WO2019113037A1 - Measurement of ccl20 after gluten exposure - Google Patents

Abstract

Provided herein are methods including methods of identifying a subject having, suspected of having or at risk for having Celiac disease comprising determining a level of CCL20 in a sample from a subject.

Description

MEASUREMENT OF CCL20 AFTER GLUTEN EXPOSURE

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application number 62/594,300, filed December 4, 2017, the contents of which are incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

Celiac disease is an autoimmune-like disorder of the small intestine that occurs in people of all ages and affects approximately 1% of people in Europe and North America. Celiac disease generally 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 Celiac disease is important in order to ensure that those affected by Celiac disease receive proper treatment.

SUMMARY OF THE INVENTION

The disclosure relates, at least in part, to determining a level of a cytokine or chemokine in order to identify Celiac disease in subjects having or suspected of having Celiac disease, or for use in determining efficacy of Celiac disease treatment. Aspects of the disclosure relate to methods of identifying (e.g., diagnosing) a subject as having or at risk of having Celiac disease by determining a level of Chemokine (C-C motif) ligand 20 (CCL20), also referred to as MIP-3a, in a sample from the subject.

Aspects of the disclosure relate to a method, comprising determining a level of CCL20 in a subject (e.g., a subject that has or is suspected of having Celiac disease), wherein the subject has been administered a composition comprising gluten peptides.

In some embodiments, the method further comprises assessing the likelihood the subject has Celiac disease, and assessing the efficacy of a treatment for the Celiac disease. In some embodiments, the treatment for the Celiac disease is a gluten peptide therapy, e.g., Nexvax2.

In some embodiments, the composition comprising gluten peptides comprises gluten protein. In some embodiments, the composition comprising gluten peptides comprises 3 grams of gluten. In some embodiments, the composition comprising gluten peptides comprises at least 3 grams of gluten. In some embodiments, the composition comprising gluten peptides is administered orally, e.g., using any one of the oral forms provided herein, at any one of the times and/or intervals provided herein. In some embodiments, the composition comprising gluten peptides is a foodstuff. In some embodiments, the composition comprising gluten peptides is a liquid composition (e.g., a liquid composition comprising gluten protein). In some embodiments, the composition comprising gluten peptides comprises the peptides of NexVax2. In some embodiments, the composition comprising gluten peptides comprises any one of the compositions provided in U.S. Patent No. 8,835,603 and U.S. Patent No. 9,464,120, which compositions are incorporated herein by reference. In some embodiments, the composition comprising gluten peptides (e.g.,

NexVax2) is administered intradermally to the subject, using any one of the intradermal forms provided herein, at any one of the times and/or intervals provided herein.

In some embodiments, the subject is following a gluten-free diet. In some

embodiments, the method further comprises obtaining a sample (e.g., plasma or serum sample) from the subject, at any one of the time points provided herein (e.g., 1 hour to 6 hours after the subject has been administered the composition comprising gluten peptides).

In some embodiments, the levels of one or more additional cytokines or chemokines are determined. In some embodiments, the one or more additional cytokines or chemokines is/are IL-2, IL-8 and/or IL-10.

In some embodiments, an elevated level of CCL20 and/or of the one or more additional cytokines or chemokines compared to a control level, respectively, indicates that the subject has Celiac disease, and the step of assessing comprises comparing the level of CCL20 and/or the level of the one or more additional cytokines or chemokines to one or more control levels of CCL20 and/or the one or more additional cytokines or chemokines, respectively. In some embodiments, the control level is any one of the control levels provided herein. In some embodiments, the control level is a baseline level (e.g., a level of the CCL20 and/or a level of the one or more additional cytokines or chemokines, respectively, prior to administration of the composition comprising gluten peptides). In some embodiments, the level(s) as compared to a baseline level that is indicative of Celiac disease is any one of the fold changes, or greater, as provided herein.

In some embodiments, the method further comprises recording whether or not the subject has Celiac disease, an indicator thereof, or an indicator of the efficacy of a treatment of the subject, based on the determining and/or assessing. In some embodiments, the method further comprises treating, suggesting a treatment, or giving information in regard to a treatment to the subject, wherein the treating or treatment comprises administration of a composition comprising gluten peptides (e.g., Nexvax2) to the subject. In some embodiments, the subject has received treatment for Celiac disease (e.g., administration of a composition comprising gluten peptides such as Nexvax2).

In some embodiments, an elevated level of the CCL20 and/or the one or more additional cytokines or chemokines, respectively, compared to a control level indicates that the subject has an unsatisfactory therapeutic response to a treatment, and the step of assessing comprises comparing the level of CCL20 and/or the level of the one or more additional cytokines or chemokines to one or more control levels, respectively.

In some embodiments, the level of the CCL20 and/or the level of the one or more additional cytokines or chemokines is measured with an assay that comprises an immuno- based assay (e.g., an ELISA, a multiplex bead-based assay, or an electrochemiluminsence assay), or with an assay that comprises a cytokine/chemokine assay such as a Proseek cytokine/chemokine assay or the like. In some embodiments, the electrochemiluminsence assay comprises an MSD® MULTI-SPOT assay, or a multispot electrochemiluminsence based cytokine assay (e.g., MSD® V-Plex). In some embodiments, the MSD® MULTI SPOT assay comprises a Chemokine Panel 1 (human) kit, or a Proinflammatory Panel 1 (human) kit.

In some embodiments, the subject has a homozygous HLA-DQ2.5 genotype or a non- homozygous HLA-DQ2.5 genotype (e.g., heterozygous HLA-DQ2.5 genotype). In some embodiments, the subject is homozygous for HLA-DQAl*05, HLA-DQBl*02, or both HLA-DQAl*05 and HLA-DQBl*02. In some embodiments, the subject is not homozygous for HLA-DQAU05, HLA-DQBU02, or both HLA-DQAU05 and HLA-DQBU02. In some embodiments, the subject has gluten sensitivity.

In some embodiments, the method further comprises treating, suggesting a treatment, or giving information in regard to a treatment that is a non-Celiac treatment to the subject. In some embodiments, the method does not comprise treating, suggesting a treatment, or giving information in regard to a treatment that is a Celiac treatment to the subject or comprises not suggesting or giving information in regard to a Celiac treatment to the subject.

Aspects of the disclosure relate to a kit for carrying out any one of the methods provided herein comprising an agent for measuring the level of CCL20, and further comprises an agent for measuring one or more additional cytokines or chemokines. In some embodiments, the one or more additional cytokines or chemokines is/are IL-2, IL-8 and/or IL-10.

In some embodiments, the agent for measuring the CCL20 and/or the one or more additional cytokines or chemokines, respectively, is a binding partner for the CCL20 and/or the one or more additional cytokines or chemokines, respectively, or an agent that recognizes such binding partner, respectively.

BRIEF DESCRIPTION OF THE DRAWINGS

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 table showing example cytokine/chemokine fold change in subjects assessed by MSD assay.

DETAILED DESCRIPTION OF THE INVENTION

Celiac disease (CD, also sometimes referred to as cceliac disease, c(o)eliac sprue, non- tropical sprue, endemic sprue, gluten enteropathy or gluten-sensitive enteropathy, and gluten intolerance) is an autoimmune disorder of the small intestine caused by ingestion of gluten- containing foods that occurs in people of all ages, ranging from middle infancy onward, and affects approximately 1% of people in Europe and North America. In many of those affected, Celiac disease is unrecognized.

