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WO2020198434A1 - Utilisation d'il-2 pour le diagnostic d'une maladie coeliaque - Google Patents

Utilisation d'il-2 pour le diagnostic d'une maladie coeliaque Download PDF

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Publication number
WO2020198434A1
WO2020198434A1 PCT/US2020/024890 US2020024890W WO2020198434A1 WO 2020198434 A1 WO2020198434 A1 WO 2020198434A1 US 2020024890 W US2020024890 W US 2020024890W WO 2020198434 A1 WO2020198434 A1 WO 2020198434A1
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subject
protein
level
lloq
composition
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Robert P. Anderson
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Immusant Inc
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Immusant Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/06—Gastro-intestinal diseases
    • G01N2800/065—Bowel diseases, e.g. Crohn, ulcerative colitis, IBS

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
  • the disclosure relates, at least in part, to determining a level of interleukin-2 (IL-2) 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 interleukin-2 (IL-2) in a sample from the subject.
  • IL-2 interleukin-2
  • aspects of the disclosure provide a method, comprising providing a blood sample from a subject that has or is suspected of having Celiac disease, wherein the subject has been administered a composition comprising gluten peptides prior to the blood sample being obtained from the subject, and measuring a level of IL-2 protein in the blood sample using an assay, wherein a level of IL-2 protein that is greater than or equal to a Lower Limit of Quantification (LLOQ) for the assay indicates that the subject has or is likely to have Celiac disease and wherein a level of IL-2 protein that is less than the LLOQ indicates that the subject does not have or is not likely to have Celiac disease.
  • LLOQ Lower Limit of Quantification
  • a level of IL-2 protein that is greater than or equal to the LLOQ indicates that the subject requires further testing to determine whether the subject has or is likely to have Celiac disease.
  • the blood sample is obtained from the subject 1 to 6 hours after the subject has been administered the composition comprising gluten peptides. In some embodiments, the blood sample is obtained from the subject 3 to 4 hours after the subject has been administered the composition comprising gluten peptides. In some embodiments, the blood sample is serum sample. In some embodiments, the LLOQ is less than or equal to 0.5 pg/mL, e.g., less than or equal to 0.4 pg/mL, less than or equal to 0.3 pg/mL, less than or equal to 0.2 pg/mL, or less than or equal to 0.1 pg/mL.
  • the LLOQ is between 0.1 and 0.5 pg/mL, e.g., between 0.1 and 0.5 pg/mL, between 0.2 and 0.5 pg/mL, between 0.3 and 0.5 pg/mL, between 0.4 and 0.5 pg/mL, between 0.1 and 0.4 pg/mL, between 0.2 and 0.4 pg/mL, between 0.3 and 0.4 pg/mL, between 0.1 and 0.3 pg/mL, between 0.2 and 0.3 pg/mL, or between 0.1 and 0.2 pg/mL.
  • the LLOQ is 0.5 pg/mL.
  • the method further comprises identifying the subject as having or likely to have Celiac disease if the level of IL-2 protein is greater than or equal to the LLOQ or identifying the subject as not having or not likely to have Celiac disease if the level of IL-2 protein is less than the LLOQ.
  • the method further comprises comparing the level of IL-2 protein to a baseline level of IL-2 protein in a control blood sample from the subject.
  • the comparing comprises providing the control blood sample from a subject, wherein the subject has not been administered a composition comprising gluten peptides prior to the control blood sample being obtained from the subject, measuring a baseline level of IL-2 protein in the control blood sample using the assay, and comparing the level of IL-2 protein to the baseline level of IL-2 protein.
  • a level of IL-2 protein that is greater than or equal to the LLOQ, a baseline level of IL-2 protein that is greater than or equal to the LLOQ and a ratio of the level of the IL-2 protein to the baseline level of IL-2 protein being less than 2 indicates an equivocal result, which optionally may require additional testing, e.g., re-performing a method as described herein for assessing IL-2 protein levels in the subject (e.g., according to any one of the above-mentioned embodiments or any other embodiments described herein), performing HLA-DQ genotyping, and/or performing a gluten challenge for 2 or more weeks followed by endoscopic duodenal biopsy and/or serology specific for transglutaminase-2 or deamidated gliadin peptide.
  • a level of IL-2 protein that is greater than or equal to the LLOQ, a baseline level of IL-2 protein that is greater than or equal to the LLOQ and a ratio of the level of the IL-2 protein to the baseline level of IL-2 protein being greater than or equal to 2 indicates that the subject has or is likely to have Celiac disease.
  • a level of IL-2 protein that is greater than or equal to the LLOQ and a baseline level of IL-2 protein that is less than the LLOQ indicates that the subject has or is likely to have Celiac disease.
  • the baseline level of IL-2 protein and the level of IL-2 protein are measured at different times. In some embodiments, the baseline level of IL-2 protein and the level of IL-2 protein are measured at the same time (e.g., in the same assay).
