WO2018220106A1 - Molécules de biomarqueurs pour la sarcopénie et leurs utilisations - Google Patents

Molécules de biomarqueurs pour la sarcopénie et leurs utilisations Download PDF

Info

Publication number
WO2018220106A1
WO2018220106A1 PCT/EP2018/064328 EP2018064328W WO2018220106A1 WO 2018220106 A1 WO2018220106 A1 WO 2018220106A1 EP 2018064328 W EP2018064328 W EP 2018064328W WO 2018220106 A1 WO2018220106 A1 WO 2018220106A1
Authority
WO
WIPO (PCT)
Prior art keywords
molecule
sarcopenia
seq
protein
sample
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/EP2018/064328
Other languages
English (en)
Inventor
Yves Henrotin
Anne Cornet
Benoit Boulanger
Céline PIRLOT
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Artialis SA
Original Assignee
Artialis SA
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 Artialis SA filed Critical Artialis SA
Publication of WO2018220106A1 publication Critical patent/WO2018220106A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

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
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96402—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from non-mammals
    • G01N2333/96405—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from non-mammals in general
    • G01N2333/96408—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from non-mammals in general with EC number
    • G01N2333/96416—Aspartic endopeptidases (3.4.23)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/70—Mechanisms involved in disease identification
    • G01N2800/7042—Aging, e.g. cellular aging

