in the art. In some embodiments, the nucleic acid binding partners bind to a part of or an entire nucleic acid sequence of at least one cytokine, e.g., IFN-γ, the sequence(s) provided herein or being identifiable using the Genbank IDs described herein or as otherwise known in the art.

Assays for detecting protein levels include, but are not limited to, immunoassays (also referred to herein as immune-based or immuno-based assays, e.g., Western blot, ELISA, and ELISpot assays), Mass spectrometry, and multiplex bead-based assays. Binding partners for protein detection can be designed using methods known in the art and as described herein. In some embodiments, the protein binding partners, e.g., antibodies, bind to a part of or an entire amino acid sequence of at least one cytokine, e.g., IFN-γ, the sequence(s) provided herein or being identifiable using the Genbank IDs described herein or as otherwise known in the art. Other examples of protein detection and quantitation methods include multiplexed immunoassays as described for example in U.S. Patent Nos. 6939720 and 8148171, and published U.S. Patent Application No. 2008/0255766, and protein microarrays as described for example in published U.S. Patent Application No. 2009/0088329.

In some embodiments, measuring a level of at least one cytokine comprises an enzyme-linked immunosorbent assay (ELISA) or enzyme-linked immunosorbent spot (ELISpot) assay. ELISA and ELISpot assays are well known in the art (see, e.g., U.S. Patent Nos. 5,939, 281, 6,410,252, and 7,575,870; Czerkinsky C, Nilsson L, Nygren H, Ouchterlony O, Tarkowski A (1983) "A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody- secreting cells". J Immunol Methods 65 (1-2): 109-121 and Lequin R (2005). "Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA)". Clin. Chem. 51 (12): 2415-8).

An exemplary ELISA involves at least one binding partner, e.g., an antibody or antigen -binding fragment thereof, with specificity for the at least one cytokine, e.g., IFN-γ. The sample with an unknown amount of the at least one cytokine can be immobilized on a solid support (e.g., a polystyrene microtiter plate) either non- specifically (via adsorption to the surface) or specifically (via capture by another binding partner specific to the same at least one cytokine, as in a "sandwich" ELISA). After the antigen is immobilized, the binding

4010157 partner for the at least one cytokine is added, forming a complex with the immobilized at least one cytokine. The binding partner can be attached to a detectable label as described herein (e.g., a fhiorophor or an enzyme), or can itself be detected by an agent that recognizes the at least one cytokine binding partner that is attached to a detectable label as described herein (e.g., a fhiorophor or an enzyme). If the detectable label is an enzyme, a substrate for the enzyme is added, and the enzyme elicits a chromogenic or fluorescent signal by acting on the substrate. The detectable label can then be detected using an appropriate machine, e.g., a fluorimeter or spectrophotometer, or by eye.

An exemplary ELISpot assay involves a binding agent for the at least one cytokine (e.g., an anti- IFN-γ) that is coated aseptically onto a PVDF (polyvinylidene fluoride)-backed microplate. Cells of interest (e.g., peripheral blood mononuclear cells) are plated out at varying densities, along with antigen (e.g., a gluten peptide as described herein), and allowed to incubate for a period of time (e.g., about 24 hours). The at least one cytokine secreted by activated cells is captured locally by the binding partner for the at least one cytokine on the high surface area PVDF membrane. After the at least one cytokine is immobilized, a second binding partner for the at least one cytokine is added, forming a complex with the

immobilized at least one cytokine. The binding partner can be linked to a detectable label (e.g., a fhiorophor or an enzyme), or can itself be detected by an agent that recognizes the binding partner for the at least one cytokine (e.g., a secondary antibody) that is linked to a detectable label (e.g., a fluorophor or an enzyme). If the detectable label is an enzyme, a substrate for the enzyme is added, and the enzyme elicits a chromogenic or fluorescent signal by acting on the substrate. The detectable label can then be detected using an appropriate machine, e.g., a fluorimeter or spectrophotometer, or by eye.

In some embodiments, a level of at least one cytokine is measured using an ELISA. As an exemplary method, at least one gluten peptide as defined herein is dried onto the inner wall of a blood collection tube. A negative control tube containing no antigen is provided. A positive control tube containing a mitogen is also provided. Blood from a subject is drawn into each of the three tubes. Each tube is agitated to ensure mixing. The tubes are then incubated at 37 degrees Celsius, preferably immediately after blood draw or at least within

4010157 about 16 hours of collection. After incubation, the cells are separated from the plasma by centrifugation. The plasma is then loaded into an ELISA plate for detection of levels of at least one cytokine (e.g., IFN-γ) present in the plasma. A standard ELISA assay as described above can then be used to detect the levels of the at least one cytokine present in each plasma sample.