  • the method comprises identifying the subject as in need of additional testing if the level of IL-2 protein is greater than or equal to the LLOQ, the baseline level of IL-2 protein is greater than or equal to the LLOQ and the ratio of the level of the IL-2 protein to the baseline level of IL-2 protein is less than 2.
  • the additional testing comprises re-performing a method as described herein for assessing IL-2 protein levels in the subject (e.g., according to any one of the above-mentioned embodiments or any other embodiments described herein), performing HLA-DQ genotyping, and/or performing a gluten challenge for 2 or more weeks followed by endoscopic duodenal biopsy and/or serology specific for transglutaminase-2 or deamidated gliadin peptide.
  • the method comprises identifying the subject as having or likely to have Celiac disease if the level of IL-2 protein is greater than or equal to the LLOQ, the baseline level of IL-2 protein is greater than or equal to the LLOQ and the ratio of the level of the IL-2 protein to the baseline level of IL-2 protein is greater than or equal to 2. In some embodiments, the method comprises identifying the subject as having or likely to have Celiac disease if the level of IL-2 protein is greater than or equal to the LLOQ and the baseline level of IL-2 protein is less than the LLOQ.
  • the composition comprising gluten peptides comprises gluten protein (e.g., wheat, rye, and/or barley). In some embodiments, the composition comprises about 3 grams of gluten. In some embodiments, the composition comprises between about 1- 6 grams of gluten. In some embodiments, the composition comprising gluten peptides is administered orally to the subject. In some embodiments, the administration is self administration by the subject. In some embodiments, the composition comprising gluten peptides is a foodstuff. In some embodiments, the foodstuff is a liquid composition.
  • 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 comprises the peptides of NexVax2.
  • the composition comprising gluten peptides is administered intradermally or subcutaneously to the subject. In some embodiments, the administration is self administration by the subject.
  • the subject is following a gluten-free diet prior to being administered the composition comprising gluten peptides.
  • the method further comprises recording whether or not the subject has or is likely to have Celiac disease, 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.
  • the treating or treatment comprises administration of a second composition comprising gluten peptides to the subject.
  • the second 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 subject has received treatment for Celiac disease, wherein the treatment comprised administration of a third composition comprising gluten peptides to the subject.
  • the third 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 assay is an immuno-based assay, wherein the LLOQ is the LLOQ for the immune-based assay.
  • the immuno-based assay comprises an ELISA, an electrochemiluminsence assay or a multiplex bead-based assay, wherein the LLOQ is the LLOQ for the ELISA, electrochemiluminsence assay or multiplex bead-based assay, respectively.
  • assay is an electrochemiluminsence assay that comprises an MSD® MULTI-SPOT assay, or a multispot electrochemiluminsence based cytokine assay (e.g., MSD V-Plex), wherein the LLOQ is the LLOQ for the MSD® MULTI-SPOT assay or the multispot electrochemiluminsence based cytokine assay, respectively.
  • MSD V-Plex multispot electrochemiluminsence based cytokine assay
  • the assay is a proximity extension assay, wherein the LLOQ is the LLOQ for the proximity extension assay.
  • the method further comprises assessing whether the subject has a homozygous HLA-DQ2.5 genotype or a non-homozygous HLA-DQ2.5 genotype.
  • the non-homozygous HLA-DQ2.5 genotype is a heterozygous HLA- DQ2.5 genotype.
  • the subject has a homozygous HLA-DQ2.5 genotype.
  • the subject has a non-homozygous HLA-DQ2.5 genotype.
  • the subject is homozygous for HLA-DQA1*05, HLA-DQB1*02, or both HLA-DQA1*05 and HLA-DQB1*02.
  • the subject is not homozygous for HLA-DQA1*05, HLA-DQB1*02, or both HLA-DQA1*05 and HLA- DQB1*02.
  • the assessing comprises a nucleic-acid-based assay.
  • the nucleic-acid-based assay is a sequencing assay or a probe-based assay, for example reverse sequence specific oligonucleotide (SSO) hybridization may be used to determine DQA, -DQB, -DPA and -DPB locus types (see, e.g., Dunckley. HLA typing by SSO and SSP methods. (2012) Methods Mol Biol.882:9-25).
  • the subject has Celiac disease. In some embodiments, the subject is suspected of having Celiac disease. In some embodiments, the subject has gluten sensitivity.
  • the method further comprises treating, suggesting a treatment, or giving information in regard to a treatment that is a Celiac treatment to the subject if the subject is identified as having or likely to have Celiac disease.
  • 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 if the subject is identified as not having or not likely to have Celiac disease.
  • kits for carrying out any one of the methods provided herein comprising ones of more agents for measuring a level of IL-2.
  • the one or more agents for measuring IL-2 is one or more binding partners for IL-2 or one or more agents that recognize such one or more binding partners.