Celiac disease generally occurs in genetically susceptible individuals who possess either HLA-DQ2 encoded by HLA- (M/ *05 and HLA -DQBl *02 (accounting for about 90% of individuals), variants of HLA- <22, or HLA- QS. Without wishing to be bound by theory, such individuals are thought to 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.

Celiac disease can be diagnosed by small bowel biopsy showing villous atrophy, crypt hyperplasia and raised intra-epithelial lymphocytes, and supported by the presence of Celiac disease-specific serology (IgA specific for transglutaminase and/or IgA and IgG specific for deamidated gliadin peptide). Intestinal histology and serological abnormalities normalize or can improve within weeks to months of adopting gluten-free diet. In general, Celiac disease can be excluded if certain alleles encoding HLA-DQAl*05, DQBl*02 and DQB 1*0302 are not present. In patients who have adopted a gluten-free diet (GFD) without definitive diagnosis, reintroduction of gluten into the diet can be necessary to make a firm diagnosis of Celiac disease. Reintroduction of >3g/day gluten (about 1.5 slices of wheat bread) daily leads to intestinal tissue damage in the majority of patients with Celiac disease usually strictly adherent to gluten free diet.

Small bowel biopsy typically requires an endoscopy, which is inconvenient and may be inconclusive if biopsies are not performed at multiple sites in the duodenum, processed meticulously and interpreted correctly. Requiring small bowel biopsy may also delay treatment because of the importance of continuing to consume gluten until after the procedure. Furthermore, Celiac disease cannot be diagnosed in patients who have excluded gluten from their diet if serology and histology show typical diagnostic features.

Accordingly, aspects of the disclosure relate to methods of identifying Celiac disease in a subject having or that is suspected of having Celiac disease and can represent an improvement to the aforementioned techniques. In some embodiments, the methods provided herein comprise determining a level of CCL20 in a sample from a subject.

Methods

One aspect of the disclosure relates to methods for identification or diagnosis of a subject, such as a subject having or suspected of having Celiac disease. The methods involve determining (e.g., measuring) a level (or amount) of CCL20 in a sample from the subject. In some embodiments, the method may also involve determining a level of IL-2, IL-8 and/or IL- 10 in a sample from the subject. As used herein“determining” refers to any method by which the respective information can be acquired. Such methods can be direct or indirect. Thus, the respective information can be acquired by experimental methods. The respective information can also be acquired by being given or provided with the information, such as in a report, or other materials.

In some embodiments, any one of the methods further comprises comparing the level(s) in the sample with a control level. The comparing may be accomplished with the assistance of a software program on a computer. In some embodiments, the comparing comprises a statistical analysis, such as a paired T-test.

In some embodiments, assessing comprises identifying the subject as having or at risk of having Celiac disease if the cytokine or chemokine level (e.g., the CCL20 level) is elevated compared to a control level; or not having or not at risk of having Celiac disease if the level is reduced compared to the control level or the same as the control level. Control levels are further described herein. The control levels can be any one of the levels by which an increase was determined as provided herein. In some embodiments of any one of the methods provided herein, the method further comprising treating or suggesting a treatment if the subject is identified as having or likely to have Celiac disease. In some embodiments of any one of the methods provided herein, the method further comprises recommending a gluten-free diet and/or providing information in regard thereto to the subject. In some embodiments of any one of the methods provided herein, the method further comprises administering a treatment, or providing information in regard thereto, to the subject. Treatments are described herein or otherwise known in the art. In some embodiments, the treatment is a composition comprising a gluten peptide as described herein, e.g., Nexvax2. In some embodiments, the treatment comprises a gluten-free diet.

In some embodiments, any one of the methods described herein further comprises recording a level as provided herein, whether or not the subject has Celiac disease based on the assessing or an indicator thereof and/or an indicator of an assessment of treatment efficacy. In some embodiments, any one of the methods described herein further comprises transmitting, such as to a database, a level as provided herein, whether or not the subject has Celiac disease based on the assessing or an indicator thereof and/or an indicator of an assessment of treatment efficacy. The transmitting may be accomplished, e.g., via a computer or network of computers.

CCL20

Chemokine (C-C motif) ligand 20 (CCL20) or liver activation regulated chemokine (LARC) or Macrophage Inflammatory Protein-3 (MIP3A) is generally considered a small cytokine, strongly chemotactic for lymphocytes and weakly attracts neutrophils, belonging to the CC chemokine family.

Exemplary sequences for CCL20 are shown below.

AY150053.1: Homo sapiens macrophage inflammatory protein-3-alpha (CCL20) gene, promoter region and partial cds

1 gttatttgac atttgctgtg ctgactagct actgctgata ggttttcttt ccctcaacaa

61 ttctgaggct ctatattgag ttatattagt acatcatcat ggagagttaa aggtaggtaa

121 ggattatttt ctgaactgca atattgatta aagccatgtg aatgtataag attcttagaa

181 gagttgacat taaatcaagg tgaagctgag gtttgagcct tacttaaagg ctgatatttt

241 ccactctaac tgcggacagt actgtagcac tgttatagta cctgctctga atgttagtct

301 agcaactcag ggtcttcttc atgacagctg aacctcaacc atgtgatggt aaatgtgtag 361 cagagtatgc ctggcatccc acctgctcct cctccccctc ctccttgact ggttctggaa 421 agcaaatagg gtgtaacaat aggagttctg gaatgttcct gtgtggggct gacctttgta

481 tcgctgttaa tcctctattt tcagacacaa aaatgattaa gttaaaactg gatgaaagtc

541 ttttctgggt cacagggctg agctgctttt gctctttgca aatacaaaga atttaacagg

601 attctcccct tctcaacttc ctgtccccca ccctgacctt cgcaccttcc caatatgagg

661 aaaaagcagg aagttttcct tgcgggtttt ttttatgatg acatgatggg gccagttgat

721 caatggggaa aaccccatgt ggcaacacgc cttctgtgta cattcccaat atttgctata

781 aatagggcca tcccaggctg ctgtcagaat ataacagcac tcccaaagaa ctgggtactc

841 aacactgagc agatctgttc tttgagctaa aaaccatg (SEQ ID NO:l )

NM_00l 130046.1: Homo sapiens C-C motif chemokine ligand 20 (CCL20), transcript variant 2, mRNA

1 agaatataac agcactccca aagaactggg tactcaacac tgagcagatc tgttctttga

61 gctaaaaacc atgtgctgta ccaagagttt gctcctggct gctttgatgt cagtgctgct

121 actccacctc tgcggcgaat cagaagcaag caactttgac tgctgtcttg gatacacaga

181 ccgtattctt catcctaaat ttattgtggg cttcacacgg cagctggcca atgaaggctg

241 tgacatcaat gctatcatct ttcacacaaa gaaaaagttg tctgtgtgcg caaatccaaa

301 acagacttgg gtgaaatata ttgtgcgtct cctcagtaaa aaagtcaaga acatgtaaaa

361 actgtggctt ttctggaatg gaattggaca tagcccaaga acagaaagaa ccttgctggg

421 gttggaggtt tcacttgcac atcatggagg gtttagtgct tatctaattt gtgcctcact

481 ggacttgtcc aattaatgaa gttgattcat attgcatcat agtttgcttt gtttaagcat

541 cacattaaag ttaaactgta ttttatgtta tttatagctg taggttttct gtgtttagct

601 atttaatact aattttccat aagctatttt ggtttagtgc aaagtataaa attatatttg

661 ggggggaata agattatatg gactttcttg caagcaacaa gctatttttt aaaaaaaact

721 atttaacatt cttttgttta tattgttttg tctcctaaat tgttgtaatt gcattataaa

781 ataagaaaaa tattaataag acaaatattg aaaataaaga aacaaaaagt tcttctgtta

841 aaaaaaaa (SEQ ID NO: 2)

