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WO2018047068A1 - Peptide utile dans le diagnostic différentiel de la rectocolite hémorragique - Google Patents

Peptide utile dans le diagnostic différentiel de la rectocolite hémorragique Download PDF

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Publication number
WO2018047068A1
WO2018047068A1 PCT/IB2017/055356 IB2017055356W WO2018047068A1 WO 2018047068 A1 WO2018047068 A1 WO 2018047068A1 IB 2017055356 W IB2017055356 W IB 2017055356W WO 2018047068 A1 WO2018047068 A1 WO 2018047068A1
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WIPO (PCT)
Prior art keywords
peptide
ulcerative colitis
seq
patients
sequence
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Ceased
Application number
PCT/IB2017/055356
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English (en)
Inventor
Margherita Morpurgo
Giacomo Carlo STURNIOLO
Sonia FACCHIN
Liboria DIGIGLIO
Renata D'INCA'
Andrea Buda
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Universita degli Studi di Padova
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Universita degli Studi di Padova
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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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • 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
    • 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

  • the present invention relates to a peptide that is able to selectively bind to biopsies of inflamed intestinal tissue taken from patients affected by ulcerative colitis, for the differential diagnosis of this pathology, thus allowing to distinguish it from Crohn's disease.
  • a method for diagnosing ulcerative colitis, and a diagnostic kit are also described.
  • IBD Inflammatory bowel disease
  • UC ulcerative colitis
  • CD Crohn's disease
  • Ulcerative colitis essentially affects the rectum, sometimes it can extend, partially or totally, to the colon, while it never affects other parts of the intestine.
  • the mucosa of the area(s) affected by UC is inflamed and ulcerated, and this results in the onset of all the characteristic symptoms.
  • IBD is an idiopathic disease and, as such, its origin is not uniquely determined. Causes are sought in the living environment, genetic make-up, intestinal microbiota composition, and organism immune response.
  • UC and CD The differential diagnosis of UC and CD is based on clinical laboratory, radiological, endoscopic, and histological evidence. However, due to histologic overlapping features in 10-15% of patients (especially pediatric patients (4)) and a similar localization of the inflammation, the differentiation between these two pathologies is not always certain. In addition, currently available genetic or serological markers do not provide conclusive information: at a genetic level, 163 genetic loci associated with IBD have been identified, of which only 23 are specific for UC, and 30 for CD (1 ), and it was observed that different genetic factors predispose to IBD through different inflammatory mechanisms (2, 3).
  • Serological testing to search for ASCA (5) and pANCA (6) antibodies are also characterized by low specificity and sensitivity (7).
  • other recently identified serologic markers e.g. anti-pancreatic antibodies (8, 9) anti-erythrocyte antibodies (10), anti- endothelial cell antibodies (1 1 )
  • the low specificity of the currently available serological markers means that, to date, there is no universally recognized diagnostic test for the differential diagnosis of either CD or UC.
  • the aim of the present invention is to provide a method suitable for rapid and early differential diagnosis of either CD or UC, useful to avoid the risk of prescribing inappropriate treatments, thus causing health issues to the patient, and increasing the costs for the treatment of the disease.
  • the invention therefore concerns the use of a peptide having the sequence of SEQ ID NO.1 as a biological marker.
  • the inventors have surprisingly found that such a protein enables the diagnosis of patients affected by ulcerative colitis distinguishing these patients from those affected by Crohn's disease or various types of intestinal inflammation.
  • the peptide according to the present invention is able to selectively bind to biopsies of inflamed intestinal tissue taken from patients affected by UC, but not from CD, or non-IBD inflammation, supplementing the currently available diagnostic tools for IBD pathologies, especially when the current diagnostic approaches are not able to distinguish between the two pathologies.
  • the present invention also relates to a method for the diagnosis of ulcerative colitis, comprising the steps of:
  • step c. contacting the sample of step a. with the substrate of step b.; and d. detecting the reaction signal, wherein when a signal is detected, ulcerative colitis is identified.
  • the invention relates to a diagnostic kit comprising the peptide having the sequence of SEQ ID NO.1 , acting as an antigen, and a reagent for separate, simultaneous, and subsequent use for the diagnosis of ulcerative colitis.
  • UC ulcerative colitis
  • colitis ulcerosa ulcerative rectocolitis
  • ulcerative rectocolitis are meant to include the chronic inflammatory disease that primarily affects the rectum, and may involve part or all of the colon. The causes of this inflammation are still unknown. The main clinical symptoms are diarrhea, often with blood and mucus, and abdominal pain;