  • 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-D(Mi *05 and HLA -DQB1 *02 (accounting for about 90% of individuals), variants of HLA-D ⁇ 22, or HLA-Obd. 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 is currently positively 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 -DQA1 *05,
  • DQB1 *02 and DQB1 *0302 are not present.
  • the presence of these alleles does not necessarily mean that the patient has Celiac disease, as only some patients having these alleles have Celiac disease.
  • GFD gluten-free diet
  • reintroduction of gluten into the diet can be necessary to make a firm diagnosis of Celiac disease based on the current diagnostic method. Reintroduction of about 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.
  • 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 interleukin 2 (IL-2) in a sample from a subject, and comparing this level of IL-2 to a Lower Limit of Quantification (LLOQ), and optionally further to a baseline level of IL-2 in a control sample.
  • IL-2 interleukin 2
  • LLOQ Lower Limit of Quantification
  • 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 providing a blood sample from a subject that has or is suspected of having Celiac disease.
  • the subject has been administered a composition comprising gluten peptides prior to the blood sample being obtained from the subject.
  • the administration may be self-administration or may be administration by another, e.g., a medical professional.
  • the methods further comprise determining or measuring a level of IL-2 in a sample from the subject.
  • the method may also involve determining (e.g., measuring ) a level of IL-2, which may be done, in some embodiments, using methods described previously (see, e.g., International Application Publication No. WO2015/164747 and International Application No. PCT/US2018/063805, both of which are incorporated by reference herein) or using methods described 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.
  • the method further comprises comparing the level of IL-2 protein in the sample with a Lower Limit of Quantification (LLOQ) level.
  • LLOQ is the lowest analyte (e.g., IL-2 protein) concentration that is within the validated quantitative range of an assay.
  • the LLOQ is determined empirically by establishing the lowest concentration of analyte that can be measured with precision (the % coefficient of variation (CV) ⁇ 25%).
  • the LLOQ is determined as part of a method described herein. In some embodiments, the LLOQ is pre-determined. In some embodiments, if the level of IL-2 protein is greater than or equal to the LLOQ, the subject has or is likely to have Celiac disease and if the level of IL-2 protein is less than the LLOQ, the subject does not have or is not likely to have Celiac disease. In further embodiments, the subject is identified as having or is likely to have Celiac disease if the level of IL-2 protein is greater than or equal to the LLOQ, or identified as not having or not likely to have Celiac disease if the level of IL-2 protein is less than or equal to the LLOQ.
  • 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.
  • the subject requires further testing if the level of IL-2 protein is greater than or equal to the LLOQ.
  • the method further comprises exposing the subject to further testing to determine whether the subject has or is likely to have Celiac disease, if the level of IL-2 protein is greater than or equal to the LLOQ.
  • the further testing comprises determining or measuring a baseline level of IL-2 in a control blood sample from a subject; and comparing the level of IL-2 protein in the sample with the baseline level of IL-2 protein in the control.
  • a level of IL-2 protein that is greater than or equal to the LLOQ, a baseline level of IL-2 protein that is greater than or equal to the LLOQ and/or a ratio of the level of the IL-2 protein to the baseline level of IL-2 protein being less than a threshold value indicates an equivocal result and/or that the subject requires additional testing (e.g., additional testing such as re performing a method as described herein for assessing IL-2 protein levels in the subject (e.g., according to any one of the above-mentioned embodiments or any other embodiments described herein), performing HLA-DQ genotyping, and/or performing a gluten challenge for 2 or more weeks followed by endoscopic duodenal biopsy and/or serology specific for transglutaminase-2 or deamidated gliadin peptide); (ii) a level of IL-2 protein that is greater than or equal to the LLOQ, a baseline level of IL-2 protein that is greater than or equal to the
  • the blood sample is obtained from the subject 1 to 6 hours after the subject has been administered the composition comprising gluten peptides. In some embodiments, the blood sample is obtained from the subject 2 to 5 hours after the subject has been administered the composition comprising gluten peptides. In some embodiments, the blood sample is obtained from the subject 3 to 4 hours after the subject has been administered the composition comprising gluten peptides. In some embodiments, the blood sample is serum sample.
  • control blood sample is a blood sample obtained from the subject prior to administration of the composition comprising gluten peptides. In some embodiments, the control blood sample is a blood sample obtained from a control subject, such as a subject that has not been administered a composition comprising gluten peptides.
  • the assessing comprises identifying the subject as having or at risk of having Celiac disease or as in need of further testing if the level of IL-2 protein is greater than or equal to the LLOQ; or not having or not at risk of having Celiac disease if the level of IL-2 protein that is less than the LLOQ.