P78556.1: C-C motif chemokine 20

1 mcctksllla almsvlllhl cgeseaasnf dcclgytdri lhpkfivgft rqlanegcdi

61 naiifhtkkk lsvcanpkqt wvkyivrils kkvknm (SEQ ID NO: 3)

NR_001123518.1: C-C motif chemokine 20 isoform 2 precursor [Homo sapiens]

1 mcctksllla almsvlllhl cgeseasnfd cclgytdril hpkfivgftr qlanegcdin 61 aiifhtkkkl svcanpkqtw vkyivrllsk kvknm (SEQ ID NO: 4)

Aspects of the disclosure relate to methods that comprise determining (e.g., measuring) a level of CCL20 in a sample from a subject, such as a subject having or suspected of having Celiac disease. Such methods are described herein.

Measuring a CCL20 level, or other cytokine or chemokine, can be accomplished using any assay known in the art (see, e.g., Molecular Cloning: A Laboratory Manual, M. Green and J. Sambrook, Fourth Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2012, Current Protocols in Molecular Biology, F.M. Ausubel, et ah, eds., John Wiley & Sons, Inc., New York). Levels of CCL20, or other cytokine or chemokine, include levels of mRNA and/or levels of protein.

Assays for detecting mRNA 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 to 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 locations, such as an Affymetrix microarray (Affymetrix®, Santa Clara, CA)). In some embodiments, the nucleic acid binding partners bind to a part of or an entire nucleic acid sequence of CCL20, or other cytokine or chemokine, such as a sequence provided herein.

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. In some

embodiments, the protein binding partners, e.g., antibodies, bind to a part of or an entire amino acid sequence of CCL20, or other cytokine or chemokine, such as a sequence provided herein. 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 CCL20, or other cytokine or chemokine, 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 CCL20, or other cytokine or chemokine. The sample is loaded into a 96-well plate containing the beads and the sample is incubated to allow binding to the beads. A second biotinylated binding partner for CCL20, or other cytokine or chemokine, is added after the CCL20, or other cytokine or chemokine, binds 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 CCL20, or other cytokine or chemokine, is then determined based on the level of fluorescence. An exemplary system for running a multiplex bead-based asay 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, measuring a level of CCL20, or other cytokine or chemokine, 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 ELISpot assay involves a binding agent for CCL20, or other cytokine or chemokine, (e.g., an antibody) 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 a 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 CCL20, or other cytokine or chemokine, is immobilized, a second binding partner for CCL20, or other cytokine or chemokine, 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 fluorophor 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.

An exemplary ELISA involves at least one binding partner, e.g., an antibody or antigen-binding fragment thereof, with specificity for a particular antigen. The sample with an unknown amount of antigen 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 antigen, as in a "sandwich" ELISA). After the antigen is immobilized, the binding partner for CCL20, or other cytokine or chemokine, is added, forming a complex with the immobilized CCL20, or other cytokine or chemokine. The binding partner can be attached to a detectable label as described herein (e.g., a fluorophor or an enzyme), or can itself be detected by an agent that recognizes the CCL20, or other cytokine or chemokine, binding partner that is attached to a detectable label as described herein (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, measuring a level of CCL20, or other cytokine or chemokine, comprises an electrochemiluminsence assay. In some embodiments, the

electrochemiluminsence assay comprises an MSD® MULTI-SPOT assay, or a multispot electrochemiluminsence based cytokine assay (e.g., MSD® V-Plex). In some embodiments, the MSD® MULTI-SPOT assay comprises a Chemokine Panel 1 (human) kit, or a

Proinflammatory Panel 1 (human) kit. In some embodiments, measuring a level of CCL20, or other cytokine or chemokine, comprises a cytokine/chemokine assay such as a Proseek cytokine/chemokine assay or the like.

Diagnostic Methods

One aspect of the disclosure relates to methods of identifying (e.g., diagnosing) subjects, such as subjects having or suspected of having Celiac disease.

In some embodiments, the method comprises determining a level of at least one cytokine or chemokine (e.g., CCL20) in a subject that has or is suspected of having Celiac disease, wherein the subject has been administered a composition comprising gluten peptides as described herein. In some embodiments of any one of the methods provided, the composition comprising gluten peptides comprises gluten protein, such as 3 grams of gluten. In some embodiments of any one of the methods provided, the composition comprising gluten peptides comprises gluten protein, such as 6 grams of gluten. In some embodiments of any one of the methods provided, the composition comprising gluten peptides comprises gluten protein, such as between 3 and 6 grams of gluten. In some embodiments of any one of the methods provided, the composition comprising gluten peptides is a foodstuff. In some embodiments of any one of the methods provided, the composition comprising gluten peptides is a liquid composition, e.g., comprising gluten protein, such as between 3 and 6 grams of gluten. In some embodiments of any one of the methods provided, the composition comprising gluten peptides is administered by oral administration. As used herein, administration includes direct as well as indirect administration. Thus, administering is meant to include instances where the subject is directed to self-administer, such as consume, the gluten peptide composition. In some embodiments of any one of the methods provided, the subject is following a gluten free diet.

Suitable forms of oral administration include foodstuffs (e.g., baked goods such as breads, cookies, cakes, etc. and liquid compositions), 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 in the art for the manufacture of pharmaceutical compositions or foodstuffs and such compositions may contain one or more agents including, for example, sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations.

In some embodiments of any one of the methods provided, the method further comprises assessing the likelihood the subject has Celiac disease. In some embodiments, assessing comprises comparing the level of the at least one cytokine or chemokine (e.g., CCL20) to a control level of the at least one cytokine or chemokine (e.g., a control level of CCL20). Levels as used herein can be absolute or relative amounts. In some embodiments, assessing comprises determining the ratio of the level of the at least one cytokine or chemokine (e.g., CCL20) to the control level. In some embodiments, the control level of the at least one cytokine or chemokine (e.g., CCL20) is a baseline level of the cytokine or chemokine. In some embodiments, the baseline level is the level of the cytokine or chemokine (e.g., CCL20) in the subject prior to the administration of the gluten peptide composition. In some embodiments of any one of the methods provided herein, the method can further comprise the step of determining a baseline level of the cytokine or chemokine (e.g., CCL20) in the subject. In some embodiments of any one of the methods provided, an elevated level of the at least one cytokine or chemokine (e.g., CCL20) compared to a control level, such as a baseline level, of the at least one cytokine or chemokine indicates that the subject has or is likely to have Celiac disease. In some embodiments of any one of the methods provided herein, the method further comprises recording whether or not the subject has or is likely to have Celiac disease based on the level or ratio.

In some embodiments of any one of the methods provided, the at least one cytokine or chemokine is CCL20 with or without also IL-2, IL-8 and/or IL-10.

