WO2012126531A1 - Méthode de diagnostic du syndrome coronaire aigu (sca) - Google Patents

Méthode de diagnostic du syndrome coronaire aigu (sca) Download PDF

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Publication number
WO2012126531A1
WO2012126531A1 PCT/EP2011/054567 EP2011054567W WO2012126531A1 WO 2012126531 A1 WO2012126531 A1 WO 2012126531A1 EP 2011054567 W EP2011054567 W EP 2011054567W WO 2012126531 A1 WO2012126531 A1 WO 2012126531A1
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WO
WIPO (PCT)
Prior art keywords
biomarker
proteins
exosomes
fluid
coronary syndrome
Prior art date
Application number
PCT/EP2011/054567
Other languages
English (en)
Inventor
Dominique De Kleijn
Gerard Pasterkamp
Leonardus Timmers
Siu Kwan Sze
Original Assignee
Cavadis B.V.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cavadis B.V. filed Critical Cavadis B.V.
Priority to PCT/EP2011/054567 priority Critical patent/WO2012126531A1/fr
Priority to EP12722288.3A priority patent/EP2676141A2/fr
Priority to PCT/EP2012/000714 priority patent/WO2012110253A2/fr
Publication of WO2012126531A1 publication Critical patent/WO2012126531A1/fr

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Classifications

    • 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
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction

Definitions

  • the present invention relates to the diagnosis of acute ischemic coronary syndromes (ACS) .
  • the invention further relates to kits and biomarkers for use in the method .
  • Chest-pain can result from many causes: gastric discomfort (e.g.
  • indigestion indigestion
  • pulmonary distress pulmonary embolism
  • dyspnea pulmonary embolism
  • dyspnea pulmonary embolism
  • musculoskeletal pain pulled muscles, bruises
  • ACS acute ischemic coronary syndrome
  • Acute coronary syndrome is usually one of three diseases involving the coronary arteries: ST elevation myocardial infarction (30%) , non ST elevation myocardial infarction (25%) , or unstable angina (38%) . These types are named according to the appearance of the electrocardiogram (ECG/EKG) as non-ST segment elevation myocardial infarction (NSTEMI) and ST segment elevation myocardial infarction (STEMI).
  • ACS is usually associated with coronary thrombosis.
  • the physician has to decide if the patient is having a life threatening ischemic ACS or not.
  • rapid treatment by opening up the occluded coronary artery is essential to prevent further loss of myocardial tissue.
  • cardiac biomarkers have become an essential tool to define if a patient has a myocardial necrosis related to myocardial infarction.
  • Favorable features of biomarkers of necrosis are high concentrations in the myocardium and absence in non- myocardial tissue, release into the blood within a
  • ischemia is further defined by the addition of at least 1 of the following criteria: ischemic ST and T-wave changes, new left bundle-branch block, new Q waves, PCI-related marker elevation, or imaging showing a new loss of
  • troponins can be detected in blood as early as 2 to 4 h after the onset of symptoms, elevation can be delayed for up to 8 to 12 h. This timing of elevation is similar to that of Creatine Kinase-MB but persists longer, for up to 5 to 14 days.
  • the occlusion itself is often the result of a thrombotic event.
  • This atherosclerotic plaque rupture brings the thrombogenic content of the plaque in contact with the blood initiating thrombosis and subsequent occlusion.
  • microvesicles Next to this, the ischemic event immediately activates endothelial cells that attract platelets that also become activated. This activation of endothelial cells and platelets is accompanied by the release of microvesicles that are secreted into the blood.
  • vesicles are formed with a selection of lipids, protein and RNA from the secreting cell and are released as an intact vesicle. They are generally called microvesicles and have a size between 20 and 1000 nm. From these microvesicles, exosomes are the best described
  • particles having a size between 50 and 100 nm having a size between 50 and 100 nm.
  • apoptopic cardiomyocytes secrete vesicles in the blood that are called apoptopic bodies.
  • proteomic analyses were performed on human plasma samples.
  • the procedure was hampered, however, by the presence of high-abundant plasma proteins such as albumin and immune-globulins, which complicated the detection of potentially interesting low-abundant proteins. Therefore sub-fractions of plasma were investigated for the presence of proteins that may have diagnostic value for the
  • Protein secretion out of the cells can occur directly after production (constitutive pathway) or is first stored in the cell and released after a trigger (regulatory pathway) .
  • Secretion not only occurs via the above mentioned pathways with individual proteins but also occurs via vesicles containing a large number of proteins and RNA. These so-called microvesicles are formed with a selection of lipids, protein and RNA from the secreting cell and are released as an intact vesicle ranging in size between 20 and 1000 nm.
  • Vesicles in the size of 50-100 nm are called exosomes and the release of exosomes has been described for various cell types, including reticulocytes, B- and T- lymphocytes, dendritic cells, mast cells, platelets, macrophages and alveolar lung cells.
  • T cells T cells
  • platelets dendritic cells
  • mast cells secretion of exosomes is regulated by specific stimuli. While early studies focused on their secretion from diverse cell types in vitro,
  • exosomes have now been identified in body fluids such as urine, amniotic fluid, malignant ascites, broncho-alveolar lavage fluid, synovial fluid, breast milk, saliva and blood. Exosomes have a wide range of biological functions,
  • exosomes express an array of proteins that reflect the originating host cell and that they contain valuable information regarding ongoing (patho) physiologic processes in the human body including information on the occurrence of ACS.
  • the present invention provides a method for the diagnosis of ACS in a patient, based on the detection of particular proteins in, on or attached to exosomes, which proteins are herein after referred to as biomarkers or differentially present proteins.
  • the proteins can be detected either in or on or attached to isolated exosomes and in or on exosomes that are still present in a body fluid, in particular serum.
  • any biomarker with diagnostic value may be used.
  • specific markers were identified in/on plasma exosomes that have diagnostic value for ACS.
  • the invention thus provides a method for the diagnosis of ACS comprising detecting a biomarker in an exosome sample or micro-vesicles of smaller or larger size from said subject, wherein said sample comprises at least one protein selected from the group of 3 proteins consisting of: Serpin F2 (IPI : IPI00879231, SWISSPRO :A2AP_HUMAN) , CD14 ( I PI : I PI 00029260 ,
  • index numbers databases accessions
  • the referenced index numbers include reference to fragments, isoforms and modifications thereof, hence the present invention foresees the use of fragments of the proteins as well as modifications and derivatives of the proteins disclosed herein as biomarkers in the context of the various aspects of the present invention.
  • a biomarker comprises one protein or a set of multiple proteins. Such a biomarker is also identified herein as a profile or protein profile.
  • a profile may comprise 1, 2 or 3 of the proteins Serpin F2 (IPI : IPI00879231, SWISSPROT :A2AP_HUMAN) , CD14
  • IPI IPI00032293, SWISSPROT : CYTC_HUMAN
  • biomarker protein instead of detecting the complete biomarker protein, one may detect peptide fragments of said biomarker proteins which are derived from the biomarker proteins by fragmentation
  • peptide fragment refers to peptides having between 5 and 50 amino acids. These peptide fragments preferably provide a unique amino acid sequence of the protein, and are associated with the cardiovascular events as disclosed herein.
  • the proteins and/or peptide fragment may optionally be detected as chemically modified proteins and/or peptides, such chemical modification may for instance be selected from the group consisting of glycosylation, oxidation,
  • the biomarkers can be found in exosomes but also physically connected or linked to exosomes, which means both in or on their surface.
  • the biomarker can be either attached to the membrane, e.g. expressed on or in the membrane surface or anchored therein, or be in loose connection therewith, i.e. adhered to the exosome without being physically attached to or in the membrane.
  • biomarkers may also be part of the membrane.
  • Cystatin C is not attached to the membrane but rather adheres to it.
  • CD14 is anchored in the membrane, and Serpin F2 has been associated with the
  • the biomarkers that are attached, anchored or adhered to the exosome can also be detected in samples of body fluid, in particular in serum.
  • the biomarker protein or a peptide fragment thereof is detected in, on or attached to exosomes that are preferably found in body fluids like serum, plasma or blood.
  • Figure 1 Area under the curve analysis for Troponin (Trop, solid curve) measured in blood taken at intake of the patient with chest pain and Troponin plus Serpin F2
  • the Athero-Express is a longitudinal vascular biobank study, which includes biomaterials from patients undergoing carotid and femoral end-arterectomy in two Dutch hospitals (UMC Utrecht and St. Antonius Hospital).
  • Plasma and tissue samples were obtained from all patients before (blood) or during end-arterectomy.
  • Exosomes were isolated from frozen human plasma by filter separation followed by ultracentrifugation .
  • exosomal proteins such as CD9 and CD81 were detected in the exosome pellet using western blotting. FACS analysis with beads of defined sizes demonstrated that the pellet contains mostly particles of 50-100 nm which is in accordance with the size of exosomes.
  • the exosome pellets collected in the Athero-Express biobank plasma were after ultracentrifugation dissolved in 40 ⁇ 6% SDS in HPLC pure water. Plaque protein was, after grinding the plaque material without any blood remains to powder, also extracted with 6% SDS. Digestion and subsequent labeling, HPLC separation and mass spectrometry analysis was identical for plaque and exosome proteins. The protein content was determined by 2-D Quant Kits. After protein reduction and alkylation, the protein mixture was diluted 20 times with 50 mM triethylammonium bicarbonate (TEAB) and protein digestion was initiated by adding trypsin in a 1:40 trypsin-to-protein ratio. The protein digests were desalted using a Sep-Pak C18 cartridge and dried in a Speedvac.
  • TEAB triethylammonium bicarbonate
  • digests were labeled with iTRAQ reagents according to the manufacturer's protocol. Briefly, digested proteins were reconstituted in 30 ⁇ of dissociation buffer and mixed with 70 ⁇ of ethanol-suspended iTRAQ reagents (one iTRAQ reporter tag per protein sample, mass tag 114-117 Dalton) . Labeling reactions were carried out at RT for 1 hr before all the samples were mixed into a single tube and dried using a Speedvac.
  • the dried fraction was re-constituted in 100 ⁇ of 0.1% formic acid. Each sample was analyzed two times using a Q-Star Elite mass spectrometer, coupled to an online
  • Quantitative proteomics were performed on exosomes from 50 patients that suffered an ACS during follow up
  • Quantitative data were available from 2 pooled events samples (Group 1 in duplo) and 2 pooled control samples (Group 2 in duplo) . Based on pilots, it was determined that a ratio of 1.2 and above means that there is significantly higher level of the protein in the event while a ratio of 0.8 and lower is a significant lower level in the event. First selection was based on proteins with identical duplo' s (both below 0.8, both above 1.2 or both between 0.8 and 1.2).
  • Second selection was based on proteins with lower (events/controls ⁇ 0.8) or higher (events/controls >1.2) expression in group 1 vs. group 2. This revealed a list of 116 proteins.
  • Athero-Express cohort 40 carotid end- arterectomy patients were selected of which 20 had a
  • Quantitative proteomics was performed on plaque samples as for the exosome proteomics. However, since 40 individual plaques were analyzed, four plaque extracts were run simultaneously each differently labeled by the iTraq reagent (114, 115, 116, 117 resp.) . Each run consisted of two plaque extracts of patients that suffered a
  • an excel file was generated containing the protein ID and the relative value of the two event/control pairs for each of the protein IDs.
  • the plaque is the origin of atherosclerotic disease leading to cardiovascular events. For this, it is very likely that plaque proteins related to future cardiovascular events can also be found in exosomes especially the plaque proteins that are related to the pathways over-represented in exosome proteins that differ between patients suffering from a cardiovascular events and healthy controls. Having established that 3 canonical pathways (acute phase,
  • Selection was based on the presence of proteins that are related to the 3 atherosclerosis related canonical pathways and for which 2 antibodies and a recombinant protein were available.
  • markers were selected based on over-representation of 3
  • 34 proteins were chosen for Luminex bead assay development. For 17 proteins out of those 34 proteins (including Cystatin C, Serpin F2 and CD14), a reproducible and quantitative Luminex bead assay was set up that could be used for measuring the protein content in exosomes isolated from individual serum samples.
  • Biomarkers will be retrospectively tested after defrosting of deep frozen blood taken on presentation .
  • QICS will investigate whether a combined use of specific symptoms and signs, electrocardiography, routine and new laboratory measures, adjunctive imaging including electron beam (EBT) computed tomography (CT) and contrast multi-slice CT (MSCT) will have a high diagnostic yield for patients with acute chest pain.
  • EBT electron beam
  • CT computed tomography
  • MSCT contrast multi-slice CT
  • Cystatin C, Serpin F2 and CD14 were measured using Luminex multiplex technology on/in or attached to exosomes that were isolated with ExoquickTM from 250 ul of serum of individual QICS patients.
  • ROC analyses were performed to determine the ability of the marker, in conjunction with a risk score, to
  • Serpin F2 (238 samples) showed a p-value of p ⁇ 0.001 between ACS and non-ACS while CD14 gave a p-value of 0.002 (238 samples)
  • Serpin F2 The strongest marker Serpin F2 was analyzed to see if it had additional value in diagnosing acute coronary syndrome on top of Troponin levels measured at the intake of the patient.
  • ROC curves ( Figure 1) show that the area under the curve increases from 0.835 for Troponin alone to 0.881 for Troponin plus Serpin F2.
  • Serpin F2 plus the maximum levels of troponin measured (Tropmax) is significantly different from Tropmax alone and Serpin F2 plus High sensitive (Hs ) - Troponin is also significantly different from Hs-Troponin alone showing the added value of Serpin F2.
  • test result variable (s) predicted probability trop has at least one tie between the positive actual state group and the negative actual state group.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
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Abstract