Definitions

  • the present invention relates to the identification of novel protein biomarkers for the detection and monitoring of sarcopenia in humans. It additionally describes methods and devices to assay said biomarker molecules in human samples.
  • Sarcopenia is a recently identified syndrome linked to loss of muscular strength, function, and mass (Cruz-Jentoft AJ, et al. Age Ageing 2010; 39: 412-23.; Sayer AA, et al. Age Ageing 2013; 42: 145- 150). It is classified as a pathology since 2016.
  • Sarcopenia is linked with aging, and according to WHO reports on aging and lifestyle, it can be estimated that sarcopenia currently affects over 50 million people worldwide and is likely to affect ca. 200 million individuals in 40 years' time. Sarcopenia affects only skeletal muscle and then, appears without chronic disease.
  • sarcopenia concerns the loss of muscle mass and function associated with aging.
  • a detailed review of the two conditions can be found in the publication by AN and Garcia (Gerontology, 2014; 60(4), p.294-305) who report that sarcopenia assessment should include muscle mass study (by methods such as DEXA), muscle strength study (handgrip strength), and muscle function study (e.g. gait speed over 6 meters) (see Table 2 in Ali and Garcia).
  • the present invention provides novel biomarker molecules of sarcopenia which are quantifiable in readily available biological fluids, especially in serum samples.
  • the present invention additionally discloses a method for the diagnosis or prognosis of a subject suspected to have or having sarcopenia.
  • the present invention also concerns a method for determining the efficacy of a treatment for sarcopenia in a patient.
  • the invention also provides a compound for use in the quantification of a novel biomarker molecule of the invention.
  • the invention proposes a kit for practicing the diagnostic/prognostic method of the invention.
  • the invention additionally discloses a method of raising antibodies to a biomarker molecule of the invention for use in the methods and kits of the invention. Detailed description of the invention
  • Sarcopenia is a syndrome linked to loss of muscular strength, function, and mass.
  • “Sarcopenia”, as used herein, requires the presence of low muscle mass and either low muscular strength or low physical performance, as further clinically defined by the European Working Group on Sarcopenia in Older People (EWGSOP; Cruz-Jentoft AJ, et al. Age Ageing 2010; 39: 412-23) and further requires that the subject does not have any underlying disease which could affect muscle mass or muscular strength.
  • “sarcopenia”, as used herein, does not include patients suffering from cachexia, from cancer, from renal diseases, from liver diseases, from chronic inflammatory disease, or from an infectious disease. Patients suffering from sarcopenia may, however, concomitantly suffer from osteoporosis.
  • treatment for sarcopenia refers to an approach to limit, reduce or suppress the symptoms generated by sarcopenia.
  • These approaches comprise but are not limited to: physical exercises and training, diet (modification of protein intake, food supplementation, vitamin D%) or drugs (for example targeting molecules involved in mitochondrial regulation or in myokines signalling, selective androgen receptor modulator (SARMs), growth hormones, myostatin inhibitors, ).
  • SARMs selective androgen receptor modulator
  • growth hormones myostatin inhibitors
  • prepro Cathepsin D refers to the full length sequence of the unprocessed protein which comprises 412 amino acids. This full length sequence is presented herein as Seq ID N°1. It is referenced as P07339 in the UniProt knowledgebase (www.uniprot.org). Both prepro Cathepsin D and pro Cathepsin D (after removal of amino acids 1-20 of Seq ID N°1 which correspond to the secretion signal peptide) are inactive forms of the protein devoid of enzymatic activity, with a molecular weight greater than 48kDa.
  • Pro Cathepsin D is processed into an "active intermediate form" (of about 48 kDa, corresponding to amino acids 65 to 412 of Seq ID N°1 ).
  • the active intermediate form is transformed into the mature active form composed of both a heavy and a light chain.
  • the heavy chain has a theoretical molecular weight of 34kDa and may be detected by Western Blot at a weight of from about 25 to about 34 kDa. It corresponds to amino acids 169 to 412 of Seq ID N°1 while the light chain has a molecular weight of about 14 kDa and corresponds to amino acids 65 to 162 of Seq ID N°1.
  • non-mature Cathepsin D protein thus includes the following forms of the protein: pro Cathepsin D (inactive form) as well as the active intermediate form of the protein.
  • mature active Cathepsin D protein thus refers to the heavy and the light chain obtained after processing of the active intermediate form.
  • active Cathepsin D protein refers to both the mature active Cathepsin D protein and the active intermediate form of the protein.
  • Biological fluid refers to blood, plasma, serum, synovial fluid, a cell culture supernatant fluid, or a cell extract.
  • sarcopenia status refers to any distinguishable manifestation of sarcopenia, as defined above.
  • sarcopenia status includes, without limitation, the presence or absence of sarcopenia in a subject, the risk of a subject developing sarcopenia, the stage of the disease, the progression of the disease, and the effectiveness or response of a patient to an intervention for the prevention or the treatment of sarcopenia.
  • biomarker molecule refers to a molecule selected from the peptides of Seq ID N° 2, 3, 4, 5, 6 and 7, the non-mature Cathepsin D protein, and fragments thereof.
  • These molecules include the non-mature Cathepsin D protein and fragments thereof, such as those of Seq ID N°2 to 21.
  • Preferred fragments include the tryptic fragments of Seq ID N° 2, 3, 4, 5, 6 and 7.
  • the concentration of the biomarker molecules of the invention in a sample may be determined by suitable assays using the information herein provided.
  • a suitable assay may include one or more of the following methods: an enzyme assay, an immunoassay, mass spectrometry, HPLC (including nano 2D UPLC such as using a nanoAcquity device from Waters, www.waters.com), electrophoresis or an antibody microarray, or any combination thereof.
  • an immunoassay may be an enzyme linked immunoassay (ELISA), a sandwich assay, a competitive assay, a radioimmunoassay, a Western Blot, an immunoassay using a biosensor, an immunoprecipitation assay, an agglutination assay, a turbidity assay or a nephelometric assay.
  • mass spectrometry it may be Matrix Assisted Laser Desorption/lonization Time-of-Flight (MALDI TOF), liquid chromatography quadruple ion trap electrospray (LCQ-MS), or surface enhanced laser desorption ionization/time of flight (SELDI TOF) mass spectrometry.
  • MALDI TOF Matrix Assisted Laser Desorption/lonization Time-of-Flight
  • LCQ-MS liquid chromatography quadruple ion trap electrospray
  • SELDI TOF surface enhanced laser desorption ionization/time of flight
  • One preferred method of detection is the use of an immunoassay, utilizing an antibody, which binds to an epitope on non-mature Cathepsin D protein or on fragments thereof.
  • Assay forms in which such an antibody can be applied include, but are not limited to, ELISA, microarray, radioimmunoassay (RIA), fluorescence activated cell sorting (FACS), Western blotting, chromatography, immunofluorescence and histochemistry.
  • MRM Multiple Reaction Monitoring
  • SRM Selected Reaction Monitoring
  • MRM is a highly sensitive and selective method for the targeted quantitation of protein/peptide abundances in complex biological samples.
  • SRM Selected Reaction Monitoring
  • MRM is highly selective (targeted), allowing the skilled person to fine tune an instrument to specifically look for peptides, or protein fragments, of interest.