  • the assessing comprises identifying the subject (a) as being in need of additional testing (e.g., additional testing such as re-performing a method as described herein for assessing IL-2 protein levels in the subject (e.g., according to any one of the above-mentioned embodiments or any other embodiments described herein), performing HLA-DQ genotyping, and/or performing a gluten challenge for 2 or more weeks followed by endoscopic duodenal biopsy and/or serology specific for transglutaminase-2 or deamidated gliadin peptide) if the level of IL-2 protein is greater than or equal to the LLOQ, the baseline level of IL-2 protein is greater than or equal to the LLOQ and the ratio of the level of the IL-2 protein to the baseline level of IL-2 protein is less than a threshold value; or (b) as having or at risk of having Celiac disease if the level of IL-2 protein is greater than or equal to the LLOQ, the baseline level of IL-2 protein is greater than
  • the threshold value is between 1.5 and 10, e.g., 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10. In some embodiments, the threshold value is 2.
  • the baseline level of IL-2 protein and the level of IL-2 protein are measured at different times (e.g., at least 1 hour apart or at least 1 day apart from each other). In some embodiments, the baseline level of IL-2 protein and the level of IL-2 protein are measured at the same time (e.g., in the same assay such as the same immunoassay).
  • the composition comprising gluten peptides comprises gluten protein. In some embodiments, the composition comprises wheat, rye, and/or barley. Such compositions are further described herein.
  • 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 comprises the peptides of NexVax2. Such compositions are further described 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 may be those described herein or otherwise known in the art. In some embodiments, the treatment is a composition comprising gluten peptides as described herein, e.g., Nexvax2. In some embodiments, 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.
  • Assays for detecting IL-2 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.
  • 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.
  • Example IL-2 protein sequences are provided below.
  • measuring a level of IL-2 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 IL-2.
  • 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 IL-2 is added after the IL-2 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 IL-2 is then determined based on the level of fluorescence.
  • An exemplary system for running a multiplex bead-based assay is the MAGPIX® system available from Luminex® Corporation (see, e.g., US Patent Nos.
  • measuring a level of IL-2 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 IL-2 (e.g., an antibody) that is coated aseptically onto a PVDF (polyvinylidene fluoride) -backed microplate.
  • a binding agent for IL-2 e.g., an antibody
  • PVDF polyvinylidene fluoride
  • Cells of interest e.g., peripheral blood mononuclear cells
  • a peptide as described herein 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 IL-2 secreted by activated cells is captured locally by the binding partner for the IL-2 on the high surface area PVDF membrane.
  • a second binding partner for IL-2 is added, forming a complex with the immobilized IL-2.
  • 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).
  • 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 IL-2.
  • the sample with an unknown amount of IL-2 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 IL-2, as in a "sandwich" ELISA).
  • a solid support e.g., a polystyrene microtiter plate
  • the binding partner for IL-2 is added, forming a complex with the immobilized IL-2.
  • 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 IL-2 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 IL-2 comprises an
  • the electrochemiluminsence assay comprises an MSD® MULTI-SPOT assay (Meso Scale Discovery, Rockville, Md), or a multispot electrochemiluminsence based cytokine assay (e.g., MSD® V-Plex, Meso Scale Discovery, Rockville, Md, such as the V-PLEX Proinflammatory panel or V-PLEX Thl7 panel).
  • MSD® MULTI-SPOT assay comprises a Chemokine Panel 1 (human) kit, or a Proinflammatory Panel 1 (human) kit.
  • measuring a level of IL-2 comprises a cytokine/chemokine assay such as a proximity extension assay (PEA) such as a Proseek® Multiplex Inflammation I 96x96 panel (Olink Proteomics, Uppsala, Sweden), a Proseek® Multiplex Immune Response (v.302) panel (Olink Proteomics, Uppsala, Sweden), or the like.
  • a cytokine/chemokine assay such as a proximity extension assay (PEA) such as a Proseek® Multiplex Inflammation I 96x96 panel (Olink Proteomics, Uppsala, Sweden), a Proseek® Multiplex Immune Response (v.302) panel (Olink Proteomics, Uppsala, Sweden), or the like.
  • measuring a level of IL-2 comprises an electrochemiluminsence assay (e.g., a multispot
  • electrochemiluminsence based cytokine assay and a proximity extension assay (PEA), optionally wherein the PEA is performed before or after the electrochemiluminsence assay.
  • PEA proximity extension assay
  • 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
  • compositions comprising gluten peptides comprises gluten protein (e.g., wheat, rye, and/or barley and/or comprising one or more of gliadin, hordein, and secalin).
  • gluten protein e.g., wheat, rye, and/or barley and/or comprising one or more of gliadin, hordein, and secalin.
  • Other compositions comprising gluten peptides are described elsewhere herein.
  • the composition comprising gluten peptides comprises gluten protein (e.g., wheat, rye, and/or barley and/or comprising one or more of gliadin, hordein, and secalin), such as between 1 and 10 grams, 1 and 6 grams, 2 and 6 grams, or 3 and 6 grams of gluten.
  • gluten protein e.g., wheat, rye, and/or barley and/or comprising one or more of gliadin, hordein, and secalin
  • the composition comprising gluten peptides is a foodstuff.
