May 22th 2008Budapest 2 OBEKON - Proteomika oRichter Genedon NyRt oMTAKémiai Kutatóközpont, Szerkezeti Kémia Intézet oTargetEx Kft. oBioSystems International.

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2 May 22th 2008Budapest 2 OBEKON - Proteomika oRichter Genedon NyRt oMTAKémiai Kutatóközpont, Szerkezeti Kémia Intézet oTargetEx Kft. oBioSystems International Kft

3 May 22th 2008Budapest 3 (van den Greef, Current Opinion in Chemical Biology 2004, 8:559–565) Betegség kialakulása biomarkerek  diagnoszikumok Genetic susceptibility “Multivariate Index Assays”

4 May 22th 2008Budapest 4 Általános séma Globális genomika adathalmaz + Más biológiai klinikai adat E-Integráció Hipotézis medált -molekuláris tudósok - betegellátás Hipotézis nélküli -genomtudomány -a holnap orvosa  Személyreszabott kezelés

5 May 22th 2008Budapest 5 Laboratóriumi vizsgálat oMa -Éhgyomri vércukor -Terheléses vércukor -Koleszterin, Triglicerid profil -Terheléses vércukor -antidiabetikum, testmozgás javasolt -Vércukor kontroll -Sztatin kezelés oHolnap -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 0.8  P450 polimorfizmus SNP (nt 425 AG/AG) -Szűrés – családtagok >25 év -Sztatin hatékonyság P>0.6

6 May 22th 2008Budapest 6 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

7 Proteomics - global - sensitive proteome analysis  OBEKON  type II diabetes early diagnostics

8 May 22th 2008Budapest 8 General Strategy for Proteome Characterization Purification Peptides Mass Spectrometry Database Search 1-DE2-DESolution Characterization MALDI-TOF MS (peptide mass mapping)  -(LC)-ESI-MS/MS (SEQUEST) Identification PTM Quantification

9 May 22th 2008Budapest 9 Multidimensional Protein Identification Technology (MudPIT) SCX RP Mass spectrometer Identify Proteins in Mixture 2D Chromatographic Separation of Peptides Protein MixtureDigested Peptides 1.Sample preparation 2. Digest 3. Purification of peptides 4.Preparation of column 5. 2DLC/LC

10 May 22th 2008Budapest 10 Challenges in blood proteome diagnostics oSix 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. oGlycoproteins, a subset of blood proteins, can be specially treated and captured for downstream proteomic analysis.

11 May 22th 2008Budapest 11 Protein Biomarker technology strategies Biomarker discovery strategies advantagedisadvantageEasy link to clinical assay 2D gel  peptide mass fingerprinting simpleScalability and sensitivity are low no Ciphergen protein chip simpleReproducibility, sensitivity, TOF-MS is inadequate no LC-MS with label (ICAT, SILAC, iTRAC) Sensitive, but less then ELISA Expensive, complex to apply to many samples, insensitive to PTMs, plasma remains a challenge no Peptidomics,sensitiveGlobal nature of the strategy is lost no MLACsimpleGlobal nature of the strategy is lost no

12 May 22th 2008Budapest 12 mAB proteomics workflow Current, MS based workflow for translation of protein biomarker discovery into clinical practice

13 May 22th 2008Budapest 13 Antibody strategies Antibody mediated biomarker discovery strategies advantagedisadvantageEasy link to clinical assay Monospecific antibodies (Uhlen et. al) scalableGlobality is questionable, reproducibility of polyclonal antibodies, insensitive to PTMs yes Recombinant antibodies and phage discplay globalityScalability at the screening level, affinity no? BSI’s mAb mediated biomarker discovery and validation Sensitive, global, natural antigen and PTM-s are seen, disease specific Not apparent (some antibodies may not work with denatured antigen) yes

14 May 22th 2008Budapest 14 Well chosen clinical collection Cntrl Pos. Enrichment of low abundance proteins Prep. of a mAB panel against virtually all proteins HTS (ELISA, etc.) HTS (ELISA, etc.) 10,000 mAbs 200 Primary Hits 10 Leads In < 1 yr ! HT MS of biomarker candidates Validation on clinical collection Diagnostics development, Clinical drug trial, Regulatory approval of Dx Kit Diagnostics development, Clinical drug trial, Regulatory approval of Dx Kit BSI patent pending Cntrl Pos. Assay prototype Plasma collection “Normalized antigen prep” + LC/MS Hybridoma technology & Screening Individual ELISAs, Protein ID, Data integration BSI- Patent pending Dx development and test in clinical trial Theranostics BSI- Patent pending Technology Workflow

15 May 22th 2008Budapest 15 Normalization of the plasma proteome (BSI patent pending) Plasma collection “Normalized antigen prep” + LC/MS Hybridoma technology & Screening Individual ELISAs, Protein ID, Data integration BSI- Patent pending Dx development and test in clinical trial BSI- Patent pending

16 May 22th 2008Budapest 16 AZ IMMUNIZÁLÁSHOZ FELHASZNÁLT PREPARÁTUMOK TÖMEGSPEKTROMETRIÁS ANALÍZISE-”PRIMER BIOMARKEREK” 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. Kontrol plazma Obez plazma 20 16 26 Kontrol plazma Obez plazma 18 14 19 A B

17 May 22th 2008Budapest 17 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 patients Control tracer – pooled 20 matched controls  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 Tracers: total plasma depleted plasma normalized plasma

18 May 22th 2008Budapest 18 LC diagnostics candidates LC/Ctrl NSCLC/Ctrl

19 May 22th 2008Budapest 19 mAb multimarker panel can correctly classify squamous cell carcinomas Adeno Squamous SCLC Control Patient with contradictory cytology/histol ogy A panel of 4 hybridomas can classify squamous cell carcinoma

20 May 22th 2008Budapest 20 72 55 40 33 24 17 170 130 100 STEP 1: Multi-immunoaffinity column 1. 2. 3. 4. 5. 6. 7. 8. STD Visible band Abscence or low intensity band STEP 2: Single mAbs 72 55 40 33 24 17 170 130 100 Ft: Elution: 72 55 40 33 24 17 170 130 100 Eluate Protein ID-1

21 May 22th 2008Budapest 21 Sequence search against human proteins in SwissProt with the motif KHL(T/S)SA Motif identification from phage sequences Eptiope-ID / phage display 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)

22 May 22th 2008Budapest 22 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)

23 May 22th 2008Budapest 23 CD26 – dipeptidyl peptidase IV oMechanism of action was discovered via an unbiased mAB approach oNew generation type II diabetes drug

24 May 22th 2008Budapest 24 Kádas János, Élesné Tóth Katalin, Tajcs Veronika, Pál Angéla BioSystems International Kft, Debrecen, Magyarország Mariana Kuras William Hempel, Nadege Tardieu, Anne Jullien, Carole Malderez, Yan Kiefert, Paco Samb, Guttman András, 2BioSystems International SAS, Evry, Franciaország