In some embodiments, measuring a T cell response comprises a multiplex bead-based assay. An exemplary multiplex bead-based assay involves use of magnetic beads that are internally dyed with fluorescent dyes to produce a specific spectral address. Binding partners (e.g., antibodies) are conjugated to the surface of beads to capture cytokines. The sample is loaded into a 96-well plate containing the beads and the sample is incubated to allow binding of cytokines to the beads. A second biotinylated binding partner for the cytokines are added after the cytokines bind to the beads. A streptavidin-conjugated detectable label is then bound to the biotin. Light emitting diodes are used to illuminate the samples, causing the fluorescent dyes in the beads to fluoresce, as well as the detectable label to fluoresce. The concentration of the cytokines are then determined based on the level of fluorescence. An exemplary system for running a multiplex bead-based assay is the MAGPIX® system available from Luminex® Corporation (see, e.g., US Patent Nos. US 8,031,918, US

8,296,088, US 8,274,656, US 8,532,351, US 8,542,897, US 6,514,295, US 6,599,331, US 6,632,526, US 6,929,859, US 7,445,844, US 7,718,262, US 8,283,037, and US 8,568,881, all of which are incorporated by reference herein, and in particular the systems provided herein).

In some embodiments, a T cell response measurement in a sample obtained after administration of a composition comprising a gluten peptide to the subject is detected using any of the methods above or any other appropriate method and is then compared to a control T cell response, e.g., a T cell response measurement in a sample obtained prior to a gluten challenge described herein. In some embodiments, a control T cell response is measured using any of the methods above or any other appropriate methods. In some embodiments, the control T cell response is a negative control T cell response. Exemplary negative controls include, but are not limited to, a T cell response in a sample that has been contacted with a non- T cell-activating peptide (e.g., a peptide not recognized by T cells present in a sample

4010157 from a subject), such as a non-CD4 + -T cell-activating peptide, or a T cell response in sample that has not been contacted with a T cell- activating peptide (e.g., contacting the sample with a saline solution containing no peptides), such as a CD4 + T cell- activating peptide.

5 Samples

Samples, as used herein, refer to biological samples taken or derived from a subject, e.g., a subject having or suspected of having Celiac disease. Examples of samples include tissue samples or fluid samples. Examples of fluid samples are whole blood, plasma, serum, and other bodily fluids that comprise T cells. In some embodiments, the sample comprises T o cells. In some embodiments, the sample comprises T cells and monocytes and/or

granulocytes. In some embodiments, the sample comprising T cells comprise whole blood or peripheral blood mononuclear cells (PBMCs). The T cell may be a CD4 + T cell, e.g., a gluten-reactive CD4 + T cell. In some embodiments, the methods described herein comprise obtaining or providing the sample. In some embodiments, a first and second sample are5 contemplated. In some embodiments, the first sample is obtained from a subject after

administration of a composition comprising gluten (e.g., comprising 1.5 grams of wheat flour protein, 1 gram of barley flour protein, and 0.5 grams of rye flour protein). In some embodiments, the second sample is prior to administration of the composition. Additional samples, e.g., third, fourth, fifth, etc., are also contemplated if additional measurements of a T o cell response are desired. Such additional samples may be obtained from the subject at any time, e.g., before or after administration of the composition.

Subjects

A subject may include any subject that is suspected of having Celiac disease. In some 5 embodiments, the subject may include any subject that has or is suspected of having Celiac disease. Preferably, the subject is a human. In some embodiments, the subject has one or more HLA-DQA and HLA-DQB susceptibility alleles encoding HLA-DQ2.5 (DQA1 *05 and DQB1 *02), HLA-DQ2.2 (DQA1 *02 and DQB1 *02) or HLA-DQ8 (DQA1 *03 and

DQB1 *0302). In some embodiments, the subject is HLA-DQ2.5 positive (i.e., has both

