WO2019113037A1 - Mesure de ccl20 après exposition au gluten - Google Patents

Mesure de ccl20 après exposition au gluten Download PDF

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Publication number
WO2019113037A1
WO2019113037A1 PCT/US2018/063805 US2018063805W WO2019113037A1 WO 2019113037 A1 WO2019113037 A1 WO 2019113037A1 US 2018063805 W US2018063805 W US 2018063805W WO 2019113037 A1 WO2019113037 A1 WO 2019113037A1
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subject
gluten
chemokines
composition
level
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PCT/US2018/063805
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English (en)
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Robert P. Anderson
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Immusant, Inc.
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Publication of WO2019113037A1 publication Critical patent/WO2019113037A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/35—Allergens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents

Definitions

  • 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.
  • 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.
  • C-C motif Chemokine
  • 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.
  • a subject e.g., a subject that has or is suspected of having Celiac disease
  • the method further comprises assessing the likelihood the subject has Celiac disease, and assessing the efficacy of a treatment for the Celiac disease.
  • the treatment for the Celiac disease is a gluten peptide therapy, e.g., Nexvax2.
  • 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.
  • 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.
  • 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.
  • the subject is following a gluten-free diet. In some embodiments, the subject is following a gluten-free diet. In some
  • 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).
  • a sample e.g., plasma or serum sample
  • 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.
  • 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
  • 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.
  • the control level is any one of the control levels provided herein.
  • 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).
  • 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.
  • 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.
  • 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.
  • the subject has received treatment for Celiac disease (e.g., administration of a composition comprising gluten peptides such as Nexvax2).
  • 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
  • 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.
  • 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.
  • the electrochemiluminsence assay comprises an MSD® MULTI-SPOT assay, or a multispot electrochemiluminsence based cytokine assay (e.g., MSD® V-Plex).
  • the MSD® MULTI SPOT assay comprises a Chemokine Panel 1 (human) kit, or a Proinflammatory Panel 1 (human) kit.
  • the subject has a homozygous HLA-DQ2.5 genotype or a non- homozygous HLA-DQ2.5 genotype (e.g., heterozygous HLA-DQ2.5 genotype).
  • the subject is homozygous for HLA-DQAl*05, HLA-DQBl*02, or both HLA-DQAl*05 and HLA-DQBl*02.
  • the subject is not homozygous for HLA-DQAU05, HLA-DQBU02, or both HLA-DQAU05 and HLA-DQBU02.
  • the subject has gluten sensitivity.
  • 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.
  • kits 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.
  • the one or more additional cytokines or chemokines is/are IL-2, IL-8 and/or IL-10.
  • 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.
  • FIG. 1 is a table showing example cytokine/chemokine fold change in subjects assessed by MSD assay.
  • 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.
  • 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.
  • 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.
  • the methods provided herein comprise determining a level of CCL20 in a sample from a subject.
  • 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.
  • the method may also involve determining a level of IL-2, IL-8 and/or IL- 10 in a sample from the subject.
  • determining refers to any method by which the respective information can be acquired. Such methods can be direct or indirect.
  • 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.
  • 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.
  • the comparing comprises a statistical analysis, such as a paired T-test.
  • 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.
  • the method further comprising treating or suggesting a treatment if the subject is identified as having or likely to have Celiac disease.
  • the method further comprises recommending a gluten-free diet and/or providing information in regard thereto to the subject.
  • 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.
  • the treatment is a composition comprising a gluten peptide as described herein, e.g., Nexvax2.
  • the treatment comprises a gluten-free diet.
  • 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.
  • 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.
  • Chemokine (C-C motif) ligand 20 CCL20
  • liver activation regulated chemokine LOC
  • MIP3A Macrophage Inflammatory Protein-3
  • AY150053.1 Homo sapiens macrophage inflammatory protein-3-alpha (CCL20) gene, promoter region and partial cds
  • NM_00l 130046.1 Homo sapiens C-C motif chemokine ligand 20 (CCL20), transcript variant 2, mRNA
  • NR_001123518.1 C-C motif chemokine 20 isoform 2 precursor [Homo sapiens]
  • 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; Karlinoth P, et al. Pathol Res Pract. 1994, 190: 1017-25), and oligonucleotide microarray (e.g., by hybridization of
  • 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.
  • immunoassays also referred to herein as immune-based or immuno-based assays, e.g., Western blot, ELISA, and ELISPOT assays
  • Mass spectrometry e.g., MS spectrometry, and multiplex bead-based assays.
  • the protein binding partners e.g., antibodies
  • 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.
  • 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
  • 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).
  • measuring a level of CCL20, or other cytokine or chemokine comprises an enzyme-linked immunosorbent assay (ELISA) or enzyme-linked
  • 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.
  • a binding agent for CCL20, or other cytokine or chemokine e.g., an antibody
  • PVDF polyvinylidene fluoride
  • Cells of interest e.g., peripheral blood mononuclear cells
  • 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.
  • 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).
  • 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).
  • 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.
  • measuring a level of CCL20, or other cytokine or chemokine comprises an electrochemiluminsence assay.
  • the electrochemiluminsence assay comprises an electrochemiluminsence assay.
  • electrochemiluminsence assay comprises an MSD® MULTI-SPOT assay, or a multispot electrochemiluminsence based cytokine assay (e.g., MSD® V-Plex).
  • the MSD® MULTI-SPOT assay comprises a Chemokine Panel 1 (human) kit, or a
  • 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.
  • One aspect of the disclosure relates to methods of identifying (e.g., diagnosing) subjects, such as subjects having or suspected of having Celiac disease.
  • 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.
  • the composition comprising gluten peptides comprises gluten protein, such as 3 grams of gluten.
  • the composition comprising gluten peptides comprises gluten protein, such as 6 grams of gluten.
  • the composition comprising gluten peptides comprises gluten protein, such as between 3 and 6 grams of gluten.
  • 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.
  • the method further comprises assessing the likelihood the subject has Celiac disease.
  • 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.
  • assessing comprises determining the ratio of the level of the at least one cytokine or chemokine (e.g., CCL20) to the control level.
  • control level of the at least one cytokine or chemokine is a baseline level of the cytokine or chemokine.
  • 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.
  • the method can further comprise the step of determining a baseline level of the cytokine or chemokine (e.g., CCL20) in the subject.
  • 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.
  • the method further comprises recording whether or not the subject has or is likely to have Celiac disease based on the level or ratio.
  • the at least one cytokine or chemokine is CCL20 with or without also IL-2, IL-8 and/or IL-10.