  • CD Crohn's disease
  • Crohn's disease means a chronic inflammatory disease that can affect the entire gastrointestinal tract. causes are still unknown. It is characterized by intestinal ulcers, often alternating with healthy intestinal tracts, and, if not properly treated, can lead to complications, such as stenoses or fistulas, that may require surgery. Symptoms may vary from abdominal pain, to chronic diarrhea, weight loss, or low-grade fever. It may also affect the anal region with fistulas or abscesses. In most cases, immunosuppressive therapy and regular monitoring enable to control the disease and its progression.
  • Figure 1 shows the graph of the differential affinity results of the phage carrying the peptide of SEQ ID NO: 1 (WPANTLK peptide): UC-ph, in mucosa samples of patients affected by various inflammatory pathologies of the colon.
  • CDSR Crohn's disease in remission Sigma
  • CDSI Crohn's disease Inflammed Sigma
  • CDIr Crohn's disease inflamed Ileum
  • CDII ulcerative colitis in remission
  • UCR ulcerative colitis in remission
  • UI ulcerative colitis in remission
  • UI ulcerative colitis in remission
  • UI ulcerative colitis in remission
  • UI ulcerative colitis
  • Figure 2A Scrambled and Synthetic UC Peptide (SEQ ID NO 1 : WPANTLK),
  • Figure 2B Multimerization process using colloidal poly-avidin nanoparticles (Ananas nanotech),
  • Figure 2C Diagram of the procedure followed for the bioaffinity experiment on a biopsy from a patient affected by UC, followed by ELISA quantification.
  • Figure 3 shows the graph of the effective S/P values obtained by performing the ELISA test with a derivative of the CD-pep peptide (SEQ ID NO: 1 : WPANTLK) multimerized on ANANAS nanoparticles, and performing the test on mucosa of patients (total 67), affected by UC in an inflamed state (21 ) or in remission (15), CD in an inflamed state (12) or in remission (15), and non-IBD intestinal inflammatory disease (4).
  • FIG. 4 Confocal fluorescence microscopy (40X) of biopsies of UC inflamed mucosa (4A, 4B, and 4C) treated with a derivative of UC-pep peptide (SEQ ID NO 1 : WPANTLK), multimerized on ANANAS nanoparticles.
  • the box 4D represents a magnification (64X) of the area in the box of panel 4C.
  • the peptide of SEQ ID NO:1 is present on the phage, and in the diagnostic test, in a cyclic form, given the presence of two cysteines at the head and tail of the sequence.
  • the recognition specificity for the inflamed mucosa of patients affected by UC was then validated by using the isolated phage UC-ph, and counting the number of phages that bind to the mucosal biopsies of patients affected by different types of intestinal inflammation.
  • the invention therefore concerns a peptide having the sequence of SEQ ID NO.1 , or a fluorescent derivative thereof, for use as a biological marker.
  • the inventors have surprisingly found that such a protein enables the diagnoses of patients affected by ulcerative colitis, distinguishing such patients form those affected by
  • IBD Inflammatory Bowel Diseases
  • UC ulcerative colitis
  • CD Crohn's disease
  • This invention relates to the identification of a peptide sequence being for the first time able to selectively bind to biopsies of inflamed intestinal tissue taken from patients affected by UC (SEQ ID NO:1 : WPANTLK), but not by CD or non-IBD inflammation, and the use of this peptide or derivatives thereof in diagnostic testing for the the rapid and selective diagnosis of this pathology.
  • this method could supplement currently available diagnostic tools for IBD pathologies, especially when the current diagnostic approaches are not able to distinguish between the two pathologies.
  • the peptide according to the present invention is therefore a biological marker to distinguish between patients affected by ulcerative colitis and patients affected by Crohn's diseases.
  • the present invention also relates to a method for the diagnosis of ulcerative colitis, comprising the steps of:
  • step c. contacting the sample of step a. with the substrate of step b.; and d. detecting the reaction signal, wherein when a signal is detected, ulcerative colitis is identified.
  • the method according to the invention enables to distinguish patients affected by ulcerative colitis from those affected by Crohn's disease, or different types of intestinal inflammation.
  • said peptide having the sequence of SEQ ID NO.1 is immobilized on the surface of the solid support of step b.
  • the peptide according to the present invention is able to selectively bind to biopsies of inflamed intestinal tissue taken from patients affected by UC, but not by CD or non-IBD inflammation, supplementing the currently available diagnostic tools for IBD pathologies, especially when the current diagnostic approaches are not able to distinguish between the two pathologies.
  • Such a solid support allows the multimerization of the peptide, and it is selected from the group consisting of nanoparticles, polyfunctionalizable polymers, or by adsorption on plastic surfaces (e.g. polystyrene).
  • the detection step d. is carried out by a technique selected from the group consisting of ELISA assay, radioimmunoassays (RIA), immunoassays, preferably Western blot and LINE blot, assays with protein or DNA microarrays, and surface plasmon resonance detection (SPR).