  • the composition comprising gluten peptides is a liquid composition, e.g., comprising gluten protein (e.g., wheat, rye, and/or barley and/or comprising one or more of gliadin, hordein, and secalin), such as between 1 and 10 grams, 1 and 6 grams, 2 and 6 grams, or 3 and 6 grams of gluten.
  • the composition comprising gluten peptides is administered by oral administration. As used herein, administration includes direct as well as indirect administration.
  • administering is meant to include instances where the subject is directed to self-administer, such as consume, the gluten peptide composition.
  • the subject is following a gluten free diet prior to administration of the composition comprising gluten peptides.
  • 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.
  • a level of IL-2 protein is determined or measured in a blood sample obtained from a subject after the administration of the gluten peptide composition to the subject.
  • the sample is obtained from the subject within 1-24 hours, such as within 1-12, 1-10, 1-8 or 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 within 1-8, 2-8, 3-8, 4-8, 2-7, 2-6, 3-8, 3-7, 3-6, 4-8, 4-7, 4-6, 3-5, or 3-4 hours after the subject has been administered the gluten peptide composition.
  • a baseline level of IL-2 protein is determined or measured in a control blood sample from a subject obtained prior to the administration of the gluten peptide composition to the subject.
  • the control blood sample is obtained at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours,
  • control blood sample is obtained within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, or 48 hours of the administration of the gluten peptide composition.
  • the method comprises (a) providing a blood sample from a subject that has or is suspected of having Celiac disease, wherein the subject has been administered a treatment and wherein the subject has been administered a composition comprising gluten peptides prior to the blood sample being obtained from the subject; (b) measuring a level of interleukin-2 (IL-2) protein in the blood sample using an assay and (c) assessing efficacy of the treatment.
  • Celiac disease e.g., responsiveness to a therapeutic gluten peptide composition such as Nexvax2
  • the method comprises (a) providing a blood sample from a subject that has or is suspected of having Celiac disease, wherein the subject has been administered a treatment and wherein the subject has been administered a composition comprising gluten peptides prior to the blood sample being obtained from the subject; (b) measuring a level of interleukin-2 (IL-2) protein in the blood sample using an assay and (c) assessing efficacy of the treatment.
  • IL-2 interleukin-2
  • the method comprises administering a treatment for Celiac disease to a subject in need thereof if the subject has a level of IL-2 protein (e.g., a level of IL-2 protein in a blood sample obtained from the subject after the subject has been administered a composition comprising gluten peptides) that is that is greater than or equal to a LLOQ (e.g., 0.5 pg/mL) for an assay.
  • a level of IL-2 protein e.g., a level of IL-2 protein in a blood sample obtained from the subject after the subject has been administered a composition comprising gluten peptides
  • LLOQ e.g., 0.5 pg/mL
  • the method comprises administering a treatment for Celiac disease to a subject in need thereof if the subject has a level of IL-2 protein (e.g., a level of IL-2 protein in a blood sample obtained from the subject after the subject has been administered a composition comprising gluten peptides) that is that is greater than or equal to a LLOQ (e.g., 0.5 pg/mL) for an assay and a baseline level of IL-2 protein (e.g., a level of IL-2 protein in a control blood sample from the subject) that is less than the LLOQ.
  • a level of IL-2 protein e.g., a level of IL-2 protein in a blood sample obtained from the subject after the subject has been administered a composition comprising gluten peptides
  • a LLOQ e.g., 0.5 pg/mL
  • a baseline level of IL-2 protein e.g., a level of IL-2 protein in a control blood sample from the subject
  • the method comprises administering a treatment for Celiac disease to a subject in need thereof if the subject has a level of IL-2 protein (e.g., a level of IL-2 protein in a blood sample obtained from the subject after the subject has been administered a composition comprising gluten peptides) that is greater than or equal to a LLOQ (e.g., 0.5 pg/mL) for an assay, a baseline level of IL-2 protein (e.g., a level of IL-2 protein in a control blood sample from the subject) that is greater than or equal to the LLOQ, and a ratio of the level of the IL-2 protein to the baseline level of IL-2 protein that is greater than or equal to a threshold value.
  • a level of IL-2 protein e.g., a level of IL-2 protein in a blood sample obtained from the subject after the subject has been administered a composition comprising gluten peptides
  • a LLOQ e.g., 0.5 pg/mL
  • the threshold value is between 1.5 and 10, e.g., 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10.
  • the threshold value is 2.
  • the treatment is Nexvax2.
  • the treatment is a composition comprising gluten peptides, such as comprising any one of the compositions provided in U.S. Patent No. 8,835,603 and U.S. Patent No. 9,464,120.
  • a level of IL-2 protein that is greater than or equal to a LLOQ for the assay indicates that a treatment for Celiac disease is or is likely ineffective and a level of IL-2 protein that is less than the LLOQ indicates that the treatment is or is likely effective.