  • the level of the at least one cytokine or chemokine is measured in a sample, e.g., a serum or plasma, obtained from the subject.
  • a sample e.g., a serum or plasma
  • 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).
  • 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).
  • 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.
  • 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.
  • 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
  • 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.
  • control level is a baseline level.
  • 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).
  • 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.
  • the treating or treatment comprises administration of a composition comprising a gluten peptide to the subject, e.g., administration of Nexvax2 to the subject.
  • the subject has received treatment, such as with a gluten peptide therapy, e.g., Nexvax2.
  • a gluten peptide therapy e.g., Nexvax2
  • 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)
  • 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.
  • measuring the level of the at least one cytokine or chemokine comprises an immuno-based assay.
  • the immuno-based assay comprises an ELISA or a multiplex bead-based assay.
  • 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).
  • the assay comprises an MSD® MULTI SPOT assay.
  • 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.
  • the method further comprises performing other testing.
  • the other testing comprises a serology test, genotyping, an intestinal biopsy, and/or a T cell response test.
  • the method further comprises performing one or more additional tests on the subject.
  • 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.
  • 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.
  • a level of IFN-g at or above a cut-off level e.g., at or above 7.2 pg/ml
  • 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.
  • the treatment comprises a gluten-free diet.
  • 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).
  • the subject is following a gluten free diet.
  • 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.
  • 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 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.
  • the sample comprises plasma or serum.
  • the methods comprise obtaining or providing the sample.
  • 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).
  • 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.
  • 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.
  • 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.
  • a subject may include any subject that has or is suspected of having Celiac disease.
  • the subject is a human.
  • 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).
  • the subject is HLA-DQ2.5 positive (i.e., has both susceptibility alleles DQA1 *05 and DQB1 *02).
  • 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.
  • PCR polymerase chain reaction
  • 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).
  • gluten sensitivity also referred to as non-Celiac gluten sensitivity
  • 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.
  • 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.
  • 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).
  • a control level is a level in a sample from a control subject (or subjects).
  • 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.
  • 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.
  • a control subject is a healthy individual not having or suspected of having Celiac disease.
  • the control level is a pre determined threshold.
  • 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.
  • 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).
  • 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).
  • 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.
  • the gluten peptide(s) is a protein.
  • the gluten peptide(s) is not full-length protein but a portion thereof.
  • the disclosure provides a composition comprising gluten peptides.
  • the composition is a composition comprising gluten protein.
  • 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
  • the composition comprising gluten peptide
  • methods described herein further comprise other testing of a subject (e.g., based on the results of the methods described 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).
  • 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.
  • the kit comprises at least one agent for identifying or measuring levels of CCL20 and/or one or more additional cytokines or chemokines.
  • the agent is a binding partner for the CCL20 and/or one or more additional cytokines or chemokines.
  • the agent is one that recognizes a binding partner for the CCL20 and/or other cytokine or chemokine.
  • the binding partner is any molecule that binds specifically to a cytokine or chemokine provided herein (e.g., CCL20).
  • 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.
  • “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.
  • the binding partner is an antibody.
  • 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.
  • 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
  • the binding partner is an MHC tetramer.
  • 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).
  • the binding partner is any molecule that binds specifically to an cytokine or chemokine nucleic acid, such as mRNA.
  • “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.
  • the binding partner that binds specifically to an mRNA is a nucleic acid, e.g., a probe.
  • the agent is a complementary nucleic acid.
  • the kit further comprises a first and second binding partner for a cytokine or chemokine provided herein.
  • the first and second binding partners are antibodies.
  • the first and second binding partners are nucleic acids, such as nucleic acid probes.
  • 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.
  • 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.
  • 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.
  • 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.
  • the kit further comprises any one of the gluten peptide compositions provided herein (e.g., a liquid composition comprising gluten protein).
  • a composition comprising gluten protein
  • such composition is contained within a container in the kit.
  • such composition is contained within a solution separate from the container, such that the composition may be added to the container prior to administration.
  • 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.
  • the container is present in the kit in duplicate or triplicate.
  • the kit further comprises a container suitable for injection of such a composition to a subject, e.g., a syringe.
  • 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 VacutainerTM or urine collection container from Becton Dickinson).
  • the container further contains an anti-coagulant, such as heparin, EDTA, citrate, sodium polyanethol sulfonate, or oxalate.
  • the container is structured to hold a defined volume, e.g., 1 mL or 5 mL.
  • the container is present in the kit in duplicate or triplicate.
  • the kit further comprises a negative control, e.g., a
  • the kit further comprises a positive control, e.g., a composition comprising a chemokine or cytokine at a known concentration or level.
  • the kit comprises any combination of the components mentioned above.
  • 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.
  • the instructions include the methods described herein. Instructions can be in any suitable form, e.g., as a printed insert or a label.
  • 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.
  • 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.
  • NPL001 contains Gluten peptides NPL001, NPL002, and NPL003.
  • the sequences for NPL001, NPL002, and NPL003 are as follows, NPL001:
  • NPL002 pyroEQPFPQPEQPFPWQP- amide (SEQ ID NO: 6)
  • 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.
  • PCR polymerase chain reaction
  • Multispot electrochemiluminescence based cytokine assays 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
  • 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.
  • 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.
  • 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.
  • cytokines/chemokines had significant increases within 4 hours, but there was variability in the number of subjects exhibiting those increases. At 6 hours following
  • 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.
  • 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.
  • 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.
  • 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.

