Diapositive 1

1
(No Transcript)
2
OBEKON - Proteomika

• Richter Genedon NyRt
• MTAKémiai Kutatóközpont, Szerkezeti Kémia Intézet
• TargetEx Kft.
• BioSystems International Kft

3
Betegség kialakulása biomarkerek ?
diagnoszikumok
Genetic susceptibility
Multivariate Index Assays
(van den Greef, Current Opinion in Chemical
Biology 2004, 8559565)
4
Általános séma
Hipotézis medált -molekuláris tudósok -
betegellátás Hipotézis nélküli -genomtudomány
-a holnap orvosa ? Személyreszabott kezelés
Globális genomika adathalmaz Más biológiai
klinikai adat
E-Integráció
5
Laboratóriumi vizsgálat

• 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 gt3, Zlt0.001,
  kritikus csúcs 12q31 xx gén
• Diéta hatékonyság Pgt0.8
• Orális antidiabetikum (xx) hatékonyság Plt0.2, yy
  Pgt0.8 ? P450 polimorfizmus SNP (nt 425 AG/AG)
• Szurés családtagok gt25 év
• Sztatin hatékonyság Pgt0.6

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- sensitiveproteome
analysis? OBEKON ? type II diabetes early
diagnostics
8
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
9
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
10
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.

11
Protein Biomarker technology strategies
Biomarker discovery strategies advantage disadvantage Easy link to clinical assay
2D gel ? peptide mass fingerprinting simple Scalability and sensitivity are low no
Ciphergen protein chip simple Reproducibility, 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, sensitive Global nature of the strategy is lost no
MLAC simple Global nature of the strategy is lost no
12
mAB proteomics workflow
Current, MS based workflow for translation of
protein biomarker discovery into clinical
practice
13
Antibody strategies
Antibody mediated biomarker discovery strategies advantage disadvantage Easy link to clinical assay
Monospecific antibodies (Uhlen et. al) scalable Globality is questionable, reproducibility of polyclonal antibodies, insensitive to PTMs yes
Recombinant antibodies and phage discplay globality Scalability at the screening level, affinity no?
BSIs 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
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 lt 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
15
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
BSI- Patent pending
16
(No Transcript)
17
Screening of the libraries with biotinylated
pooled depleted plasmaLC 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

18
LC diagnostics candidates
LC/Ctrl
NSCLC/Ctrl
19
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
20
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
21
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)
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
CD26 dipeptidyl peptidase IV

• Mechanism of action was discovered via an
  unbiased mAB approach
• New generation type II diabetes drug

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