L'invention concerne une méthode de diagnostic de la survenue d'un syndrome coronaire aigu chez un sujet, comprenant une étape consistant à déterminer la présence d'un biomarqueur révélateur de la survenue d'un syndrome coronaire aigu dans un échantillon d'exosomes prélevé chez le sujet. Ledit échantillon d'exosomes est constitué d'exosomes isolés à partir d'un liquide organique, ledit liquide organique contenant des exosomes et étant choisi parmi le sérum, le plasma, le sang, l'urine, le liquide amniotique, les ascites malignes, le liquide de lavage broncho-alvéolaire, le liquide synovial, le lait de femme, la salive et, tout particulièrement, le sérum.
PCT/EP2011/054567 2011-02-18 2011-03-24 Méthode de diagnostic du syndrome coronaire aigu (sca) WO2012126531A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
PCT/EP2011/054567 WO2012126531A1 (fr) 2011-03-24 2011-03-24 Méthode de diagnostic du syndrome coronaire aigu (sca)
EP12722288.3A EP2676141A2 (fr) 2011-02-18 2012-02-17 Biomarqueurs exosomaux pour la prédiction d'événements cardiovasculaires
PCT/EP2012/000714 WO2012110253A2 (fr) 2011-02-18 2012-02-17 Biomarqueurs exosomaux pour des événements cardiovasculaires