Abstract

La présente invention concerne des méthodes comprenant des méthodes d'identification d'un patient atteint, suspecté d'être atteint ou exposé à une maladie cœliaque consistant à déterminer un niveau de CCL20 dans un échantillon en provenance d'un patient.
PCT/US2018/063805 2017-12-04 2018-12-04 Mesure de ccl20 après exposition au gluten WO2019113037A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090311214A1 (en) * 2008-06-11 2009-12-17 University Of Maryland, Baltimore Compositions and methods for the innate immune response pathway
WO2017049035A1 (fr) * 2015-09-17 2017-03-23 Amgen Inc. Prédiction de réponse clinique à des antagonistes d'il23 utilisant des biomarqueurs de la voie de l'il23
US20170218453A1 (en) * 2014-09-29 2017-08-03 Immusant, Inc. Use of hla genetic status to assess or select treatment of celiac disease

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090311214A1 (en) * 2008-06-11 2009-12-17 University Of Maryland, Baltimore Compositions and methods for the innate immune response pathway
US20170218453A1 (en) * 2014-09-29 2017-08-03 Immusant, Inc. Use of hla genetic status to assess or select treatment of celiac disease
WO2017049035A1 (fr) * 2015-09-17 2017-03-23 Amgen Inc. Prédiction de réponse clinique à des antagonistes d'il23 utilisant des biomarqueurs de la voie de l'il23

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