  • a level of IL-2 protein that is greater than or equal to the LLOQ indicates that the subject requires further testing to determine whether the treatment is effective and the method further comprises comparing the level of IL-2 protein to a baseline level of IL-2 protein in a control blood sample from the subject.
  • Control samples are described elsewhere herein.
  • the comparing comprises: (d) providing the control blood sample from a subject, wherein the subject has not been administered a composition comprising gluten peptides prior to the control blood sample being obtained from the subject; (e) measuring a baseline level of IL-2 protein in the control blood sample using the assay; and (f) comparing the level of IL-2 protein to the baseline level of IL-2 protein.
  • a level of IL-2 protein that is greater than or equal to the LLOQ, a baseline level of IL-2 protein that is greater than or equal to the LLOQ and a ratio of the level of the IL-2 protein to the baseline level of IL-2 protein being less than a threshold value indicates an equivocal result and/or that the subject requires additional testing (e.g., additional testing such as re-performing a method as described herein for assessing IL-2 protein levels in the subject (e.g., according to any one of the above-mentioned embodiments or any other embodiments described herein), performing HLA-DQ genotyping, and/or performing a gluten challenge for 2 or more weeks followed by endoscopic duodenal biopsy and/or serology specific for transglutaminase-2 or deamidated gliadin peptide); (ii) a level of IL-2 protein that is greater than or equal to the LLOQ, a baseline level of IL-2 protein that is greater than or equal to the L
  • the threshold value is between 1.5 and 10, e.g., 1.5, 1.6, 1.7. 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10. In some embodiments, the threshold value is 2.
  • the subject is following a gluten free diet.
  • the method comprises treating, adjusting a treatment, suggesting a treatment, or giving information in regard to a treatment to the subject based on a level of IL-2.
  • the method comprises stopping, continuing, or increasing a treatment if the level of IL-2 protein is greater than or equal to a LLOQ for an assay.
  • the method comprises stopping, continuing or decreasing a treatment if the level of IL-2 protein is less than the LLOQ.
  • a level of IL-2 protein that is greater than or equal to the LLOQ indicates that the subject requires further testing to determine whether the treatment should be adjusted and the method further comprises comparing the level of IL-2 protein to a baseline level of IL-2 protein, e.g., as described above.
  • the treating or treatment comprises administration of a second composition comprising gluten peptides to the subject.
  • the second composition comprises 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 treatment further comprises administration of a third composition comprising gluten peptides to the subject.
  • the third 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 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.
  • 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, such as Nexvax2. Samples
  • 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 blood samples.
  • the sample comprises 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, 6, 7 or 8 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.
  • the sample is obtained, e.g., within 1 hour to 24 hours, within 1 hour to 8 hours, within 1 hour to 7 hours, within 1 hour to 6 hours, within 2 hours to 8 hours, within 2 hours to 7 hours, within 2 hours to 6 hours, within 2 hours to 4 hours, within 3 hours to 8 hours, within 3 hours to 7 hours, within 3 hours to 6 hours, within 3 hours to 5 hours, within 4 hours to 8 hours, within 4 hours to 7 hours, within 4 hours to 6 hours, within 5 hours to 6 hours, or within 3 to 4 hours of administration of the composition. In some embodiments of any one of the methods provided, the sample is obtained within 3 hours to 4 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).
  • the subject has a homozygous HLA-DQ2.5 genotype. In some embodiments, the subject has a non-homozygous HLA-DQ2.5 genotype, e.g., a heterozygous HLA-DQ2.5 genotype. In some embodiments, the subject is homozygous for HLA-DQA1*05, HLA-DQB1*02, or both HLA-DQA1*05 and HLA-DQB1*02. In some embodiments, the subject is not homozygous for HLA-DQA1*05, HLA-DQB1*02, or both HLA-DQA1*05 and HLA-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 DQB1 *02), HLA-DQ2.2 ( DQA1 *02 and DQB1 *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 (see, e.g., Dunckley.
  • PCR polymerase chain reaction
  • the subject is on a gluten-free diet.
  • the subject is a subject having been administered a treatment as described herein (e.g., Nexvax2).
  • the subject has been on a gluten-free diet for a period of time, e.g., at least one week, at least one month or at least one year.
  • 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.
  • 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:10.3390/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 (DQA1*05 and DQB1*02), DQ2.2 (DQA1*02 and DQB1*02) or DQ8 (DQA1*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).
  • a control level is the LLOQ of an assay.
  • the term“gluten peptide” includes any peptide comprising a sequence derived from, or encompassed within, one or more of gluten proteins, such as alpha (a), beta (b), g (g) and omega (w) 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 (e.g., wheat, rye, and/or barley and/or comprising one or more of gliadin, hordein, and secalin).