  • MS techniques for biomarker quantitation can be found in the published literature, such as in Lemoine et al., 2012 ("The current status of clinical proteomics and the use of MRM and MRM 3 for biomarker validation", Expert Rev. Mol. Diagn. , 12(4), pages 333-342), in Calderon-Celis et al., 2017 ("Standardization approaches in absolute quantitative proteomics with mass spectrometry", Mass Spec Rev. 2017; pages 1-23) or in Boja et al., 2014 (“Analytical Validation Considerations of Multiplex Mass-Spectrometry-Based Proteomic Platforms for Measuring Protein Biomarkers", J. Proteome Res.; 13; pages 5325-5332).
  • the invention provides a method for detecting the amount of non-mature Cathepsin D protein or fragments thereof in biological fluids.
  • a biological fluid sample is contacted with a binding agent (as further defined herein) specific towards an epitope within the amino acid sequence of non-mature Cathepsin D protein, essentially all non-mature Cathepsin D protein or fragments thereof in the sample containing this epitope will be bound by such a binding agent.
  • the amount of protein or fragments thereof bound by the binding agent will be detected by methods which the skilled artisan will develop using the information herein provided.
  • the epitope bound by binding agents reactive with non-mature Cathepsin D protein or fragments thereof may comprise five or more amino acids.
  • the present invention provides a method of determining the sarcopenia diagnosis of a subject, comprising the steps of:
  • step (i) determining the concentration(s) in a biological sample of one or more molecule(s) selected from the peptides of Seq ID N°2 to 7, the non-mature Cathepsin D protein, and fragments thereof; (ii) comparing the molecule concentration(s) determined in step (i) with one or more reference values, wherein an increase in the concentration(s) of the molecule(s) compared to the reference values is diagnostic of sarcopenia.
  • reference values refer to the biomarker levels that can be measured in samples from control subjects (i.e. not suffering from sarcopenia) by a skilled person, using the same sample type, purification method, and analytical method as those used to determine the sarcopenia status of a subject according to a method of the invention.
  • fragment is intended to refer to a polypeptide, a peptide or otherwise, released from mammalian non-mature Cathepsin D protein by an oxidative or enzymatic processing .
  • a fragment is different from the non-mature Cathepsin D protein by its structure and configuration and may undergo modifications such as phosphorylation, glycosylation or any other post-translational modification including, but not limited to, nitration, chlorination, sumoylation, ubiquitylation and glycation.
  • the fragment according to the present invention contributes to the identification of the sarcopenia status of the patient.
  • the present invention also provides a method of determining the prognosis of a patient with sarcopenia comprising the steps of:
  • the present invention also provides a method of determining the prognosis of sarcopenia incidence or progression in a subject comprising the steps of:
  • the present invention further provides a method of determining the efficacy of a treatment for sarcopenia in a patient, comprising the steps of:
  • step (iv) comparing the molecule(s) concentrations determined in step (i) and step (iii) with one another, and optionally with one or more reference value(s), wherein a stable concentration or a decreasing concentration of said molecule(s) indicates an efficacious treatment.
  • the treatment for sarcopenia may include, by way of non-limiting examples, physical exercises and training, diet (modification of protein intake, food supplementation, vitamin D%) or drugs (for example targeting molecules involved in mitochondrial regulation or in myokines signalling, selective androgen receptor modulator (SARMs), growth hormones, myostatin inhibitors, ).
  • drugs for example targeting molecules involved in mitochondrial regulation or in myokines signalling, selective androgen receptor modulator (SARMs), growth hormones, myostatin inhibitors, .
  • SARMs selective androgen receptor modulator
  • Other approaches for the treatment of sarcopenia are described in JE Morley from 2016 ("Pharmacologic Options for the Treatment of Sarcopenia", Calcif. Tissue Int. , 98(4), p.319-33).
  • the present inventors have shown in the Examples that the levels of the one or more molecule(s) selected from the peptides of Seq ID N°2 to 7, the non-mature Cathepsin D protein and fragments thereof, are correlated with the sarcopenia status of the patient, the skilled person will directly understand that the efficacy of a given treatment for sarcopenia may be monitored by following the concentration in a biological sample of said molecule(s) over time as the treatment is taking place.
  • a decrease in concentration of one or more of said molecule(s) of 5% or more, of 10% or more, of 20% or more, or of 30% or more, compared to the concentration of said molecule(s) before initiation of the treatment, or at an earlier treatment time is indicative of efficacy of said treatment.
  • Efficacy of a treatment may be monitored by analysing samples taken from a patient at various time points following initiation of the treatment. By monitoring changes in the concentration of the biomarker molecule(s) over time and comparing these concentrations to normal and/or reference values, efficacy of the treatment may be determined.
  • reference concentration(s) may include the concentration of the biomarker molecule in the patient when a sample was first taken and analysed, or the concentration of the biomarker molecule in the patient when a sample was last taken, or both.
  • the present invention additionally provides a method of treating a patient suffering from sarcopenia, comprising the steps of: (i) determining the concentration in a biological sample of one or more molecule(s) selected from the peptides of Seq ID N°2 to 7, the non-mature Cathepsin D protein and fragments thereof;
  • step (iv) comparing the molecule(s) concentrations determined in step (i) and step (iii) with one another, and optionally with one or more reference value(s);
  • step (v) adjusting the treatment regimen according to the result of the comparison of step (iv);
  • the treatment regimen is increased when the molecule level(s) are stable or increasing.
  • the treatment regimen is increased refers to a situation where the administered treatment is considered not efficacious enough, and a stronger treatment needs to be administered.
  • the treatment comprises physical exercise, more intense and/or more frequent physical exercise is instructed.
  • the present invention also provides a means of selecting a subject for treatment, comprising the steps of:
  • step (ii) comparing the molecule concentration(s) determined in step (i) with one or more reference values, wherein an increase in the concentration(s) of the molecule(s) compared to the reference values is indicative that said subject has to be selected for treatment.
  • the protein, or fragment, sequence, detected comprise a number of modifications, such as post-translational modifications, and/or appear in a non-native form, such as an unfolded form.
  • the biological sample to be assessed is first processed with trypsin, and the protein fragments to be detected are proteolytic fragments, such as those of Seq ID N°2 to 7.
  • the present invention thus provides a method of detecting a molecule selected from the peptides of Seq ID N°2 to 7, the non-mature Cathepsin D protein and fragments thereof, wherein the method uses a purposely-designed immunoassay or a specific mass spectrometry assay, such as an MRM- based assay.
  • Any disclosed method may be used in conjunction with the assessment of clinical symptoms and/or imaging results and/or the concentration of one or more other biomarkers.
