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OBEKON - Proteomika

Richter Genedon NyRt MTAKémiai Kutatóközpont, Szerkezeti Kémia Intézet TargetEx Kft. BioSystems International Kft. OBEKON - Proteomika. Betegség kialakulása biomarkerek  diagnoszikumok. Genetic susceptibility. “Multivariate Index Assays”.

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OBEKON - Proteomika

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  1. Richter Genedon NyRt MTAKémiai Kutatóközpont, Szerkezeti Kémia Intézet TargetEx Kft. BioSystems International Kft OBEKON - Proteomika Budapest

  2. Betegség kialakulása biomarkerek  diagnoszikumok Genetic susceptibility “Multivariate Index Assays” (van den Greef, Current Opinion in Chemical Biology 2004, 8:559–565) Budapest

  3. Általános séma • Hipotézis medált • -molekuláris tudósok • - betegellátás • Hipotézis nélküli • -genomtudomány • -a holnap orvosa •  Személyreszabott kezelés Globális genomika adathalmaz + Más biológiai klinikai adat E-Integráció Budapest

  4. Laboratóriumivizsgálat • Ma • Éhgyomri vércukor • Terheléses vércukor • Koleszterin, Triglicerid profil • Terheléses vércukor • antidiabetikum, testmozgás javasolt • Vércukor kontroll • Sztatin kezelés • Holnap • Metabolikus státusz II típusú diabétesz - genetikai prediszpozíció : + LOD >3, Z<0.001, kritikus csúcs 12q31– xx gén • Diéta hatékonyság P>0.8 • Orális antidiabetikum (xx) hatékonyság P<0.2, yy P>0.8  P450 polimorfizmus SNP (nt 425 AG/AG) • Szűrés – családtagok >25 év • Sztatin hatékonyság P>0.6 Budapest

  5. MammaPrint™ *(Agendia) / gen expression profiling for breast cancer • assesses clearly the individual risk of breast cancer recurrence • provides valuable information to decide on the right treatment • is cleared by the FDA and has been proven in more than 7,000 tests • no additional invasive procedures Budapest

  6. Proteomics- global- sensitiveproteome analysis OBEKON  type II diabetes early diagnostics

  7. General Strategy for Proteome Characterization Purification 1-DE 2-DE Solution Peptides MALDI-TOF MS (peptide mass mapping) -(LC)-ESI-MS/MS (SEQUEST) Mass Spectrometry Characterization • Identification • PTM • Quantification Database Search Budapest

  8. SCX RP Multidimensional Protein Identification Technology (MudPIT) 3. Purification of peptides 1.Sample preparation 5. 2DLC/LC 4.Preparation of column 2. Digest 2D Chromatographic Separation of Peptides Protein Mixture Digested Peptides Mass spectrometer Identify Proteins in Mixture Budapest

  9. Challenges in blood proteome diagnostics • Six high abundant proteins constitute about 80% of all serum proteins (e.g., albumin alone is about 51%) making virtually impossible to visualize proteins that hold possible important diagnostic information, but present in blood at lower levels. • Glycoproteins, a subset of blood proteins, can be specially treated and captured for downstream proteomic analysis. Budapest

  10. Protein Biomarker technology strategies Budapest

  11. Current, MS based workflow for translation of protein biomarker discovery into clinical practice mAB proteomics workflow Budapest

  12. Antibody strategies Budapest

  13. Technology Workflow “Normalized antigen prep”+ LC/MS Dx development and test in clinical trial Individual ELISAs,Protein ID, Data integration BSI- Patent pending Hybridoma technology & Screening Plasmacollection BSI- Patent pending Assay prototype Theranostics 10,000 mAbs 200 Primary Hits 10 Leads Cntrl Cntrl Pos. Pos. In < 1 yr ! BSI patent pending Well chosen clinical collection Enrichment of low abundance proteins Prep. of a mAB panel against virtually all proteins HTS (ELISA, etc.) HT MS of biomarker candidates Validation on clinical collection Diagnostics development, Clinical drug trial, Regulatory approval of Dx Kit Budapest

  14. Normalization of the plasma proteome (BSI patent pending) “Normalized antigen prep”+ LC/MS Dx development and test in clinical trial Individual ELISAs,Protein ID, Data integration BSI- Patent pending Hybridoma technology & Screening Plasmacollection Budapest BSI- Patent pending

  15. AZ IMMUNIZÁLÁSHOZ FELHASZNÁLT PREPARÁTUMOK TÖMEGSPEKTROMETRIÁS ANALÍZISE-”PRIMER BIOMARKEREK” Obez plazma Kontrol plazma A 20 26 16 Obez plazma Kontrol plazma B 18 19 14 2.ábra: Venn diagramm;Kontrol donorokból és betegekből származó, egy (A) és két (B) lépésben (Agilent és Normalizáló oszlopkromatográfia) depletált plazmamintákból azonosított fehérjék száma. A mindkét csoportban azonosított fehérjék a diagramm átfedő részében láthatóak. Az egyes mintára specifikusak a diagramm két oldalsó részében jelennek meg. Budapest

  16. Signal with LC tracer (Vmax, norm.) Signal with control tracer (Vmax, norm.) Screening of the libraries with biotinylated pooled depleted plasma:LC tracer – pooled 20 patientsControl tracer – pooled 20 matched controls • Tracers: • total plasma • depleted plasma • normalized plasma • The pooling in order to avoid the biological variability at the initial screening stage • The threshhold (1.5) defined by the sensitivity of the screening assay: direct capture ELISA • The redundancy of the generated mAb libraries is minimized by the immunization procedure and a redundancy screening of the generated hybridomas Budapest

  17. LC diagnostics candidates LC/Ctrl NSCLC/Ctrl Budapest

  18. mAb multimarker panel can correctly classify squamous cell carcinomas A panel of 4 hybridomas can classify squamous cell carcinoma Adeno Squamous SCLC Control Patient with contradictory cytology/histology Budapest

  19. 170 170 170 130 130 130 100 100 100 72 72 72 55 55 55 40 40 40 33 33 33 24 24 24 17 17 17 Protein ID-1 STEP 2: Single mAbs STEP 1: Multi-immunoaffinity column Ft: Eluate Elution: Abscence or low intensity band Visible band 1. 2. 3. 4. 5. 6. 7. 8. STD Budapest

  20. Eptiope-ID / phage display Sequence search against human proteins in SwissProt with the motif KHL(T/S)SA Motif identification from phage sequences E-MAP: Epitope-mediated antigen prediction Bastas et al. (2007) MCP Papers in Press. Published on September 25, 2007 as Manuscript M700107-MCP200 OBEKON Tagetex Kft (Hungary) Budapest

  21. Multiplex assays (fluorescence) Identical results with BSI mABs and tracers on two multiplexing platforms (comparison of singal intensity a set of mABs with two different tracers) Collaboration with Telechem Inc (CA) , Randox Ltd (IRL). and BMD SAS (France) Budapest

  22. CD26 – dipeptidyl peptidase IV • Mechanism of action was discovered via an unbiased mAB approach • New generation type II diabetes drug Budapest

  23. Kádas János, Élesné Tóth Katalin, Tajcs Veronika, Pál Angéla BioSystems International Kft, Debrecen, Magyarország Mariana KurasWilliam Hempel, Nadege Tardieu, Anne Jullien, Carole Malderez, Yan Kiefert, Paco Samb, Guttman András, 2BioSystems International SAS, Evry, Franciaország Budapest

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