4010157 susceptibility alleles DQAl *05 and DQB1 *02). In some embodiments, a subject may have a family member that has one or more HLA-DQA and HLA-DQB susceptibility alleles encoding HLA-DQ2.5 (DQA1 *05 and DQB1 *02), HLA-DQ2.2 (DQA1 *02 and DQB1 *02) or HLA- DQ8 (DQAl *03 and DQB1 *0302). Subjects can be tested for the presence of the HLA-DQA and HLA-DQB susceptibility alleles encoding HLA-DQ2.5 (DQAl *05 and DQB1 *02), DQ2.2 (DQAl *02 and DQB1 *02) or DQ8 (DQAl *03 and DQB1 *0302). Exemplary sequences that encode the DQA and DQB susceptibility alleles include HLA-DQA 1*0501 (Genbank accession number: AF515813.1) HLA-DQA1*0505 (AH013295.2), HLA- DQB 1*0201 (AY375842.1) or HLA-DQB 1*0202 (AY375844.1). Methods of genetic testing are well known in the art (see, e.g., Bunce M, et al. Phototyping: comprehensive DNA typing for HLA-A, B, C, DRB1, DRB3, DRB4, DRB5 & DQB1 by PCR with 144 primer mixes utilizing sequence- specific primers (PCR-SSP). Tissue Antigens 46, 355-367 (1995); Olerup O, Aldener A, Fogdell A. HLA-DQB 1 and DQAl typing by PCR amplification with sequence-specific primers in 2 hours. Tissue antigens 41, 119-134 (1993); Mullighan CG, Bunce M, Welsh KI. High-resolution HLA-DQB 1 typing using the polymerase chain reaction and sequence- specific primers. Tissue-Antigens. 50, 688-92 (1997); Koskinen L, Romanos J, Kaukinen K, Mustalahti K, Korponay-Szabo I, et al. (2009) Cost-effective HLA typing with tagging SNPs predicts celiac disease risk haplotypes in the Finnish, Hungarian, and Italian populations. Immunogenetics 61: 247-256.; and Monsuur AJ, de Bakker PI, Zhernakova A, Pinto D, Verduijn W, et al. (2008) Effective detection of human leukocyte antigen risk alleles in celiac disease using tag single nucleotide polymorphisms. PLoS ONE 3: e2270). Subjects that have one or more copies of a susceptibility allele are considered to be positive for that allele. Detection of the presence of susceptibility alleles can be

accomplished by any nucleic acid assay known in the art, e.g., by polymerase chain reaction (PCR) amplification of DNA extracted from the patient followed by hybridization with sequence-specific oligonucleotide probes or using leukocyte-derived DNA (Koskinen L, Romanos J, Kaukinen K, Mustalahti K, Korponay-Szabo I, Barisani D, Bardella MT, Ziberna F, Vatta S, Szeles G et al: Cost-effective HLA typing with tagging SNPs predicts Celiac disease risk haplotypes in the Finnish, Hungarian, and Italian populations. Immunogenetics

4010157 2009, 61(4):247-256; Monsuur AJ, de Bakker PI, Zhernakova A, Pinto D, Verduijn W, Romanos J, Auricchio R, Lopez A, van Heel DA, Crusius JB et al: Effective detection of human leukocyte antigen risk alleles in Celiac disease using tag single nucleotide

polymorphisms. PLoS ONE 2008, 3(5):e2270).

Controls and Control Subjects

In some embodiments, any one of the methods provided herein comprise measuring a T cell response in a sample obtained from a subject after administration of a composition comprising a gluten (e.g., comprising a ratio of 3:2: 1 of wheat flour protein, barley flour protein, and rye flour protein by weight in grams, respectively, such as 1.5 grams of wheat flour protein, 1 gram of barley flour protein, and 0.5 grams of rye flour protein) and comparing the T cell response to one or more control T cell responses. In some

embodiments, the control T cell response is a T cell response in a sample obtained from the same subject prior to administration of the composition comprising gluten (e.g., comprising a ratio of 3:2: 1 of wheat flour protein, barley flour protein, and rye flour protein by weight in grams, respectively, such as 1.5 grams of wheat flour protein, 1 gram of barley flour protein, and 0.5 grams of rye flour protein).

However, other or further controls are also contemplated. For example, a control T cell response may be a T cell response in a sample from a control subject (or subjects). In some embodiments, a control subject has one or more HLA-DQA and HLA-DQB

susceptibility alleles encoding HLA-DQ2.5 (DQA1 *05 and DQB1 *02), DQ2.2 (DQA1 *02 and DQBl *02) or DQ8 (DQAl *03 and DQBl *0302) described herein but does not have Celiac disease. In some embodiments, a control subject does not have any of the HLA-DQA and HLA-DQB susceptibility alleles encoding HLA-DQ2.5 (DQA1 *05 and DQB1 *02), DQ2.2 (DQAl *02 and DQBl *02) or DQ8 (DQAl *03 and DQBl *0302) described herein. In some embodiments, a control subject is a healthy individual not having or suspected of having Celiac disease. In some embodiments, a control T cell response is a pre-determined value from a control subject or subjects, such that the control T cell response need not be measured every time the methods described herein are performed.