  • the method of the invention is carried out in vitro.
  • the reference value, to which the determined concentration of the biomarker molecule is compared is the concentration of the same molecule in one or more "normal" subjects that do not have any detectable sarcopenia, or any clinical symptoms of sarcopenia, and have so called "normal values" of the biomarker molecule.
  • the reference value may be a previous value for the biomarker molecule obtained from a specific subject. This kind of reference value may be used if the method is to be used to monitor progression of sarcopenia, or to monitor the response of a patient to a particular treatment.
  • an increase in the concentration of the biomarker molecule may be indicative of the sarcopenia status in the subject.
  • an increase in the concentration of the biomarker molecule may be indicative, or diagnostic, of sarcopenia in the subject.
  • an at least about 5%, at least about 10%, at least about 20%, at least about 30% at least about 50%, at least about 70% increase in the concentration of a biomarker molecule according to the invention in a sample from a subject compared to a reference sample from a normal subject is diagnostic of sarcopenia.
  • an increase in the concentration of a biomarker molecule according to the invention of 2 fold or more in a sample from a subject compared to a reference sample from a normal subject is diagnostic of sarcopenia.
  • binding agent refers to any molecule capable of specifically binding a biomarker molecule of the invention.
  • binding agent includes, but is not limited to, aptamers (both DNA and peptide aptamers), affimers, synthetic binding proteins (such as polypeptides having a specific domain of protein A as a backbone - affibodiesTM), antibodies or any fragment derived thereof, such as Fab, Fab' and F(ab')2, Fd, single-chain Fvs, single-chain antibodies, disulfide- linked Fvs and fragments comprising either a VT or VFI domain, a heavy chain antibody, a single domain antibody, a minibody, the variable domain derived from camelid heavy chain antibodies, the variable domain of the new antigen receptors derived from shark antibodies, a protein scaffold including an alphabody, protein A, protein G, designed ankyrin-repeat domains, fibronectin type III repeats, anticalins, knottin
  • the biomarker molecule or an immunogenic fragment thereof, is used as an antigen for immunisation.
  • the peptide is emulsified in an adjuvant medium, preferably incomplete Freund's adjuvant and injected subcutaneously or into the peritoneal cavity of a mammalian host, preferably a rodent, more preferably a rabbit, even more preferably a mouse.
  • an adjuvant medium preferably incomplete Freund's adjuvant
  • injected subcutaneously or into the peritoneal cavity of a mammalian host preferably a rodent, more preferably a rabbit, even more preferably a mouse.
  • a carrier protein to enhance immunogenic properties of the antigenic peptide.
  • Useful carriers are proteins such as keyhole limpet hemocyanin (KLH), edestin, albumins, such as bovine or human serum albumin (BSA or HSA), tetanus toxoid, and cholera toxoid, polyaminoacids, such as poly-(D-lysine-D-glutamic acid).
  • KLH keyhole limpet hemocyanin
  • edestin albumins
  • BSA or HSA bovine or human serum albumin
  • tetanus toxoid such as bovine or human serum albumin (BSA or HSA)
  • tetanus toxoid such as bovine or human serum albumin (BSA or HSA)
  • tetanus toxoid such as bovine or human serum albumin (BSA or HSA)
  • tetanus toxoid such as bovine or human serum albumin (BSA or HSA)
  • Antisera are subsequently screened for their ability to bind an epitope within the biomarker molecule sequence. Antisera from the most promising hosts may be used in their crude form or purified.
  • Monoclonal antibodies may be generated from immunised mice or other animals with the most promising antibody titre, by fusing lymphocytes isolated from the spleen or nodes of these mice or animals with a myeloma cell line.
  • the generated hybridoma clones are screened for antibodies with reactivity toward an epitope within the biomarker molecule sequence, and cell lines can be established for production and purification of monoclonal antibodies.
  • Antibodies may be synthetic, monoclonal, polyclonal, oligoclonal, bispecific, chimeric and/or humanised.
  • the antibody may be complete or a fragment thereof, such as, Fv, Fab and F(ab)2 fragments.
  • binding agents may be selected from existing libraries of binding agents by known techniques such as, e.g. phage display.
  • One or more of the binding agents used may comprise a tag or a label, such as a radioactive, fluorescent, chemiluminescent tag or a label, a dye, an enzyme, or a histidine tag or label, or any other suitable label or tag known in the art.
  • Mammalian body fluids such as blood, plasma, serum, synovial fluid, and subcutaneous interstitial fluid, as well as extracts from cells or tissues or supernatants from cells or tissues cultured in vitro may be used in the methods of the invention.
  • the biological fluid used is serum.
  • the biological fluid may be used as it is, or it may be purified prior to the quantification step.
  • This purification step may be accomplished using a number of standard procedures, including but not limited to, cartridge adsorption and elution, molecular sieve chromatography, dialysis, ion exchange, alumina chromatography, hydroxyapatite chromatography, and combinations thereof.
  • a suitable purification protocol is illustrated in Example 2.
  • the biological fluids to be used for measuring biomarkers levels are first prepared by depletion of the most abundant proteins before they are analysed. Preparation methods include but are not limited to: protein depletion methods, methods to dissociate protein complexes as they can occur in the biological fluid, etc... as these methods are performed by the skilled artisan using the detailed information provided herein.
  • the biological sample may be depleted in Human Serum Albumin using a commercial kit, such as the Pierce Albumin Depletion kit (catalog number: 85160; Thermo Fisher Scientific, 168 Third Avenue, Waltham, MA USA 02451 ).
  • the biological sample may be depleted using the ProteoPrep® Blue Albumin & IgG Depletion Kit (Sigma-Aldrich, 3050 Spruce Street, St. Louis, MO 63103 USA).
  • the biological sample may be depleted in the most abundant proteins using the "ProteoPrep20 Plasma Immunodepletion kit" from Sigma (http://www.sigmaaldrich.com/, Catalog No: PROT20).
  • One embodiment of the present invention constitutes a diagnostic kit for use in detection and/or monitoring of sarcopenia.
  • This includes a binding agent recognizing an epitope comprised in the sequence of a biomarker molecule of the invention.
  • antibodies of the present invention either alone or with a second antibody with specificity towards the first antibody or another part of the epitope-containing biomarker molecule.
  • the kit can be applied on mammalian body fluids or extracts of cells or tissues, preferably derived from humans.
  • a peptide between 6 and 20 amino acids, in which a succession of amino acids is equivalent to the binding epitope for one of said antibodies might be supplied either in a labelled or non-labelled form.
  • the non-mature Cathepsin D protein, or fragments thereof is supplied in a labelled or non-labelled form in the kit.
  • the peptide, the non-mature Cathepsin D protein or fragments thereof may be obtained by a synthetic or a recombinant route or a cell-free system.
  • the antibodies may be labelled by joining them, either covalently or non-covalently, with a reporter molecule. Suitable reporter molecules or labels, which may be used for ease of detection, include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • One of the non-labelled antibodies or a peptide of the kit might be immobilised, preferably on a solid surface like a microtiter plate, possibly by conjugation to a suitable protein carrier like BSA.