In some embodiments of any one of the methods provided, the level of the at least one cytokine or chemokine (e.g., CCL20) is measured in a sample, e.g., a serum or plasma, obtained from the subject. In some embodiments of any one of the methods provided, the sample is obtained from the subject within 1-24 hours, such as within 1-6 hours, of administration of the composition (e.g., a liquid composition comprising gluten protein). In some embodiments of any one of the methods provided, the sample from the subject is obtained at least 1 hour after (e.g., 1 hour after) the subject has been administered gluten peptide composition (e.g., a liquid composition comprising gluten protein). In some embodiments of any one of the methods provided, the sample from the subject is obtained at least 2 hours after (e.g., 2 hours after) the subject has been administered the gluten peptide composition. In some embodiments of any one of the methods provided, the sample from the subject is obtained at least 3 hours after (e.g., 3 hours after) the subject has been administered the gluten peptide composition. In some embodiments of any one of the methods provided, the sample from the subject is obtained at least 4 hours after (e.g., 4 hours after) the subject has been administered the gluten peptide composition. In some embodiments of any one of the methods provided, the sample from the subject is obtained at least 5 hours after (e.g., 5 hours after) the subject has been administered the gluten peptide composition. In some embodiments of any one of the methods provided, the sample from the subject is obtained at least 6 hours after (e.g., 6 hours after) the subject has been administered the gluten peptide composition. In some embodiments of any one of the methods provided, the sample from the subject is obtained 4-6 hours after the subject has been administered the gluten peptide composition.

In some embodiments of any one of the methods provided, an elevated level of the at least one cytokine or chemokine (e.g., CCL20) compared to a control level of the at least one cytokine or chemokine indicates that the subject has Celiac disease, and the step of assessing comprises comparing the level of the at least one cytokine or chemokine to a control level of the at least one cytokine or chemokine.

In some embodiments of any one of the methods provided, the control level is a baseline level. In some embodiments of any one of the methods provided, the baseline level is a level of the at least one cytokine or chemokine (e.g., CCL20) prior to administration of the composition (e.g., a liquid composition comprising gluten protein).

In some embodiments of any one of the methods provided, the method further comprises recording whether or not the subject has Celiac disease based on the assessing. In some embodiments of any one of the methods provided, the method further comprises treating, suggesting a treatment, or giving information in regard a treatment to the subject.

In some embodiments of any one of the methods provided, the treating or treatment comprises administration of a composition comprising a gluten peptide to the subject, e.g., administration of Nexvax2 to the subject.

In some embodiments of any one of the methods provided, the subject has received treatment, such as with a gluten peptide therapy, e.g., Nexvax2. In some embodiments of any one of the methods provided, an elevated level of at least one cytokine or chemokine (e.g., CCL20) compared to a control level of the at least one cytokine or chemokine indicates that the subject has an unsatisfactory therapeutic response to a treatment, such as a gluten peptide therapy (e.g., Nexvax2), and the step of assessing comprises comparing the level of the at least one cytokine or chemokine to a control level of the at least one cytokine or chemokine.

In some embodiments of any one of the methods provided, measuring the level of the at least one cytokine or chemokine (e.g., CCL20) comprises an immuno-based assay. In some embodiments of any one of the methods provided, the immuno-based assay comprises an ELISA or a multiplex bead-based assay. In some embodiments of any one of the methods provided, the immuno-based assay comprises an electrochemiluminescence assay. An exemplary electrochemiluminescence assays includes, but is not limited to, MSD-ECL Meso Scale Discovery® electrochemiluminsence (Meso Scale Diagnostics, Maryland). In some embodiments of any one of the methods provided, the assay comprises an MSD® MULTI SPOT assay. In some embodiments of any one of the methods provided, the MSD® MULTI SPOT assay comprises a Chemokine Panel 1 (human) kit. In some embodiments of any one of the methods provided, the MSD® MULTI-SPOT assay comprises a Proinflammatory Panel 1 (human) kit.

In some embodiments of any one of the methods provided herein, the method further comprises performing other testing. In some embodiments of any one of the methods provided, the other testing comprises a serology test, genotyping, an intestinal biopsy, and/or a T cell response test. In some embodiments of any one of the methods provided herein, the method further comprises performing one or more additional tests on the subject. In some embodiments of any one of the methods provided, the method further comprises contacting a sample comprising a T cell from the subject with a gluten peptide and measuring a T cell response in the sample. In some embodiments of any one of the methods provided, a T cell response is measured by measuring a level of IFN-g, where an increased level of IFN-g compared to a control level (e.g., a level of IFN-g in a sample that has not been contacted with a gluten peptide) may identify a subject as having Celiac disease. In some embodiments of any one of the methods provided, a level of IFN-g at or above a cut-off level (e.g., at or above 7.2 pg/ml) may identify a subject as having or likely as having Celiac disease.

In some embodiments of any one of the methods provided herein, the method further comprises treating or suggesting a treatment if the subject is identified as having or likely of having Celiac disease. In some embodiments of any one of the methods provided herein, the method further comprises recommending a gluten-free diet and/or providing information in regard thereto to the subject. In some embodiments of any one of the methods provided herein, the method further comprises administering a treatment, or providing information in regard thereto, to the subject. Treatments are described herein or are otherwise known in the art. In some embodiments of any one of the methods provided, the treatment is a

composition comprising a gluten peptide as described herein, e.g., Nexvax2. In some embodiments, the treatment comprises a gluten-free diet.

Therapeutic Efficacy Methods

One aspect of the disclosure relates to methods of assessing the efficacy of treatment of Celiac disease (e.g., responsiveness to a therapeutic gluten peptide composition such as Nexvax2). In some embodiments of any one of the methods provided, the subject is following a gluten free diet.

In some embodiments of any one of the methods provided, the method further comprises recording the level(s), the result(s) of the assessing and/or the treatment, or suggestion for treatment, based on the assessing. In some embodiments of any one of the methods provided, the method further comprises recording whether or not the treatment has been effective or completely effective based on the level or comparison thereof. In some embodiments of any one of the methods provided, a decreased or similar level of compared to a control level indicates that a treatment has been effective. In some embodiments of any one of the methods provided, treating comprises continuing with the treatment, or suggesting comprises suggesting the subject continue with the treatment, based on the assessing. In some embodiments of any one of the methods provided, treating comprises ceasing the treatment, or suggesting comprises suggesting the subject cease the treatment, based on the assessing. In some embodiments of any one of the methods provided, treating comprises administering a different or additional treatment, or the suggesting comprises suggesting the subject be treated with an additional or different treatment, based on the assessing. In some embodiments of any one of the methods provided, the treatment is a composition comprising a gluten peptide as described herein.

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 fluid samples. In some embodiments of any one of the methods provided, the sample comprises plasma or serum. In some embodiments of any one of the methods provided herein, the methods comprise obtaining or providing the sample. In some embodiments of any one of the methods provided herein, the sample is obtained from the subject after administration to the subject of a composition comprising a gluten peptide as described herein (e.g., a liquid composition comprising gluten protein). In some embodiments of any one of the methods provided, the sample is obtained, e.g., at least or within 1, 2, 3, 4, 5, or 6 hours after administration of the composition to the subject. In some embodiments of any one of the methods provided, the sample is obtained, e.g., within 4 hours of administration of the composition. In some embodiments of any one of the methods provided, the sample is obtained, e.g., 4 hours after administration of the composition. In some embodiments of any one of the methods provided, the sample is obtained, e.g., within 24 hours or within 6 hours of administration of the composition. In some embodiments of any one of the methods provided, the sample is obtained, e.g., within 1 hour to 24 hours, within 1 hour to 6 hours, within 2 hours to 6 hours, within 2 hours to 4 hours, within 3 hours to 5 hours, within 3 hours to 6 hours, within 4 hours to 6 hours, or within 5 hours to 6 hours of administration of the composition. In some embodiments of any one of the methods provided, the sample is obtained within 4 hours to 6 hours of administration of the composition.