4010157 Gluten Peptides and Compositions Containing Gluten Peptides

As used herein the term "gluten peptide" includes any peptides comprising an amino acid sequence derived from, or encompassed within, one or more of gluten proteins alpha 5 (a), beta (β), γ (γ) and omega (ω) gliadins, and low and high molecular weight (LMW and HMW) glutenins in wheat, B, C and D hordeins in barley, β, γ and CO secalins in rye, and optionally avenins in oats. Preferably, in some embodiments, the gluten peptide(s) stimulate a CD4+ T cell specific response.

Exemplary gluten peptides and methods for synthesizing such peptides are known in l o the art (see, e.g., PCT Publication Nos.: WO/2001/025793, WO/2003/104273,

WO/2005/105129, WO/2010/060155 and PCT Application No.: PCT/US2014/027101, all of which are incorporated herein by reference in their entirety, in particular with respect to the gluten peptides disclosed therein).

The length of the peptide may vary. In some embodiments, peptides are, e.g., 4, 5, 6,

15 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acids in length. In some embodiments, peptides are, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, or 100 or fewer amino acids in length. In some

20 embodiments, peptides are, e.g., 4-1000, 4-500, 4-100, 4-50, 4-40, 4-30, or 4-20 amino acids in length. In some embodiments, peptides are 4-20, 5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 11- 20, 12-20, 13-20, 14-20, or 15-20 amino acids in length. In some embodiments, peptides are e.g., 5-30, 10-30, 15-30 or 20-30 amino acids in length. In some embodiments, peptides are 4-50, 5-50, 6-50, 7-50, 8-50, 9-50, 10-50, 11-50, 12-50, 13-50, 14-50, or 15-50 amino acids

25 in length. In some embodiments, peptides are 8-50 amino acids in length.

In some embodiments, any one of the methods described herein comprise contacting a composition comprising a gluten peptide with a sample from a subject (e.g., a sample comprising T cells).

In some embodiments, the composition comprises at least one of: (i) a first peptide

4010157 comprising the amino acid sequence PFPQPELPY (SEQ ID NO: 1) and PQPELPYPQ (SEQ ID NO: 2), (ii) a second peptide comprising the amino acid sequence PFPQPEQPF (SEQ ID NO: 3) and PQPEQPFPW (SEQ ID NO: 4), and (iii) a third peptide comprising the amino acid sequence PIPEQPQPY (SEQ ID NO: 5). In some embodiments, the composition 5 comprises at least one of: (i) a first peptide comprising the amino acid sequence PFPQPELPY (SEQ ID NO: 1) and PQPELPYPQ (SEQ ID NO: 2), (ii) a second peptide comprising the amino acid sequence PFPQPEQPF (SEQ ID NO: 3) and PQPEQPFPW (SEQ ID NO: 4), and (iii) a third peptide comprising the amino acid sequence PIPEQPQPY (SEQ ID NO: 5) and the amino acid sequence EQPIPEQPQ (SEQ ID NO: 12). "First", "second", and "third" are o not meant to imply an order of use or importance, unless specifically stated otherwise. In some embodiments, the composition comprises the first and second peptide, the first and third peptide, or the second and third peptide. In some embodiments, the composition comprises the first and second peptide. In some embodiments, the composition comprises the first, second, and third peptide. In some embodiments, the first peptide comprises the amino 5 acid sequence LQPFPQPELPYPQPQ (SEQ ID NO: 6); the second peptide comprises the amino acid sequence QPFPQPEQPFPWQP (SEQ ID NO: 7); and/or the third peptide comprises the amino acid sequence PEQPIPEQPQPYPQQ (SEQ ID NO: 8).

Modifications to a gluten peptide are also contemplated herein. This modification may occur during or after translation or synthesis (for example, by farnesylation, prenylation, o myristoylation, glycosylation, palmitoylation, acetylation, phosphorylation (such as

phosphotyrosine, phosphoserine or phosphothreonine), amidation, derivatisation by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, and the like). Any of the numerous chemical modification methods known within the art may be utilized including, but not limited to, specific chemical cleavage by 5 cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4, acetylation,

formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, etc.