  • the kit comprises at least one agent for determining the concentration of a biomarker molecule according to the invention, in a biological sample.
  • the agent may be an enzyme, an antibody, a protein probe, a metabolite or any other suitable composition.
  • the agent for determining the concentration of the biomarker molecule is preferably labelled.
  • the kit may also comprise means for detecting the label.
  • the kit may comprise one or more capture agents for capturing the biomarker molecule in a biological sample.
  • the capture agent may be one or more antibodies.
  • the kit may comprise two antibodies for use in a sandwich assay to determine the concentration of a biomarker molecule.
  • the kit comprises two antibodies, each directed to a different epitope on the biomarker molecule.
  • One antibody is preferably the capture antibody, and the other antibody is preferably a detection antibody and may be labelled to allow its detection.
  • the capture agent may be attached to a solid support.
  • the solid support may be a chip, a microtiter plate, a bead or a resin.
  • the kit may comprise instructions for suitable operational parameters in the form of a label or separate insert.
  • the instructions may inform a user about how to collect the sample, and/or how to wash the capture agent.
  • the kit may comprise samples of the biomarker molecule to be detected, for example in the form of a synthetic peptide or polypeptide.
  • the samples of the biomarker molecule may be used as a standard for calibration and comparison and may optionally be labelled.
  • the kit may also comprise instructions to compare the concentration of the biomarker molecule detected in a sample with a calibration sample or chart.
  • the kit may also include instructions indicating what concentration of the biomarker molecule is diagnostic of sarcopenia.
  • Example 1 Recruitment of sarcopenia patients and healthy controls
  • Pairs of subjects were matched between the control group and the sarcopenia group in order to eliminate individual differences based on sex (same sex) and age (the age difference between the matched subjects could not exceed 10 years).
  • MNA Mini Nutritional Assessment
  • a blood sample of approximately 29 mL and a urine sample of approximately 10 mL were taken from each enrolled patient. It was decided to proceed with the proteomic analysis in Example 2 below on the basis of samples from female patients. A total of 10 samples in the sarcopenia group (7 from subgroup 4 and 3 from subgroup 3) and of 10 samples in the control group (6 from subgroup 1 and 4 from subgroup 2) were used.
  • Proteomic analysis was performed with serum samples collected in BD Vacutainer serum tubes (ref. 368815).
  • the samples were reduced, alkylated and reduced, and processed using the 2D- Clean up kit (GE Healthcare LifeSciences www.gelifesciences.com, Product code 80-6484-51 ) according to the manufacturer's instructions.
  • the protein pellets after the washing steps were further resolubilized in bicarbonate ammonium 50 mM.
  • the samples were digested in solution with trypsin (16 hours at 37°C with a trypsin/total proteins ratio (w/w) of 1/10, followed by 3 hours at 37°C with a ratio of 1/20 in 80% ACN). The reaction was stopped by addition of trifluoroacetic acid. The samples were evaporated to dryness in a speed vacuum.
  • a volume of 9 ⁇ _ per sample, corresponding to 0.6 ⁇ g of digested proteins was injected on the nano 2D UPLC - Orbitrap MS System.
  • the analyses were performed on a nano-UPLC (nanoAcguiXy, Waters) - ESI-Q-Orbitrap (Q Exactive, Thermo), in positive ion mode.
  • the LC method was a 2D LC method with 3 steps of 180 minutes.
  • the 3 steps are made on the column at high pH with increasing percentage of acetonitrile and the peptides eluted from the column at high pH are loaded after dilution on the low pH column.
  • the mass spectrometer method is a TopN-MSMS method where N was set to 12, meaning that the spectrometer acquires one Full MS spectrum, selects the 12 most intense peaks in this spectrum (singly charged precursors excluded) and makes a Full MS2 spectrum of each of these 12 compounds.
  • MS spectrum acquisition Mass range from 400 to 1750 w/z, Resolution of 70000, AGC target of 1 e6 or Maximum injection time of 200 ms.
  • the parameters for MS2 spectrum acquisition are: Isolation Window of 1.6 w/z, Collision energy (NCE) of 25, Resolution of 17500, AGC target of 1 e5 or Maximum injection time of 50 ms.
  • Protein identifications are considered significant if proteins are identified with at least 2 peptides per protein taking into account only an FDR ⁇ 0.01 (False Discovery Rate).
  • the Perseus software (available at http://www.perseus-framework.or , and described in Cox, J and Mann, M., BMC Bioinformatics, 2012, 13 Suppl 16:S12; and in Tyanova, S. et al. Nature Methods, 2016) was used to determine the ratio of the protein expression level in the sarcopenia group to the protein expression level in the control group and to calculate the attached statistical significance (p- value).
  • Cathepsin D a 412 amino acids long protein with a UniprotKB (http://www.uniprot.org/) accession number of P07339, was identified through six most represented tryptic sequences which are listed below as Seq ID 2 to 7.
  • Seq ID N°6 was detected in 9 out of 10 samples from the sarcopenia group and in 5 out of 10 samples from the control group. It was quantified at a 4.3 higher level in the serum of the sarcopenia group compared to the level in the serum of the control group.
  • the peptide of Seq ID N°5 has a particular status since it was never detected in the serum of control subjects, but was identified in 7 (out of 10) samples from the sarcopenia group.
  • the peptide of Seq ID N°3 finally, was detected in 10 out of 10 samples from the sarcopenia group and in 9 out of 10 samples from the control group. It was quantified at a 2.2 higher level in the serum of sarcopenia patients compared to the level in the serum of control patients.
  • Cathepsin D protein represents a useful biomarker to detect in serum samples a condition of sarcopenia, in the absence of cachexia and other underlying diseases, such as cancer.
  • Example 3 Confirmatory Elisa experiments for biomarker molecules identified in Example 2
  • Cathepsin D is an acid protease active in intracellular protein breakdown. It is synthesized as "prepro-cathepsin D” as a 412 amino acids chain, with a signal peptide of 20 amino acids which, upon cleavage in the endoplasmic reticulum yields pro-cathepsin D.
  • the primary sequence of Cathepsin D is prone to many post-translational modifications.
  • an active chain of pro-cathepsin D is formed (MW of 48 kDa), which is in turn transformed into the mature form comprising a heavy chain (34 kDa) and a light chain (14 kDa).
  • Cathepsin D levels were first assessed in the 20 samples from Example 1 using a commercial ELISA test (Abeam, 330 Cambridge Science Park, Cambridge, CB4 0FL, UK, product code: #ab213470), using a 10-times dilution of the samples.
  • Example 4 Confirmatory Western Blot experiments for biomarkers identified in Example 2
  • the secondary antibody was obtained from Jackson Immunoresearch (Jackson ImmunoResearch Europe Ltd., Unit 7, Acorn Business Centre, Oaks Drive - Newmarket, Suffolk, CB8 7SY, UK, product GAR-HRP) and was used at a 1/5000 dilution.
  • Example 5 Confirmatory Western Blot experiments for biomarkers identified in Example 2
  • the primary antibody was obtained from Abeam (330 Cambridge Science Park, Cambridge, CB4 0FL, UK, product code: #ab75852, as used herein, "Abeam antibody #ab75852”) and was used at a 1/1000 dilution.
  • a preadsorbed against human serum protein secondary antibody was selected and obtained from Abeam (Abeam PLC, 330 Cambridge Science Park, Cambridge, UK., product code: ab97080) and used at a 1/10000 dilution.