In some embodiments of any one of the methods provided herein, a second sample is obtained, e.g., a control sample. Controls and control samples are described herein. In some embodiments of any one of the methods provided, the second sample is obtained prior to administration of a composition comprising a gluten peptide as described herein (e.g., a liquid composition comprising gluten protein). In some embodiments of any one of the methods provided, additional samples are obtained, e.g., at different time points, such as during treatment of a subject.

Subjects

A subject may include any subject that has or is suspected of having Celiac disease. Preferably, the subject is a human. In some embodiments of any one of the methods provided, 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 ( DQA / *02 and DQB I *02) or HLA-DQ8 ( DQA1 *03 and DQB1 *0302). In some embodiments of any one of the methods provided, the subject is HLA-DQ2.5 positive (i.e., has both susceptibility alleles DQA1 *05 and DQB1 *02). In some embodiments of any one of the methods provided, 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 DQB I *02), HLA-DQ2.2 (DQA1 *02 and DQB I *02) or HLA-DQ8 ( DQA1 *03 and DQB1 *0302). The presence of susceptibility alleles can be detected by any nucleic acid detection method 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. In some embodiments of any one of the methods provided herein, the subject is on a gluten-free diet. In some embodiments of any one of the methods provided, the subject is a subject having been administered a treatment as described herein (e.g., Nexvax2). In some embodiments of any one of the methods provided, the subject has been on a gluten-free diet for a period of time. In some embodiments of any one of the methods provided, a subject may be determined to have been on a gluten-free diet or not by asking the subject whether they have ingested gluten during a period of time. In some embodiments of any one of the methods provided, a subject may be determined to have been on a gluten-free diet or not by performing other testing.

A subject may be one with gluten sensitivity (also referred to as non-Celiac gluten sensitivity). As used herein, a subject with gluten sensitivity exhibits one or more reactions to gluten but 1) does not have Celiac disease or wheat allergy or 2) in which neither allergic nor autoimmune mechanisms against gluten are or can be identified. Generally, gluten sensitivity is not accompanied by the concurrence of anti-tTG autoantibodies or other autoimmune comorbidities and is distinct from Celiac disease. Any one of the methods provided herein may be used to determine or confirm that a subject has gluten sensitivity and not Celiac disease with or without the current methods of making such determination or confirmation.

In some embodiments of any one of the methods provided herein, a subject with gluten sensitivity is one that meets or can meet the Salerno Experts’ Criteria (Nutrients 2015, 7, 4966-4977; doi:l0.3390/nu7064966).

Controls and Control Levels

In some embodiments, a control level is a level 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 (DQAl*05 and DQBl*02), DQ2.2 (DQAl*02 and DQBl*02) or DQ8 (DQAl*03 and DQB 1*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 {DQA1 *02 and DQB1 *02) or DQ8 (DQA1 *03 and DQB1 *0302) described herein. In some embodiments, a control subject is a healthy individual not having or suspected of having Celiac disease. In some embodiments, the control level is a pre determined threshold. In some embodiments, a control level is a pre-determined level from a control subject or subjects, such that the control level need not be measured every time the methods described herein are performed.

In some embodiments, a control level is a level in a second sample from the same subject from which the first sample was obtained (e.g., a first and second sample may be obtained from the same subject and the comparison between the first and second sample is used to determine if the subject has or is at risk of having Celiac disease). In some embodiments, the second sample is obtained from the subject prior to gluten peptide administration as described herein (e.g., administration of a liquid composition comprising gluten protein).

Gluten Peptides and Compositions Containing Gluten Peptides

As used herein the term“gluten peptide” includes any peptide comprising a sequence derived from, or encompassed within, one or more of gluten proteins, such as alpha ( 01 ), beta ( b ), g ( g ) and omega ( U) ) gliadins, and low and high molecular weight (LMW and HMW) glutenins in wheat, B, C and D hordeins in barley, b , g and omega secalins in rye, and optionally avenins in oats, including deamidated variants thereof containing one or more glutamine to glutamate substitutions. In some embodiments, the gluten peptide(s) is a protein. In other embodiments, the gluten peptide(s) is not full-length protein but a portion thereof. In some embodiments, the disclosure provides a composition comprising gluten peptides. In some embodiments, the composition is a composition comprising gluten protein. In some embodiments, the composition is Nexvax2. Nexvax2 is a peptide composition containing the following three peptides: a first peptide (NPL001) which has the amino acid sequence ELQPFPQPELPYPQPQ (SEQ ID NO: 8), wherein the N-terminal glutamate is a pyroglutamate and the carboxyl group of the C-terminal glutamine is amidated; a second peptide (NPL002) which has the amino acid sequence EQPFPQPEQPFPWQP (SEQ ID NO: 9), wherein the N-terminal glutamate is a pyroglutamate and the carboxyl group of the C- terminal proline is amidated; and a third peptide (NPL003) which has the amino acid sequence EPEQPIPEQPQPYPQQ (SEQ ID NO: 10), wherein the N-terminal glutamate is a pyroglutamate and the carboxyl group of the C-terminal glutamine is amidated In some embodiments, the composition comprising gluten peptides comprises any one of the compositions provided in U.S. Patent No. 8,835,603 and U.S. Patent No. 9,464,120, which compositions are incorporated herein by reference.

Other Testing

In some embodiments of any one of the methods provided, methods described herein further comprise other testing of a subject (e.g., based on the results of the methods described herein). As used herein,“other testing” describes use of at least one additional diagnostic method in addition to the methods provided herein. Any diagnostic method or combinations thereof for Celiac disease is contemplated as other testing. Exemplary other testing includes, but is not limited to, intestinal biopsy, serology (measuring the levels of one or more antibodies present in the serum), genotyping (see, e.g., Walker- Smith JA, et al. Arch Dis Child 1990), and measurement of a T cell response. Such other testing may be performed as part of the methods described herein or after the methods described herein (e.g., as a companion diagnostic), or before use of the methods described herein (e.g., as a first-pass screen to eliminate certain subjects before use of the methods described herein, e.g., eliminating those that do not have one or more HLA-DQA and HLA-DQB susceptibility alleles). In some embodiments of any one of the methods provided, however, no other testing is required to assess the subject’s Celiac disease status, for example, having or not having Celiac disease.

Kits Another aspect of the disclosure relates to kits. In some embodiments, the kit comprises at least one agent for identifying or measuring levels of CCL20 and/or one or more additional cytokines or chemokines. In some embodiments of any one of the kits provided, the agent is a binding partner for the CCL20 and/or one or more additional cytokines or chemokines. In some embodiments of any one of the kits provided, the agent is one that recognizes a binding partner for the CCL20 and/or other cytokine or chemokine.

Any suitable binding partner is contemplated. In some embodiments, the binding partner is any molecule that binds specifically to a cytokine or chemokine provided herein (e.g., CCL20). Any one of the kits provided may include binding partners for any one of the embodiments of pairs or sets of cytokines or chemokines 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 an antigen to be measured than to a portion of or the entirety of another molecule. In some embodiments, the binding partner is an antibody. As used herein, the term“antibody” also includes antigen-binding fragments thereof, such as Fab, F(ab)2, Fv, single chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, scFv, or dAb fragments. Methods for producing antibodies 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 ah, "Immunology" (2nd Ed.), Gower Medical Publishing, London, New York (1989), W02006/040153, WO2006/122786, and W02003/002609). 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 an MHC tetramer. 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 streptavidin).