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PCT/EP2011/054567 WO2012126531A1 (fr) 2011-03-24 2011-03-24 Méthode de diagnostic du syndrome coronaire aigu (sca)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105849554A (zh) * 2013-10-24 2016-08-10 新加坡科技研究局 利用低分子量有机两性离子的外泌体回收方法
US10500231B2 (en) 2013-03-13 2019-12-10 University Of Miami Method for isolation and purification of microvesicles from cell culture supernatants and biological fluids
CN111965366A (zh) * 2020-07-09 2020-11-20 何凤屏 一种唾液快速检测外泌体肌钙蛋白的方法及试剂盒
EP3577214A4 (fr) * 2017-02-06 2021-03-24 Goetzl, Edward, J. Exosomes dérivés cellules endothéliales et leurs utilisations

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10500231B2 (en) 2013-03-13 2019-12-10 University Of Miami Method for isolation and purification of microvesicles from cell culture supernatants and biological fluids
EP3677271A1 (fr) 2013-03-13 2020-07-08 University Of Miami Procédé pour isoler et purifier les microvésicules de surnageants de culture cellulaire et fluides biologiques
EP4218774A1 (fr) 2013-03-13 2023-08-02 University Of Miami Procédé d'isolement et de purification de microvésicules de surnageant de culture cellulaire et de fluides biologiques
US11730768B2 (en) 2013-03-13 2023-08-22 University Of Miami Method for isolation and purification of microvesicles from cell culture supernatants and biological fluids
CN105849554A (zh) * 2013-10-24 2016-08-10 新加坡科技研究局 利用低分子量有机两性离子的外泌体回收方法
EP3577214A4 (fr) * 2017-02-06 2021-03-24 Goetzl, Edward, J. Exosomes dérivés cellules endothéliales et leurs utilisations
CN111965366A (zh) * 2020-07-09 2020-11-20 何凤屏 一种唾液快速检测外泌体肌钙蛋白的方法及试剂盒

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