The phrases "protecting group" and "blocking group" as used herein, refers to modifications to the peptide which protect it from undesirable chemical reactions, particularly chemical reactions in vivo. Examples of such protecting groups include esters of carboxylic

4010157 acids and boronic acids, ethers of alcohols and acetals, and ketals of aldehydes and ketones. Examples of suitable groups include acyl protecting groups such as, for example, furoyl, formyl, adipyl, azelayl, suberyl, dansyl, acetyl, theyl, benzoyl, trifluoroacetyl, succinyl and methoxysuccinyl; aromatic urethane protecting groups such as, for example,

5 benzyloxycarbonyl (Cbz); aliphatic urethane protecting groups such as, for example, t- butoxycarbonyl (Boc) or 9-fluorenylmethoxy-carbonyl (FMOC); pyroglutamate and amidation. Many other modifications providing increased potency, prolonged activity, ease of purification, and/or increased half-life will be known to the person skilled in the art.

The peptides may comprise one or more modifications, which may be natural post- o translation modifications or artificial modifications. The modification may provide a

chemical moiety (typically by substitution of a hydrogen, for example, of a C-H bond), such as an amino, acetyl, acyl, carboxy, hydroxy or halogen (for example, fluorine) group, or a carbohydrate group. Typically, the modification is present on the N- or C-terminal.

Furthermore, one or more of the peptides may be PEGylated, where the PEG

5 (polyethyleneoxy group) provides for enhanced lifetime in the blood stream. One or more of the peptides may also be combined as a fusion or chimeric protein with other proteins, or with specific binding agents that allow targeting to specific moieties on a target cell.

A gluten peptide may also be chemically modified at the level of amino acid side chains, of amino acid chirality, and/or of the peptide backbone.

o Particular changes can be made to a gluten peptide to improve resistance to

degradation or optimize solubility properties or otherwise improve bioavailability compared to the parent gluten peptide, thereby providing gluten peptides having similar or improved therapeutic, diagnostic and/or pharmacokinetic properties. A preferred such modification, in some embodiments, includes the use of an N-terminal acetyl group or pyroglutamate and/or a 5 C-terminal amide. Such modifications have been shown in the art to significantly increase the half-life and bioavailability of peptides compared to the peptides having a free N- and C- terminus (see, e.g., PCT Publication No.: WO/2010/060155). In some embodiments, the first, second and/or third peptides comprise an N-terminal acetyl group or pyroglutamate group and/or a C-terminal amide group. In some embodiments, the first peptide comprises

4010157 the amino acid sequence ELQPFPQPELPYPQPQ (SEQ ID NO: 9), wherein the N-terminal E is a pyroglutamate; the second peptide comprises the amino acid sequence

EQPFPQPEQPFPWQP (SEQ ID NO: 10), wherein the N-terminal E is a pyroglutamate; and/or the third peptide comprises the amino acid sequence EPEQPIPEQPQPYPQQ (SEQ ID NO: 11), wherein the N-terminal E is a pyroglutamate. In some embodiments, the first peptide comprises the amino acid sequence ELQPFPQPELPYPQPQ (SEQ ID NO: 9), wherein the N-terminal E is a pyroglutamate, and wherein the peptide contains a C-terminal amide group; the second peptide comprises the amino acid sequence EQPFPQPEQPFPWQP (SEQ ID NO: 10), wherein the N-terminal E is a pyroglutamate, and wherein the peptide contains a C-terminal amide group; and/or the third peptide comprises the amino acid sequence EPEQPIPEQPQPYPQQ (SEQ ID NO: 11), wherein the N-terminal E is a pyroglutamate, and wherein the peptide contains a C-terminal amide group. In some embodiments, the first peptide consists of the amino acid sequence ELQPFPQPELPYPQPQ (SEQ ID NO: 9), wherein the N-terminal E is a pyroglutamate, and wherein the peptide contains a C-terminal amide group; the second peptide consists of the amino acid sequence EQPFPQPEQPFPWQP (SEQ ID NO: 10), wherein the N-terminal E is a pyroglutamate, and wherein the peptide contains a C-terminal amide group; and/or the third peptide consists of the amino acid sequence EPEQPIPEQPQPYPQQ (SEQ ID NO: 11), wherein the N-terminal E is a pyroglutamate, and wherein the peptide contains a C-terminal amide group.