In some embodiments, the binding partner is any molecule that binds specifically to an cytokine or chemokine nucleic acid, such as mRNA. As described herein,“binds specifically 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. In some embodiments, the agent is a complementary nucleic acid. In some embodiments of any one of the kits provided, the kit further comprises a first and second binding partner for a cytokine or chemokine provided herein. In some

embodiments, the first and second binding partners are antibodies. In some embodiments, the first and second binding partners are nucleic acids, such as nucleic acid probes. In some embodiments, the first or second binding partner is bound to a surface. The first or second binding partner may be bound to the surface covalently or non-covalently. The first or 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, 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 agent that is or recognizes a binding partner is contemplated.

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 are detectable through use of, e.g., an enzyme assay, a chromogenic 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 herein, the kit further comprises any one of the gluten peptide compositions provided herein (e.g., a liquid composition comprising gluten protein). In an embodiment, such composition is contained within a container in the kit. In some embodiments, such composition is contained within a solution separate from the container, such that the composition may be added to the container prior to administration. In some embodiments, such composition is in lyophilized form in a separate container, such that the composition may be reconstituted and added to the container prior to administration, in some embodiments. In some embodiments, the container is present in the kit in duplicate or triplicate.

In some embodiments of any one of the kits herein, the kit further comprises a container suitable for injection of such a composition to a subject, e.g., a syringe. In some embodiments, the kit further comprises a container for containing a sample obtained from a subject, e.g., a plasma or serum sample. Containers for plasma, serum, etc. are known in the art and are commercially available (e.g., a Vacutainer™ or urine collection container from Becton Dickinson). In some embodiments, the container further contains an anti-coagulant, such as heparin, EDTA, citrate, sodium polyanethol sulfonate, or oxalate. In some embodiments, the container is structured to hold a defined volume, e.g., 1 mL or 5 mL. In some embodiments, the container is present in the kit in duplicate or triplicate.

In some embodiments, the kit further comprises a negative control, e.g., a

composition that does not comprise a gluten peptide, e.g., a saline solution. In some embodiments, the kit further comprises a positive control, e.g., a composition comprising a chemokine or cytokine at a known concentration or level.

In some embodiments, the kit comprises any combination of the components mentioned above.

In some embodiments of any one of the methods provided, the kit further comprises instructions for performing any one of the methods provided herein and/or for detecting a level of at least one cytokine or chemokine in a sample from a subject having or suspected of having Celiac disease or a subject undergoing treatment for 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 ah, 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); L.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’.

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 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

Example 1; Blood Serum Biomarker Analysis After Gluten Exposure: Identification of IL-2, IL8. IL-10. and CCL20

This study evaluated blood serum and plasma cytokines and chemokines as biomarkers for gluten exposure in subjects with Celiac Disease (CeD). The goal of the evaluation was to identify cytokines and/or chemokines that exhibited a fold change increase after gluten exposure over background levels determined prior to gluten exposure.

Additionally, the goal was also to identify potential biomarkers in blood serum of CeD subjects exposed to gluten compared to CeD subjects exposed to a placebo or gluten sham.

Analysis was initially performed on plasma samples from a subset of subjects that were enrolled in a phase I clinical trial. The trial was conducted to evaluate the safety of the Nexvax2 peptide therapeutic (which contains Gluten peptides NPL001, NPL002, and NPL003). The sequences for NPL001, NPL002, and NPL003 are as follows, NPL001:

pyroELQPFPQPELPYPQPQ-amide (SEQ ID NO: 5), NPL002: pyroEQPFPQPEQPFPWQP- amide (SEQ ID NO: 6), and NPL003: pyroEPEQPIPEQPQPYPQQ-amide (SEQ ID NO: 7).

Clinical trial subjects were intradermally administered either Nexvax2 or a saline placebo. Blood plasma samples were collected before and at 2, 4, and 6 hours after the first Nexvax2 or placebo administration and at the last administration (16 doses total). Samples obtained from the clinical trial were evaluated for expression of 92 cytokines and chemokines in a high capacity Proseek Multiplex screen utilizing a polymerase chain reaction (PCR) based Proximity Extension Assay that was performed at OLINK Proteomics (Uppsala, Sweden). Additional samples were also obtained from an initial 6 subjects enrolled in a different trial. The subjects were exposed to gluten or gluten sham. Gluten exposure was an oral challenge with 3g gluten or gluten sham in a flavored slurry. Collection of blood serum was executed before and at 1, 2, 3, 4, 5, 6, 7, 8, 24, and 144 hours after oral challenge. Blood serum samples were evaluated for a select group of cytokines and chemokines utilizing the validated Meso Scale Discovery (MSD) V-Plex electrochemiluminescence immunoassay.

Multispot electrochemiluminescence based cytokine assays (MSD V-Plex) were performed in duplicate according to manufacturer’s instructions on serum samples. Samples were thawed at room temperature and centrifuged at 500 xg for 10 minutes at room

temperature. Samples were then diluted according to manufacturer’s recommendation in a separate 96-well plate and then pipetted directly into dedicated 96-well plates for multispot- based assays to measure the concentrations of different cytokines.

Briefly, Wash Buffer was added to each well of the plate then removed immediately including residual liquid by inverting plate and tapping it onto absorbent paper towels.

Calibrator standards and controls were diluted accordingly and 50 uL each were added into the designated wells. Samples (50 pL) were then transferred into the designated sample wells. Plates were sealed and incubated on a plate shaker at room temperature for 2 hours.

Following the incubation, plates were washed 3 times and detection antibodies (25 uL) were added to each well. Plates were sealed and incubated on the plate shaker for 2 hours at room temperature. Plates were then washed 3 times and 150 pL of 2X read buffer was added to each well. Plates were then run on the MSD MESO™ Sector S600 instrument plate reader. The lower limits of detection (LLOD) for each cytokine are shown in Table 1 below.

Calculated values on the lower end of the scale that were not achieved using the standard curves were reported as less than or equal to the lowest concentration in the reported LLOD range.

Table 1. Reported LLOD values for the MSD V-Plex

OLINK Proseek Analysis

The Proseek Cytokine/Chemokine assays were run successfully at OLINK, enabling the detection of 92 different analytes in human plasma. The data were analyzed using Excel and OLINK Wizard and GenEx software. All assays demonstrated large dynamic ranges and showed low pg/mL LLOD sensitivity. Data values below LLOD were either removed from Data Set or replaced by the corresponding LLOD value. All standards exhibited good reproducibility of replicates with the average calculated concentration Coefficient of

Variability (CV) being in an acceptable range. Excellent assay accuracy and precision was observed during the evaluation, with most percent recovery values calculated for the controls were between 80% and 120%.

Some sample calculated concentrations could not be resolved because the signal fell below LLOD and replaced by the corresponding LLOD value. Most samples were within the detection range. All subject data were reported or adjusted. Additional analysis of the data was performed with the fold change in cytokine/chemokine concentration in the blood plasma being calculated compared to time points prior to Nexvax2 administration.