Landscapes

  • 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)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne l'identification de nouveaux biomarqueurs protéiques pour la détection et la surveillance de la sarcopénie chez l'être humain, en l'absence de toute maladie sous-jacente susceptible d'affecter la masse musculaire ou la force musculaire. Il est en particulier démontré que des formes spécifiques de la protéine cathepsine D non mature sont significativement surexprimées chez les patients atteints de sarcopénie. La présente invention concerne également des procédés et des dispositifs pour doser lesdites molécules de biomarqueurs dans des échantillons humains et fournir des moyens destinés à générer des agents de liaison contre lesdites molécules de biomarqueurs.
PCT/EP2018/064328 2017-05-31 2018-05-31 Molécules de biomarqueurs pour la sarcopénie et leurs utilisations Ceased WO2018220106A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP17173773.7 2017-05-31
EP17173773 2017-05-31

Publications (1)

Publication Number Publication Date
WO2018220106A1 true WO2018220106A1 (fr) 2018-12-06

Family

ID=59030767

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2018/064328 Ceased WO2018220106A1 (fr) 2017-05-31 2018-05-31 Molécules de biomarqueurs pour la sarcopénie et leurs utilisations

Country Status (1)

Country Link
WO (1) WO2018220106A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2713905C1 (ru) * 2019-10-29 2020-02-11 Федеральное государственное бюджетное образовательное учреждение высшего образования "Смоленский государственный медицинский университет" министерства здравоохранения Российской Федерации Способ диагностики саркопении у лиц пожилого и старческого возраста
GR1010870B (el) * 2023-12-14 2025-02-06 Εθνικο Και Καποδιστριακο Πανεπιστημιο Αθηνων, Πεπτιδια-αναλογα της καθεψινης d ως θεραπευτικοι παραγοντες στη νοσο του alzheimer
RU2836305C1 (ru) * 2024-06-07 2025-03-12 Федеральное государственное бюджетное образовательное учреждение высшего образования "Санкт-Петербургский государственный педиатрический медицинский университет" Министерства здравоохранения Российской Федерации (ФГБОУ ВО СПбГПМУ Минздрава России) Способ лабораторной диагностики саркопении у маломобильных пациентов детского возраста