  • 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: 3), 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: 4), 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: 5), wherein the N-terminal glutamate is a pyroglutamate and the carboxyl group of the C-terminal glutamine is amidated
  • the composition comprising gluten
  • a gluten peptide may include one or more sequences of epitopes known to be recognized by a CD4+ T cell in a subject with Celiac disease, e.g., PELP (SEQ ID NO: 6), PELPY (SEQ ID NO: 7), QPELPYP (SEQ ID NO: 8), PQPELPY (SEQ ID NO: 9),
  • PELP SEQ ID NO: 6
  • PELPY SEQ ID NO: 7
  • QPELPYP SEQ ID NO: 8
  • PQPELPY SEQ ID NO: 9
  • FPQPELP (SEQ ID NO: 10), PELPYPQ (SEQ ID NO: 11), FPQPELPYP (SEQ ID NO: 12), PYPQPELPY (SEQ ID NO: 13), PFPQPELPY (SEQ ID NO: 14), PQPELPYPQ (SEQ ID NO: 15), PFPQPEQPF (SEQ ID NO: 16), PQPEQPFPW (SEQ ID NO: 17), PIPEQPQPY (SEQ ID NO: 18), EQPIPEQPQ (SEQ ID NO: 19), PQPELPYPQ (SEQ ID NO: 20), FRPEQPYPQ (SEQ ID NO: 21), PQQSFPEQQ (SEQ ID NO: 22), IQPEQPAQL (SEQ ID NO: 23), QQPEQPYPQ (SEQ ID NO: 24), SQPEQEFPQ (SEQ ID NO: 25), PQPEQEFPQ (SEQ ID NO: 26), QQPEQ
  • PFPQPEQPF (SEQ ID NO: 37), PQPEQPFPQ (SEQ ID NO: 38), PYPEQEEPF (SEQ ID NO: 39), PYPEQEQPF (SEQ ID NO: 40), PFSEQEQPV (SEQ ID NO: 41), EGSFQPSQE (SEQ ID NO: 42), EQPQQPFPQ (SEQ ID NO: 43), EQPQQPYPE (SEQ ID NO: 44), QQGYYPTSPQ (SEQ ID NO: 45), EGSFQPSQE (SEQ ID NO: 46), PQQSFPEQE (SEQ ID NO: 47), or QGYYPTSPQ (SEQ ID NO: 48) (see, e.g., Sollid LM, Qiao SW, Anderson RP, Gianfrani C, Koning F. Nomenclature and listing of celiac disease relevant gluten epitopes recognized by CD4+ T cells. Immunogenetics. 2012;64:455-60; PC
  • the composition comprising gluten peptides comprises at least one of: (i) a first peptide comprising the amino acid sequence PFPQPELPY (SEQ ID NO: 49) and PQPELPYPQ (SEQ ID NO:50), (ii) a second peptide comprising the amino acid sequence PFPQPEQPF (SEQ ID NO: 51) and PQPEQPFPW (SEQ ID NO: 52), and (iii) a third peptide comprising the amino acid sequence PIPEQPQPY (SEQ ID NO: 53).
  • the composition comprises at least one of: (i) a first peptide comprising the amino acid sequence PFPQPELPY (SEQ ID NO: 49) and PQPELPYPQ (SEQ ID NO: 50),
  • a second peptide comprising the amino acid sequence PFPQPEQPF (SEQ ID NO: 51) and PQPEQPFPW (SEQ ID NO: 52), or (iii) a third peptide comprising the amino acid sequence PIPEQPQPY (SEQ ID NO: 53) and EQPIPEQPQ (SEQ ID NO: 54).“First”,“second”, and “third” are not meant to imply an order of use or importance, unless specifically stated otherwise.
  • 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
  • kits comprises at least one agent for identifying or measuring levels of IL-2.
  • the kit comprises at least one agent for identifying or measuring levels of IL-2.
  • the agent is a binding partner for the IL-2. In some embodiments of any one of the kits provided, the agent is one that recognizes a binding partner for the IL-2.
  • the binding partner is any molecule that binds specifically to IL-2.
  • “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.
  • 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.
  • 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 kit further comprises a first and second binding partner for IL-2.
  • 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.
  • 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. 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 saline solution. In some embodiments, the kit further comprises one or more positive controls, e.g., one or more compositions comprising IL-2 at a known concentrations or levels.
  • 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 IL-2 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.
  • a double-blind gluten challenge consisting of a either 5 grams vital wheat gluten flour (Bob’s Red Mill Natural Foods Inc., Milwaukie OR), which was estimated to contain 3-grams gluten protein according to the Osbourne equation, or gluten-free fine-ground white rice flour (“McKenzies Rice Flour”, Ward McKenzies Pty Ltd, Australia) added to one serve (7 grams) gluten-free“Vitafresh Low Calorie Lime” or“Vitafresh Low Calorie Sweet Navel Orange” (Hansells Food Group, New Zealand) mixed together in 100 ml water consumed over 2 min.
  • Adverse events were recorded hourly and graded according to“The Common Terminology Criteria for Adverse Events (CTCAE) version 4.0” (FDA Guidance for Industry - Toxicity Grading Scale).