  • a technique selected from the group consisting of ELISA assay, radioimmunoassays (RIA), immunoassays, preferably Western blot and LINE blot, assays with protein or DNA microarrays, and surface plasmon resonance detection (SPR).
  • said biological sample of step a. is a biopsy, preferably an intestinal tissue biopsy, more preferably a biopsy of inflamed intestinal tissue, or patient's serum.
  • the biopsy may, for example, derive from the colon mucosa, or the pouch region of operated patients.
  • the invention relates to a diagnostic kit comprising the peptide having the sequence of SEQ ID NO.1 , acting as an antigen, and a reagent for separate, simultaneous, and subsequent use for the diagnosis of ulcerative colitis.
  • said peptide having the sequence of SEQ ID NO:1 is immobilized on a solid support, preferably on a nanoparticle, or a polyfuctionalized colloidal support.
  • Mucosal biopsies were taken from the colon or ileum of patients affected by intestinal inflammatory pathologies of different types, after informed consent. The patients were subjected to colonoscopy evaluation or, when scheduled, they underwent surgery.
  • the biopsies were stored in DMEM at 4°C, and used within 2 hours.
  • the biopsies were incubated with 4x10 10 CFU of CD-ph suspended in 1 imL of PBS buffer, under constant mild stirring. After 30 minutes, the biopsies were subjected to subsequent washing cycles (1 x PBST + 4x PBS) and dried by aspiration. 100 ⁇ _ of 0,2M glycine-HCI buffer, pH 2.2, were then added to the biopsies, and after 5 minutes the solution was aspirated and immediately neutralized with 15 ⁇ _ of 1 M TRIS buffer, pH 9.1 .
  • the bacteriophage content in the neutralized solution was measured by inoculating different dilutions of E.co// ' -agar suspension in a Petri dish, and counting the number of lysis plaques formed after 16h of incubation at 37°C.
  • Figure 1 shows the average of the values obtained carrying out the test on a cohort of different patients.
  • Mucosal biopsies taken from the colon or ileum of patients with confirmed diagnosis of intestinal inflammatory pathologies of different types were incubated with a derivative of CD-pep, or a peptide having the same amino acid composition but amino acids arranged in a different order (scrambled peptide -SCR-pep) immobilized on the surface of colloidal poly-avidin nanoparticles (ANANAS nanotech, PD, Italy) through a biotin linker (30% of biotin binding sites saturated, 100 ⁇ g/mL in avidin) suspended in 1 imL of PBS buffer, under constant mild stirring.
  • ANANAS nanotech colloidal poly-avidin nanoparticles
  • the biopsies were subjected to subsequent washing cycles (1 x PBST + 4x PBS), and then dried by aspiration. PBS percentage and ANANAS concentration may be varied, without compromising the assay functioning.
  • the nanoparticles affinity-bound to the biopsies were removed by acidic treatment with 100 ⁇ _ of 2M glycine buffer, pH 2.0, and after 5 minutes the acidic solution was transferred into a second test tube, and immediately neutralized with 15 ⁇ _ of 1 M TRIS buffer, pH 9.1 .
  • the nanoparticles content in the various washing solutions, and in the detachment solution, was obtained through an ELISA test.
  • the solutions were incubated in microwells of an ELISA plate (50 ⁇ _ ⁇ / ⁇ ), pre-conditioned so as to expose immobilized biotin on the surface. After 3 hours incubation at RT, the microwells were washed extensively, and added with 2 ⁇ g/mL (50 ⁇ _/ ⁇ ) of biotin-HRP (ANANAS nanotech, PD, Italy). After 30 minutes, the biotin-HRP solution was removed, and the plate developed with a solution of tetramethylbenzidine (TMB), blocking the enzymatic reaction after 20 minutes by addition of 0.5M H2SO4.
  • TMB tetramethylbenzidine
  • the absorbance intensity generated in each well was measured spectrophotometrically at 450 nm, and compared to that generated in the well of the respective positive control, in which the corresponding nano- assembled ANANAS-peptide was incubated at a known concentration.
  • the absorbance data were processed according to the following formula:
  • - S/P Scrambled corresponds to the same value generated by performing the test on a second biopsy with a peptide having the same amino acid composition, but whose amino acids have been aligned in inverted order.
  • Figure 2 shows the sequences of the biotinylated peptides used in the test, the multimerization procedure using ANANAS particles, and the test operation scheme based on the use of multimerized synthetic peptides combined with the ELISA method.
  • Figure 3 summarizes the effective S/P values obtained by performing the test with UC-pep e CD-pep peptides on the mucosa of patients (67 in total) affected by UC in inflamed state (21 ) or in remission (15), CD in inflamed state ( 2) or in remission (15), and non-IBD inflammatory intestinal diseases(4).
  • Confocal fluorescence microscopy images were obtained from a series of UC samples, and from biopsies of normal mucosa, treated with the multimerized peptide in the same way as in the ELISA assays, and fixed before the acidic elution phase.
  • fluorescence is localized at the epithelial surface level, while no signal is observed at the lumen cells level, clearly indicating that targets are on the surface.