Kits

Another aspect of the disclosure relates to kits. In some embodiments, the kit comprises a composition comprising gluten (e.g., comprising a ratio of 3:2: 1 of wheat flour protein, barley flour protein, and rye flour protein by weight in grams, respectively, such as 1.5 grams of wheat flour protein, 1 gram of barley flour protein, and 0.5 grams of rye flour protein). In some embodiments of any one of the kits provided, the composition is a foodstuff (e.g., baked goods such as breads, cookies, muffins, cakes, etc.). In some embodiments of any one of the kits provided, composition is provided in triplicate. In some

4010157 embodiments, the composition is provided in nonuplicate (i.e., nine of the composition are provided).

In some embodiments of any one of the kits provided, the kit comprises or further comprises a gluten peptide composition as described herein. In some embodiments of any 5 one of the kits provided, the kit comprises or further comprises: (a) a composition comprising at least one of: (i) a first peptide comprising the amino acid sequence PFPQPELPY (SEQ ID NO: 1) and PQPELPYPQ (SEQ ID NO: 2), (ii) a second peptide comprising the amino acid sequence PFPQPEQPF (SEQ ID NO: 3) and PQPEQPFPW (SEQ ID NO: 4), and (iii) a third peptide comprising the amino acid sequence PIPEQPQPY (SEQ ID NO: 5); and/or (b) an o agent for assessing a T cell response. In some embodiments of any one of the kits provided, the kit comprises or further comprises: (a) a composition comprising at least one of: (i) a first peptide comprising the amino acid sequence PFPQPELPY (SEQ ID NO: 1) and

PQPELPYPQ (SEQ ID NO: 2), (ii) a second peptide comprising the amino acid sequence PFPQPEQPF (SEQ ID NO: 3) and PQPEQPFPW (SEQ ID NO: 4), and (iii) a third peptide5 comprising the amino acid sequence PIPEQPQPY (SEQ ID NO: 5) and the amino acid

sequence EQPIPEQPQ (SEQ ID NO: 12); and/or (b) an agent for assessing a T cell response. In some embodiments of any one of the kits provided, the agent is a binding partner for a cytokine indicative of the T cell response. In some embodiments of any one of the kits provided, the kit further comprises an agent that recognizes the binding partner for, for o example, IFN-γ, IP- 10, and/or IL-2.

In some embodiments of any one of the kits provided, the composition comprising the peptides contained in the kit comprises the first and second peptide, the first and third peptide, or the second and third peptide. In some embodiments of any one of the kits provided, the composition comprises the first and second peptide. In some embodiments of 5 any one of the kits provided, the composition comprises the first, second, and third peptide.

In some embodiments, the first peptide comprises LQPFPQPELPYPQPQ (SEQ ID NO: 6); the second peptide comprises QPFPQPEQPFPWQP (SEQ ID NO: 7); and/or the third peptide comprises PEQPIPEQPQPYPQQ (SEQ ID NO: 8). In some embodiments of any one of the kits provided, the first, second and/or third peptides comprise an N-terminal acetyl group or

4010157 pyroglutamate group, and/or a C terminal amide group. In some embodiments of any one of the kits provided, the first peptide comprises ELQPFPQPELPYPQPQ (SEQ ID NO: 9), wherein the N-terminal E is a pyroglutamate; the second peptide comprises

EQPFPQPEQPFPWQP (SEQ ID NO: 10), wherein the N-terminal E is a pyroglutamate; 5 and/or the third peptide comprises EPEQPIPEQPQPYPQQ (SEQ ID NO: 11), wherein the N- terminal E is a pyroglutamate. In some embodiments of any one of the kits provided, the first peptide consists of ELQPFPQPELPYPQPQ (SEQ ID NO: 9), wherein the N-terminal E is a pyroglutamate, and wherein the peptide contains a C-terminal amide group; the second peptide consists of EQPFPQPEQPFPWQP (SEQ ID NO: 10), wherein the N-terminal E is a o pyroglutamate, and wherein the peptide contains a C-terminal amide group; and/or the third peptide consists of EPEQPIPEQPQPYPQQ (SEQ ID NO: 11), wherein the N-terminal E is a pyroglutamate, and wherein the peptide contains a C-terminal amide group.