The mean fold change at 2, 4, and 6 hours, combined, after the first dose Nexvax2 administration, allows for a ranking of the cytokines/chemokines. Subjects that were administered Nexvax2 exhibited within 6 hours a >1.5 pg/mL fold change in 27 of the 92 cytokines/chemokines tested. Among the group of 27 cytokines/chemokines, the most significant were CCL20 (MIP-3a), IL-8 and IL-2 with CCL20 ranked first with the highest fold change observed. Although variable, the peak fold change for IL-8 and IL-2 were observed at 4 hours after Nexvax2 administration. The peak fold change for CCL20 was observed at 6 hours after Nexvax2 administration.

All 4 subjects administered Nexvax2 that were evaluated in this assay were similar in CCL20, IL-8, and IL-2 responses with significant fold increases observed at 4 hours.

Additional cytokines/chemokines had significant increases within 4 hours, but there was variability in the number of subjects exhibiting those increases. At 6 hours following

Nexvax2 administration, there was increased expression of CCL20 and IL-8, but increases were observed in 3/4 subjects. The only consistent increases at 6 hours compared to 4 hours in all 4 subjects, occurred when measuring CXCL9, CCL2, and IL-6.

Evaluation of the responses in subjects administered a placebo showed very little significant changes in any of the 92 cytokines/chemokines, with the exception of IL-6.

Compared to subjects receiving Nexvax2, placebo subjects exhibited increases in IL-6, but were significantly less than those observed in Nexvax2 subjects.

Overall, the OLINK Proseek data revealed that subjects exposed to gluten via Nexvax2 administration have a significant cytokine/chemokine response within 4-6 hours after administration. That same response is not observed in subjects receiving a placebo. The most significant fold changes observed were increases in CCL20, IL-8 and IL-2. One, two or all 3 may be viable biomarkers to identify subjects exposed to gluten.

MSD Analysis

Samples from the study subjects were collected as blood serum and were evaluated in the MSD VPlex Prolnflammatory Panel and the VPlex CCL20 assay. The data were analyzed using MSD Workbench 4.0 software. The software fits the standard curves using a 4- parameter logistic fit with l/y2 weighting. All assays demonstrated large dynamic ranges and showed femtogram/mL to low pg/mL LLOD sensitivity. All standards exhibited good reproducibility of replicates with the average calculated concentration Coefficient of Variability (CV) being in the quantifiable range of 20%. Excellent assay accuracy and precision was observed during the evaluation, with percent recovery values calculated for the controls were between 80% and 120%.

Average concentrations were calculated for IL-2, IL-8, IL-10 and CCL20. Some sample calculated concentrations could not be resolved because the signal fell below the curve fit and are designated NaN. Unresolved data was adjusted and reported as equal to the LLOD value calculated on each plate. Calculated concentrations were adjusted for dilution, 2-fold for the Prolnflammatory Panel and 4-fold for CCL20. The majority of samples were within the detection range.

Additional analysis of the data was performed with the fold change in

cytokine/chemokine concentration in the blood serum being calculated compared to the 0 time point prior to oral gluten challenge. Significant fold changes were observed for IL-2 from 4-8 hours following gluten challenge in all subjects receiving gluten. Earlier significant responses were seen at 2 and 3 hours in 4 of 5 subjects receiving gluten. There was no significant increase in IL-2 observed in the subject that was challenged with the gluten sham. Significant CCL20 responses were also observed, but were presented in only 4 of 5 subjects receiving gluten. The highest fold changes were observed at 4 hours following the oral gluten challenge. There was no significant increase in CCL20 observed in the subject that was challenged with the gluten sham.

Significant IL-8 and IL-10 responses were also observed in a variable number of subjects and at varying time points after gluten challenge. The greatest IL-8 response was observed at 3 hours after gluten in 4 of 5 subjects, and at 4 hours in 5 of 5 subjects, but with overall diminished levels observed. Significant stimulation of IL-10 after gluten challenge was observed at 3 and 4 hours after gluten in 3 of 5 and 4 of 5 subjects respectively. Again, there was very little significant production of IL-8 and IL-10 in subjects receiving the gluten sham.

Data related to the MDS analysis are shown in Tables 2-4 and FIG. 1.

Conclusion

Overall, candidate biomarkers which may be utilized to identify CeD subjects have been identified. Significant responses to gluten were observed in 2 different studies utilizing 2 different forms of gluten exposure. IL-2 and CCL20 responses were stimulated and measured in the blood of subjects given either an administration of a purified and concentrated gluten peptide therapeutic, or subjects given an oral challenge of gluten in its natural state of wheat gluten. The significant responses observed with these 2 studies were also measured by 2 different methods, OLINK Proseek analysis and MSD VPlex analysis. Both methods of assay resulted in the same observation of IL-2 and CCL20 expression significantly increased in subjects exposed to gluten.

IL-8 and IL-10 responses were also observed to be increased in subjects exposed to gluten, but with some variable timing and increased levels of cytokine/chemokine concentrations.

Although limited in number, the subjects receiving either a saline administration in the Nexvax2 clinical trial or a sham gluten oral challenge in the other study, both groups exhibited no increased cytokine/chemokine responses. The data differentiates between those that are exposed to gluten and those that are not.

Table 2. IL-2, IL-8, and IL-10 concentrations in subjects assessed by MSD Proinflammatory Panel assay

Table 3. CCL20 concentrations in subjects assessed by MSD assay

Table 4. Cytokine/Chemokine fold change in subjects assessed by MSD assay

Claims

1. A method, comprising determining a level of CCL20 in a subject that has or is suspected of having Celiac disease, wherein the subject has been administered a composition comprising gluten peptides.

2. The method of claim 1, wherein the method further comprises assessing the likelihood the subject has Celiac disease.

3. The method of claim 1 or 2, wherein the method further comprises assessing the efficacy of a treatment for Celiac disease.

4. The method of claim 3, wherein the treatment is a gluten peptide therapy.

5. The method of any one of the preceding claims, wherein the composition comprising gluten peptides comprises gluten protein.

6. The method of claim 5, wherein the composition comprising gluten peptides comprises 3 grams of gluten.

7. The method of any one of the preceding claims, wherein the composition comprising gluten peptides is administered orally.

8. The method of claim 7, wherein the composition comprising gluten peptides is administered orally in any one of the oral forms provided herein.

9. The method of claim 8, wherein the composition comprising gluten peptides is administered orally in any one of the oral forms provided herein at any one of the times and/or intervals provided herein.

10. The method of any one of the preceding claims, wherein the composition comprising gluten peptides is a foodstuff.

11. The method of claim 10, wherein the foodstuff is a liquid composition.

12. The method of any one of claims 1-4, wherein the composition comprising gluten peptides comprises any one of the compositions provided in U.S. Patent No. 8,835,603 and U.S. Patent No. 9,464,120, which compositions are incorporated herein by reference.

13. The method of any one of claims 1-4, wherein the composition comprising gluten peptides comprises the peptides of NexVax2.

14. The method of any one of claims 1-4, 12 and 14, wherein the composition comprising gluten peptides is administered intradermally to the subject.

15. The method of claim 14, wherein the composition comprising gluten peptides is administered intradermally in any one of the intradermal forms provided herein.

16. The method of claim 15, wherein the composition comprising gluten peptides is administered intradermally in any one of the intradermal forms provided herein at any one of the times and/or intervals provided herein.