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000061636A2 (fr) * 1999-04-14 2000-10-19 Research Corporation Technologies, Inc. Anticorps glycosyles de façon aberrante a valeur pronostique et diagnostique pour le cancer
WO2005029088A2 (fr) * 2003-09-20 2005-03-31 Electrophoretics Limited Methode pour diagnostiquer des troubles associes a une lesion cerebrale
WO2010015659A1 (fr) * 2008-08-07 2010-02-11 Proteomika, S.L. Marqueurs du cancer et procédés permettant de les détecter
CN101735317A (zh) * 2008-11-25 2010-06-16 中国科学院北京基因组研究所 Cathepsin D抗原多肽、应用及含有该多肽的检测试剂盒
CN101885758B (zh) * 2009-05-11 2014-04-30 中国医学科学院肿瘤研究所 一种自身抗原表位、其编码基因及其用途
WO2015111008A2 (fr) 2014-01-27 2015-07-30 Novartis Ag Biomarqueurs prédictifs de l'atrophie musculaire, procédé et utilisation associés
US20160154006A1 (en) * 2013-06-07 2016-06-02 Pierce Biotechnology, Inc. Absolute Quantitation of Proteins and Protein Modifications by Mass Spectrometry with Multiplexed Internal Standards

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000061636A2 (fr) * 1999-04-14 2000-10-19 Research Corporation Technologies, Inc. Anticorps glycosyles de façon aberrante a valeur pronostique et diagnostique pour le cancer
WO2005029088A2 (fr) * 2003-09-20 2005-03-31 Electrophoretics Limited Methode pour diagnostiquer des troubles associes a une lesion cerebrale
WO2010015659A1 (fr) * 2008-08-07 2010-02-11 Proteomika, S.L. Marqueurs du cancer et procédés permettant de les détecter
CN101735317A (zh) * 2008-11-25 2010-06-16 中国科学院北京基因组研究所 Cathepsin D抗原多肽、应用及含有该多肽的检测试剂盒
CN101885758B (zh) * 2009-05-11 2014-04-30 中国医学科学院肿瘤研究所 一种自身抗原表位、其编码基因及其用途
US20160154006A1 (en) * 2013-06-07 2016-06-02 Pierce Biotechnology, Inc. Absolute Quantitation of Proteins and Protein Modifications by Mass Spectrometry with Multiplexed Internal Standards
WO2015111008A2 (fr) 2014-01-27 2015-07-30 Novartis Ag Biomarqueurs prédictifs de l'atrophie musculaire, procédé et utilisation associés

Non-Patent Citations (23)

* Cited by examiner, † Cited by third party
Title
"UniprotKB", Database accession no. P07339
ABCAM: "ab119586- Cathepsin D human ELISA kit", 6 May 2016 (2016-05-06), pages 1 - 24, XP055396915, Retrieved from the Internet <URL:http://www.abcam.com/ps/products/119/ab119586/documents/ab119586 Cathepsin D Human ELISA Kit v4 (website).pdf> [retrieved on 20170808] *
ABCAM: "Product datasheet: Anti-Cathepsin D antibody [EPR3057Y] ab75852", 1 April 2017 (2017-04-01), XP055397030, Retrieved from the Internet <URL:http://www.abcam.com/Cathepsin-D-antibody-EPR3057Y-ab75852.pdf> [retrieved on 20170808] *
ALI; GARCIA, GERONTOLOGY, vol. 60, no. 4, 2014, pages 294 - 305
BOJA ET AL.: "Analytical Validation Considerations of Multiplex Mass-Spectrometry-Based Proteomic Platforms for Measuring Protein Biomarkers", J. PROTEOME RES., vol. 13, 2014, pages 5325 - 5332
BROTTO M: "Lessons from the FNIH-NIA-FDA sarcopenia consensus summit", IBMS BONEKEY, vol. 9, 2012, pages 210
CALDERON-CELIS ET AL.: "Standardization approaches in absolute quantitative proteomics with mass spectrometry", MASS SPEC REV., 2017, pages 1 - 23
CESARI ET AL., J. CACHEXIA SARCOPENIA MUSCLE, vol. 3, 2012, pages 181 - 90
COX ET AL., NAT. BIOTECHNOL., vol. 26, 2008, pages 1367 - 72
COX, J; MANN, M., BMC BIOINFORMATICS, vol. 13, no. 16, 2012, pages 12
CRUZ-JENTOFT AJ ET AL., AGE AGEING, vol. 39, 2010, pages 412 - 23
CURCIO FRANCESCO ET AL: "Biomarkers in sarcopenia: A multifactorial approach", EXPERIMENTAL GERONTOLOGY, vol. 85, no. 8962, 12 September 2016 (2016-09-12), pages 1 - 8, XP029794755, ISSN: 0531-5565, DOI: 10.1016/J.EXGER.2016.09.007 *
JE MORLEY: "Pharmacologic Options for the Treatment of Sarcopenia", CALCIF. TISSUE INT., vol. 98, no. 4, 2016, pages 319 - 33, XP035868655, DOI: doi:10.1007/s00223-015-0022-5
KALINKOVICH ALEXANDER ET AL: "Sarcopenia - The search for emerging biomar", AGEING RESEARCH REVIEWS, vol. 22, 8 May 2015 (2015-05-08), pages 58 - 71, XP029167929, ISSN: 1568-1637, DOI: 10.1016/J.ARR.2015.05.001 *
KOLASKAR AS; TONGAONKAR PC, FEBS LETT., vol. 276, no. 1-2, 10 December 1990 (1990-12-10), pages 172 - 4
LEMOINE ET AL.: "The current status of clinical proteomics and the use of MRM and MRM3 for biomarker validation", EXPERT REV. MOL. DIAGN., vol. 12, no. 4, 2012, pages 333 - 342
MÁSA MARTIN ET AL: "Cathepsin D propeptide: mechanism and regulation of its interaction with the catalytic core", BIOCHEMI, AMERICAN CHEMICAL SOCIETY, US, vol. 45, no. 51, 26 December 2006 (2006-12-26), pages 15474 - 15482, XP008130324, ISSN: 0006-2960, [retrieved on 20061208], DOI: 10.1021/BI0614986 *
PAHOR ET AL., THE JOURNAL OF NUTRITION HEALTH AND AGING, vol. 13, 2009, pages 8
RICCARDO CALVANI ET AL: "Biomarkers for physical frailty and sarcopenia: state of the science and future developments : Biomarkers for physical frailty and sarcopenia", JOURNAL OF CACHEXIA, SARCOPENIA AND MUSCLE DEC 2013, vol. 6, no. 4, 7 July 2015 (2015-07-07), pages 278 - 286, XP055397401, ISSN: 2190-5991, DOI: 10.1002/jcsm.12051 *
SAYER AA ET AL., AGE AGEING, vol. 42, 2013, pages 145 - 150
SCHARF G ET AL., J CACHEXIA SARCOPENIA MUSCLE, vol. 3, 2012, pages 145 - 8
SUMBUL ALI ET AL: "Sarcopenia, Cachexia and Aging: Diagnosis, Mechanisms and Therapeutic Options - A Mini-Review", GERONTOLOGY, vol. 60, no. 4, 1 January 2014 (2014-01-01), CH, pages 294 - 305, XP055249215, ISSN: 0304-324X, DOI: 10.1159/000356760 *
TYANOVA, S. ET AL., NATURE METHODS, 2016