  • CCAE Cosmetic Terminology Criteria for Adverse Events
  • CeD PRO® CeD patient reported outcome questionnaire
  • Each hour a modified version of the CeD patient reported outcome questionnaire (CeD PRO®) was completed by participants to grade their worst experience for 11 symptoms over the previous one-hour graded on a whole number scale from 0 (none) to 10 (worst possible) (Leffler DA, Kelly CP, Green PH, Fedorak RN, DiMarino A, Perrow W, et al. Larazotide acetate for persistent symptoms of celiac disease despite a gluten-free diet: a randomized controlled trial. Gastroenterology.
  • IL-2 from serum was measured using electrochemiluminescence (ECL) assay kits (Meso Scale Discovery, Rockville, Maryland). Data were analyzed by Discovery Workbench 4.0. Calculated values lower than LLOQ, or values on the low end of the scale that were not achieved using the standard curves, were reported as equal to the LLOQ concentration.
  • ECL electrochemiluminescence
  • serum was also obtained from 25 healthy non-CeD patients on unrestricted diets that underwent the gluten challenge described above. Serum was obtained before (baseline) and at 4-hours after gluten challenge.
  • the IL-2 protein levels in all baseline sera for CeD patients on GLD were lower than LLOQ for the ECL assay (0.5 pg/mL) and 23 of 25 the CeD patients on GLD were greater than or equal to the LLOQ in the sera obtained 4 hours after gluten challenge.
  • the serum IL-2 levels were less than LLOQ at baseline and at 4 hours post gluten challenge.
  • a serum IL-2 level lower than the LLOQ for the ECL assay (0.5 pg/mL) at 3-4 hours post gluten challenge is consistent with a patient not having Celiac disease.
  • a serum IL-2 level greater or equal to the LLOQ at 3-4 hours post gluten challenge may indicate that the subject requires additional testing to confirm Celiac disease diagnosis.
  • the serum IL-2 level from the baseline sample may be informative to further make the determination.
  • a serum IL-2 level greater or equal to the LLOQ at 3-4 hours post gluten challenge and a baseline IL-2 level lower than the LLOQ is consistent with the subject having Celiac disease.
  • a serum IL-2 level greater or equal to the LLOQ at 3-4 hours post gluten challenge and a baseline IL-2 level greater or equal to the LLOQ may be equivocal if the fold change in IL-2 (IL-2 at 3-4 hours post gluten challenge over baseline) is less than 2.
  • additional testing may be appropriate, e.g., redoing the gluten challenge and remeasuring IL- 2, and/or performing HLA-DQ genotyping, and/or performing a gluten challenge for 2 or more weeks followed by endoscopic duodenal biopsy and/or serology specific for transglutaminase-2 or deamidated gliadin peptide.
  • a serum IL-2 level greater or equal to the LLOQ at 3-4 hours post gluten challenge and a baseline IL-2 lower than the LLOQ is likely consistent with Celiac disease, if the fold change in IL-2 (IL-2 at 3-4 hours post gluten challenge over baseline) is greater than or equal to 2.

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Abstract

L'invention concerne des procédés comprenant des procédés d'identification d'un sujet ayant, suspecté d'avoir ou présentant un risque de souffrir d'une maladie coeliaque, comprenant la détermination d'un niveau d'IL-2 dans un échantillon provenant d'un sujet.
PCT/US2020/024890 2019-03-26 2020-03-26 Utilisation d'il-2 pour le diagnostic d'une maladie coeliaque Ceased WO2020198434A1 (fr)

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US20170097346A1 (en) * 2014-04-24 2017-04-06 Immusant, Inc. Use of interleukin-2 for diagnosis of celiac disease

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US20170097346A1 (en) * 2014-04-24 2017-04-06 Immusant, Inc. Use of interleukin-2 for diagnosis of celiac disease

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Title
JASON A TYE-DIN; DZURIS JOHN L; RUSSELL AMY K; WANG SUYUE; GOLDSTEIN KAELA; WILLIAMS LESLIE J; ANDERSON ROBERT P: "Serum IL -2 and IL -8 are Elevated within 4 h after Gluten Ingestion in Celiac Disease (CED) Patients on Gluten-Free Diet (GFD) and Potential to Resolve Indeterminate Diagnoses for Patients on GFD", GASTROENTEROLOGY, vol. 152, no. 5, 30 April 2017 (2017-04-30), pages S114, XP055430715, ISSN: 0016-5085, DOI: 10.1016/S0016-5085(17)30720-5 *
MANUEL PENEDO-PITA , JAVIER PETEIRO-CARTELLE: "Increased Serum Levels of Interleukin-2 and Soluble Interleukin-2 Receptor in Celiac Disease", JOURNAL OF PEDIATRIC GASTROENTEROLOGY AND NUTRITION, vol. 12, no. 1, 1 January 1991 (1991-01-01), pages 56 - 60, XP055744375, DOI: 10.1097/00005176-199101000-00012 *

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