17. The method of any one of the preceding claims, wherein the subject is following a gluten-free diet.

18. The method of any one of the preceding claims, wherein the method further comprises obtaining a sample from the subject.

19. The method of claim 18, wherein the determining is measuring, and the measuring is performed on the sample.

20. The method of claim 18 or 19, wherein the sample is obtained from the subject at any one of the time points provided herein.

21. The method of any one of claims 18 to 20, wherein the sample is obtained from the subject 1 hour to 6 hours after the subject has been administered the composition comprising gluten peptides.

22. The method of any one of claims 18 to 21, wherein the sample from the subject is a plasma or serum sample.

23. The method of claim 22, wherein the sample from the subject is a serum sample.

24. The method of any one of the preceding claims, wherein one or more additional cytokines or chemokines are determined.

25. The method of claim 24, wherein the one or more additional cytokines or chemokines is/are IL-2, IL-8 and/or IL-10.

26. The method of claim 25, wherein the one or more additional cytokines or chemokines is IL-lO.

27. The method of claim 25, wherein the one or more additional cytokines or chemokines is IL-8.

28. The method of claim 25, wherein the one or more additional cytokines or chemokines is IL-2.

29. The method of claim 25, wherein the one or more additional cytokines or chemokines are IL-2 and IL-8.

30. The method of claim 25, wherein the one or more additional cytokines or chemokines are IL-2, IL-8 and IL-10.

31. The method of any one of the preceding claims, wherein an elevated level of CCL20 and/or the one or more additional cytokines or chemokines compared to a control level, respectively, indicates that the subject has Celiac disease, and the step of assessing comprises comparing the level of CCL20 and/or the one or more additional cytokines or chemokines to a control level, respectively.

32. The method of claim 31, wherein the control level is any one of the control levels provided herein.

33. The method of claim 31 or 32, wherein the control level is a baseline level.

34. The method of claim 33, wherein the baseline level is a level of the CCL20 and/or the one or more additional cytokines or chemokines, respectively, prior to administration of the composition comprising gluten peptides.

35. The method of any one of claims 31 to 34, wherein the level(s) as compared to a baseline level that is indicative of Celiac disease is any one of the fold changes, or greater, as provided herein.

36. The method of any one of the preceding claims, wherein the method further comprises recording whether or not the subject has Celiac disease, an indicator thereof, or an indicator of the efficacy of a treatment of the subject, based on the determining and/or assessing.

37. The method of any one of the preceding claims, wherein the method further comprises treating, suggesting a treatment, or giving information in regard to a treatment to the subject.

38. The method of claim 37, wherein the treating or treatment comprises administration of a composition comprising gluten peptides to the subject.

39. The method of claim 38, wherein the composition comprising gluten peptides comprises any one of the compositions provided in U.S. Patent No. 8,835,603 and U.S.

Patent No. 9,464,120, which compositions are incorporated herein by reference.

40. The method of any one of the preceding claims, wherein the subject has received treatment for Celiac disease.

41. The method of claim 40, wherein the treatment comprised administration of a composition comprising gluten peptides to the subject.

42. The method of claim 41, wherein the composition comprising gluten peptides comprises any one of the compositions provided in U.S. Patent No. 8,835,603 and U.S.

Patent No. 9,464,120, which compositions are incorporated herein by reference.

43. The method of any one of the preceding claims, wherein an elevated level of the CCL20 and/or the one or more additional cytokines or chemokines, respectively, compared to a control level indicates that the subject has an unsatisfactory therapeutic response to a treatment, and the step of assessing comprises comparing the level of CCL20 and/or the one or more additional cytokines or chemokines to a control level, respectively.

44. The method of any one of the preceding claims, wherein the level of the CCL20 and/or the one or more additional cytokines or chemokines is measured with an assay that comprises an immuno-based assay.

45. The method of claim 44, wherein the immuno-based assay comprises an ELISA or a multiplex bead-based assay.

46. The method of any one of claims 1 to 44, wherein the level of the CCL20 and/or the one or more additional cytokines or chemokines is measured with an assay that comprises an electrochemiluminsence assay.

47. The method of claim 46, wherein the assay comprises an MSD® MULTI-SPOT assay.

48. The method of claim 47, wherein the MSD® MULTI-SPOT assay comprises a Chemokine Panel 1 (human) kit.

49. The method of claim 47, wherein the MSD® MULTI-SPOT assay comprises a Proinflammatory Panel 1 (human) kit.

50. The method of claim 46, wherein the electrochemiluminsence assay comprises a multispot electrochemiluminsence based cytokine assay (e.g., MSD V-Plex).

51. The method of any one of the preceding claims, wherein the level of the CCL20 and/or the one or more additional cytokines or chemokines is measured with an assay that comprises a cytokine/chemokine assay such as a Proseek cytokine/chemokine assay or the like.

52. The method of any one of the preceding claims, wherein the method further comprises assessing whether the subject has a homozygous HLA-DQ2.5 genotype or a non-homozygous HLA-DQ2.5 genotype.

53. The method of claim 52, wherein the non-homozygous HLA-DQ2.5 genotype is a heterozygous HLA-DQ2.5 genotype.

54. The method of any one of the preceding claims, wherein the subject has a

homozygous HLA-DQ2.5 genotype.

55. The method of any one of claims 1 to 53, wherein the subject has a non-homozygous HLA-DQ2.5 genotype.

56. The method of any one of claims 1 to 53, wherein the subject is homozygous for HLA-DQAl*05, HLA-DQBl*02, or both HLA-DQAl*05 and HLA-DQBl*02.

57. The method of any one of claims 1 to 53, wherein the subject is not homozygous for HLA-DQAl*05, HLA-DQBl*02, or both HLA-DQAl*05 and HLA-DQBl*02.

58. The method of any one of the preceding claims, wherein the subject has Celiac disease.

59. The method of any one of the preceding claims, wherein the subject is suspected of having Celiac disease.

60. The method of any one of claims 1 to 57, wherein the subject has gluten sensitivity.

61. The method of any one of the preceding claims, wherein the method further comprises treating, suggesting a treatment, or giving information in regard to a treatment that is a non- Celiac treatment to the subject.

62. The method of any one of the preceding claims, wherein the method does not comprise treating, suggesting a treatment, or giving information in regard to a treatment that is a Celiac treatment to the subject or comprises not suggesting or giving information in regard to a Celiac treatment to the subject.

63. A kit for carrying out any one of the methods provided herein comprising an agent for measuring a level of CCL20.

64. The kit of claim 63, wherein the kit further comprises an agent for measuring one or more additional cytokines or chemokines.

65. The kit of claim 64, wherein the one or more additional cytokines or chemokines is/are IL-2, IL-8 and/or IL-10.

66. The kit of claim 65, wherein the one or more additional cytokines or chemokines is IL-10.

67. The kit of claim 65, wherein the one or more additional cytokines or chemokines is IL-8.

68. The kit of claim 65, wherein the one or more additional cytokines or chemokines is IL-2.

69. The kit of claim 65, wherein the one or more additional cytokines or chemokines are IL-2 and IL-8.

70. The kit of claim 65, wherein the one or more additional cytokines or chemokines are IL-2, IL-8 and IL-10.

71. The kit of any one of claims 63 to 70, wherein the agent for measuring the CCL20 and/or the one or more additional cytokines or chemokines, respectively, is a binding partner for the CCL20 and/or the one or more additional cytokines or chemokines, respectively, or an agent that recognizes such binding partner, respectively.