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2713905C1 (ru) * 2019-10-29 2020-02-11 Федеральное государственное бюджетное образовательное учреждение высшего образования "Смоленский государственный медицинский университет" министерства здравоохранения Российской Федерации Способ диагностики саркопении у лиц пожилого и старческого возраста
GR1010870B (el) * 2023-12-14 2025-02-06 Εθνικο Και Καποδιστριακο Πανεπιστημιο Αθηνων, Πεπτιδια-αναλογα της καθεψινης d ως θεραπευτικοι παραγοντες στη νοσο του alzheimer
WO2025125071A1 (fr) * 2023-12-14 2025-06-19 National And Kapodistrian University Of Athens Peptides de cathepsine d utilisés en tant qu'agents thérapeutiques dans la maladie d'alzheimer
RU2836305C1 (ru) * 2024-06-07 2025-03-12 Федеральное государственное бюджетное образовательное учреждение высшего образования "Санкт-Петербургский государственный педиатрический медицинский университет" Министерства здравоохранения Российской Федерации (ФГБОУ ВО СПбГПМУ Минздрава России) Способ лабораторной диагностики саркопении у маломобильных пациентов детского возраста

Similar Documents

Publication Publication Date Title
US10145853B2 (en) Biomarkers for non-alcoholic fatty liver disease, and methods for detecting non-alcoholic fatty liver disease by using such biomarkers
US20210341492A1 (en) Newborn screening for primary immunodeficiencies, cystinosis, and wilson disease
US20190100576A1 (en) Signal biomarkers
WO2018220106A1 (fr) Molécules de biomarqueurs pour la sarcopénie et leurs utilisations
JP5156997B2 (ja) Ｉｖ型コラーゲン様免疫活性ペプチド
JP6392762B2 (ja) 女性対象において癌になるリスクの予測を補助するための方法
CA2748852C (fr) Biomarqueurs associes a la nephropathie
WO2018220108A1 (fr) Molécules de biomarqueurs pour la sarcopénie et leurs utilisations
EP2859354B1 (fr) Troponine i cardiaque nitrée en tant que biomarqueur d&#39;ischémie cardiaque
JP6755649B2 (ja) 虚血性疾患の診断マーカー
WO2015158608A2 (fr) Diagnostic d&#39;une néphropathie chronique par analyse quantitative de modifications post-translationnelles des protéines plasmatiques
AU2014351090A1 (en) Biomarker for cardiac disorders
JP2014181967A (ja) 中枢神経ループス（ｎｐｓｌｅ）診断用バイオマーカー
JP7336097B2 (ja) 乳がんに関するペプチドマーカー
US8012694B2 (en) Assay for the detection of phosphorylated PTH
JP2024117521A (ja) 膵臓がんの検出方法
JP2016148623A (ja) 大腸がんの検出方法
JP5924659B2 (ja) 免疫複合体の網羅的解析方法および新規関節リウマチバイオマーカー
JP2013003066A (ja) Ｄｍｐ１の測定方法
JPWO2016013400A1 (ja) 肺癌診断用バイオマーカー
KR20190050054A (ko) 심혈관 질환 조기 진단용 조성물, 이를 포함하는 진단키트, 및 심혈관 질환 조기 진단을 위한 정보 제공방법
JP2010054245A (ja) 生体内緊張状態の緩和・誘導抑制効果の評価方法及び評価用キット、並びに、物質のスクリーニング方法
HK1212762B (zh) 预测女性对象患癌症风险或诊断其癌症的方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18726503

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18726503

Country of ref document: EP

Kind code of ref document: A1