In some embodiments of any one of the kits provided, the kit further comprises a container for whole blood. In some embodiments of any one of the kits provided, the gluten5 peptide composition is contained within the container (e.g., dried onto the wall of the

container). In some embodiments of any one of the kits provided, the composition is contained within a solution separate from the container, such that the composition may be added to the container after blood collection. In some embodiments of any one of the kits provided, the composition is in lyophilized form in a separate container, such that the o composition may be reconstituted and added to the container after blood collection, in some embodiments. In some embodiments of any one of the kits provided, the container further contains an anti-coagulant, such as heparin. In some embodiments of any one of the kits provided, the container is structured to hold a defined volume of blood e.g. 1 mL or 5 mL. In some embodiments of any one of the kits provided, the container is present in the kit in 5 duplicate or triplicate.

In some embodiments of any one of the kits provided, the kit further comprises a negative control container for whole blood and/or a positive control container for whole blood. The negative control container may be, for example, an empty container or a container containing a non- T cell-activating peptide (e.g., dried onto the wall of the

4010157 container), such as a non-CD4 + -T cell-activating peptide. The positive control container may contain, for example, a mitogen such as PHA-L (e.g., 10 units PHA-L). In some

embodiments of any one of the kits provided, the negative control container and/or positive control container are structured to hold a defined volume of blood e.g. 1 mL or 5 mL. In 5 some embodiments of any one of the kits provided, the negative control container and/or positive control container are present in the kit in duplicate or triplicate. In some

embodiments of any one of the kits provided, the kit comprises any combination of the components mentioned above.

Any suitable binding partner is contemplated. In some embodiments, the binding o partner is any molecule that binds specifically to a cytokine as provided herein. As described herein, "binds specifically" means that the molecule is more likely to bind to a portion of or the entirety of a protein to be measured than to a portion of or the entirety of another protein. In some embodiments, the binding partner is an antibody or antigen-binding fragment thereof, such as Fab, F(ab)2, Fv, single chain antibodies, Fab and sFab fragments, F(ab')2, Fd5 fragments, scFv, or dAb fragments. Methods for producing antibodies and antigen-binding fragments thereof are well known in the art (see, e.g., Sambrook et al, "Molecular Cloning: A Laboratory Manual" (2nd Ed.), Cold Spring Harbor Laboratory Press (1989); Lewin, "Genes IV", Oxford University Press, New York, (1990), and Roitt et al., "Immunology" (2nd Ed.), Gower Medical Publishing, London, New York (1989), WO2006/040153, WO2006/122786, o and WO2003/002609). Binding partners also include other peptide molecules and aptamers that bind specifically. Methods for producing peptide molecules and aptamers are well known in the art (see, e.g., published US Patent Application No. 2009/0075834, US Patent Nos. 7435542, 7807351, and 7239742). In some embodiments, the binding partner is any molecule that binds specifically to an IFN-γ mRNA. As described herein, "binds specifically 5 to an mRNA" means that the molecule is more likely to bind to a portion of or the entirety of the mRNA to be measured (e.g., by complementary base-pairing) than to a portion of or the entirety of another mRNA or other nucleic acid. In some embodiments, the binding partner that binds specifically to an mRNA is a nucleic acid, e.g., a probe.

4010157 In some embodiments of any one of the kits provided, the kit further comprises a first and second binding partner for a cytokine provided herein. In some embodiments of any one of the kits provided, the kit further comprises one or more binding partners for IP- 10, IFN-γ, and/or IL-2. In some embodiments of any one of the kits provided, the binding partners are antibodies or antigen binding fragments thereof. In some embodiments of any one of the kits provided, the second binding partner is bound to a surface. The second binding partner may be bound to the surface covalently or non-covalently. The second binding partner may be bound directly to the surface, or may be bound indirectly, e.g., through a linker. Examples of linkers, include, but are not limited to, carbon-containing chains, polyethylene glycol (PEG), nucleic acids, monosaccharide units, and peptides. The surface can be made of any material, e.g., metal, plastic, paper, or any other polymer, or any combination thereof. In some embodiments of any one of the kits provided, the first binding partner is washed over the cytokine bound to the second binding partner (e.g., as in a sandwich ELISA). The first binding partner may comprise a detectable label, or an agent that recognizes the first binding partner (e.g., a secondary antibody) may comprise a detectable label.

Any suitable agent that recognizes a binding partner is contemplated. In some embodiments, the binding partner is any molecule that binds specifically to the binding partner. In some embodiments, the agent is an antibody (e.g., a secondary antibody) or antigen-binding fragment thereof, such as Fab, F(ab)2, Fv, single chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, scFv, or dAb fragments. Agents also include other peptide molecules and aptamers that bind specifically to a binding partner. In some embodiments, the binding partner comprises a biotin moiety and the agent is a composition that binds to the biotin moiety (e.g., an avidin or strep tavidin).