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  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
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  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
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  • Physics & Mathematics (AREA)
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  • General Physics & Mathematics (AREA)
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Abstract

L'invention concerne un peptide apte à se lier de façon sélective à des biopsies de tissus intestinaux enflammés prélevés sur des patients atteints de rectocolite hémorragique, pour le diagnostic différentiel de cette pathologie, ce qui permet de faire la distinction entre la rectocolite hémorragique et la maladie de Crohn. L'invention concerne également une méthode de diagnostic de la rectocolite hémorragique, ainsi qu'une trousse de diagnostic associée.
PCT/IB2017/055356 2016-09-06 2017-09-06 Peptide utile dans le diagnostic différentiel de la rectocolite hémorragique Ceased WO2018047068A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IT102016000090019 2016-09-06
IT102016000090019A IT201600090019A1 (it) 2016-09-06 2016-09-06 Peptide per la diagnosi differenziale della Rettocolite ulcerosa

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008008349A2 (fr) * 2006-07-11 2008-01-17 Drexel University Procédés d'évaluation quantitative d'une inflammation avec des nanoparticules pour la biodétection
WO2015011675A1 (fr) * 2013-07-26 2015-01-29 Ananas Nanotech S.R.L. Complexes nano-assemblés d'acides nucléiques, d'avidine et de composés biotinylés utilisables en tant que supports d'administration intracellulaire

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008008349A2 (fr) * 2006-07-11 2008-01-17 Drexel University Procédés d'évaluation quantitative d'une inflammation avec des nanoparticules pour la biodétection
WO2015011675A1 (fr) * 2013-07-26 2015-01-29 Ananas Nanotech S.R.L. Complexes nano-assemblés d'acides nucléiques, d'avidine et de composés biotinylés utilisables en tant que supports d'administration intracellulaire

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
IBRAHIM MASOOD ET AL: "Biomarkers in the management of ulcerative colitis: a brief review Biomarker bei der Versorgung der Colitis ulcerosa: ein kurzer Überblick", GERMAN MEDICAL SCIENCE, 1 January 2011 (2011-01-01), pages 1 - 7, XP055377868, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3046642/pdf/GMS-09-03.pdf> [retrieved on 20170601] *
MARGHERITA MORPURGO ET AL: "Detection of a fluorescent-labeled avidin-nucleic acid nanoassembly by confocal laser endomicroscopy in the microvasculature of chronically inflamed intestinal mucosa", INTERNATIONAL JOURNAL OF NANOMEDICINE, 2015, pages 399, XP055259596, DOI: 10.2147/IJN.S70153 *
SAITO H ET AL: "Isolation of peptides useful for differential diagnosis of Crohn's disease and ulcerative colitis", 20030401, vol. 52, no. 4, 1 April 2003 (2003-04-01), pages 535 - 540, XP009088009, ISSN: 0017-5749, DOI: 10.1136/GUT.52.4.535 *

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