In some embodiments, the binding partner and/or the agent comprise a detectable label. Any suitable detectable label is contemplated. Detectable labels include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means, e.g., an enzyme, a radioactive label, a fluorophore, an electron dense reagent, biotin, digoxigenin, or a hapten. Such detectable labels are well- known in the art and can be detectable through use of, e.g., an enzyme assay, a chromo genie

4010157 assay, a luminometric assay, a fluorogenic assay, or a radioimmune assay. The reaction conditions to perform detection of the detectable label depend upon the detection method selected.

In some embodiments of any one of the kits provided, the kit further comprises instructions for performing a challenge method provided herein and/or for detecting a T cell response (e.g., detecting a cytokine indicative of the T cell response) in a sample from a subject having or suspected of having Celiac disease. In some embodiments of any one of the kits provided, the instructions include the methods described herein. Instructions can be in any suitable form, e.g., as a printed insert or a label.

General Techniques and Definitions

Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

Unless otherwise indicated, techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984); J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989); T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991); D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996); F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley- Interscience (1988, including all updates until present); Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988); and J.E. Coligan et al. (editors), Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

In any one aspect or embodiment provided herein "comprising" may be replaced with "consisting essentially of or "consisting of.

4010157 Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present disclosure to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the 5 remainder of the disclosure in any way whatsoever. All publications cited herein are

incorporated by reference for the purposes or subject matter referenced herein.

EXAMPLES

i o Example 1

HLA-DQ2.5-positive celiac disease subjects on gluten-free diet were used in this study. Blood was collected immediately before and 6 days after commencing 3-day oral gluten challenge. The gluten challenge consisted of 3 cookies per day, each cookie containing 1.5 grams of wheat flour protein, 1 gram of barley flour protein, and 0.5 grams of

15 rye flour protein. Whole blood or PBMCs were incubated with pools or single peptides derived from gluten or recall antigens. IFNy and IP- 10 levels were measured in plasma from the whole blood that was incubated in 96-well plates with a pool of peptides. Plasma cytokine/chemokine levels were measured by MAGPIX® multiplex bead assay (IFNy and IP- 10) or by ELISA (IFNy and IP- 10), and PBMC separated from the same blood sample were

2 o incubated in overnight IFNy ELISpot assays.

The peptide pool used was:

Peptide pool 1 (pE=pyroglutamate)

(pE)LQPFPQPELPYPQPQ-amide (SEQ ID NO: 9)

25 (pE)QPFPQPEQPFPWQP-amide (SEQ ID NO: 10)

(pE)PEQPIPEQPQPYPQQ-amide (SEQ ID NO: 11)

Results

Both IP- 10 and IFN-y levels were elevated in blood samples after gluten challenge

4010157 using either ELISA, MAGPIX®, or ELISpot (FIGs. 1 and 2). Using a cut-off of stimulation index >1.25 and cytokine level >100 pg/mL to indicate a positive result, the IP- 10 assay was positive in 10/10 subjects after gluten challenge. Using a cut-off of stimulation index >1.25 and cytokine level >7.2 pg/mL to indicate a positive result, the IFN-γ assay was positive in 5 7/10 subjects after gluten challenge. The levels of IP-10 and IFN-γ before and after gluten challenge as measured by MAGPIX® are summarized in Tables 1 and 2.

Table 1. Whole blood secretion of IFNy pre- and post- gluten challenge

Day 0 = prior to oral gluten challenge, Day 6 = 6 days after commencing the 3-day gluten l o challenge.

Table 2. Whole blood secretion of IP10 pre- and post-gluten challenge

4010157 Minimum 1.192 65.26 3.648 1230

25% 1.235 100.7 5.861 2168

Percentile

Median 1.536 274.7 13.00 6998

75% 1.876 608.8 17.99 9333

Percentile

Maximum 3.186 1453 26.15 9618

Day 0 = prior to oral gluten challenge, Day 6 = 6 days after commencing the 3-day gluten challenge.

EQUIVALENTS

While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features,

4010157 systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood to control over

5 dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

o The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple

5 elements listed with "and/or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and/or B", when used in conjunction with open-ended language such as "comprising" can refer, in one o embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in 5 a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein

4010157 shall only be interpreted as indicating exclusive alternatives (i.e. "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another

embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of and "consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.0

4010157