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Richter Genedon NyRt MTAKémiai Kutatóközpont, Szerkezeti Kémia Intézet TargetEx Kft. BioSystems International Kft. OBEKON - Proteomika. Betegség kialakulása biomarkerek diagnoszikumok. Genetic susceptibility. “Multivariate Index Assays”. - PowerPoint PPT Presentation
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May 22th 2008 Budapest 2
OBEKON - ProteomikaOBEKON - Proteomika
o Richter Genedon NyRto MTAKémiai Kutatóközpont,
Szerkezeti Kémia Intézeto TargetEx Kft.o BioSystems International Kft
May 22th 2008 Budapest 3
(van den Greef, Current Opinion in Chemical Biology 2004, 8:559–565)
Betegség kialakulása biomarkerek diagnoszikumok
Genetic susceptibility
“Multivariate Index Assays”
May 22th 2008 Budapest 4
ÁÁltalltaláános snos séémama
Globális genomika adathalmaz +
Más biológiai klinikai adat
E-Integráció
Hipotézis medált
-molekuláris tudósok- betegellátás
Hipotézis nélküli-genomtudomány-a holnap orvosa Személyreszabott kezelés
May 22th 2008 Budapest 5
LaboratóriumiLaboratóriumi vizsgálatvizsgálat
o Ma- Éhgyomri vércukor- Terheléses vércukor- Koleszterin, Triglicerid
profil- Terheléses vércukor
- antidiabetikum, testmozgás javasolt
- Vércukor kontroll- Sztatin kezelés
o Holnap- Metabolikus státuszII 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
May 22th 2008 Budapest 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
ProteomicsProteomics
- global- global- sensitive- sensitiveproteome analysisproteome analysis
OBEKON OBEKON type II diabetes early diagnostics type II diabetes early diagnostics
May 22th 2008 Budapest 8
General Strategy for General Strategy for Proteome CharacterizationProteome Characterization
Purification
Peptides
Mass Spectrometry
Database Search
1-DE 2-DE Solution
Characterization MALDI-TOF MS(peptide mass mapping)
-(LC)-ESI-MS/MS(SEQUEST)
• Identification• PTM• Quantification
May 22th 2008 Budapest 9
Multidimensional Protein Multidimensional Protein Identification Technology (MudPIT)Identification Technology (MudPIT)
SCX RP
Massspectrometer
Identify Proteinsin Mixture
2D ChromatographicSeparation of Peptides
Protein Mixture DigestedPeptides
1.Sample preparation
2. Digest
3. Purificationof peptides
4.Preparationof column
5. 2DLC/LC
May 22th 2008 Budapest 10
Challenges in blood proteome Challenges in blood proteome diagnostics diagnostics
o 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.
o Glycoproteins, a subset of blood proteins, can be specially treated and captured for downstream proteomic analysis.
May 22th 2008 Budapest 11
Protein Biomarker technology strategiesProtein 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
May 22th 2008 Budapest 12
mAB proteomics mAB proteomics workflowworkflow
Phase
Discovery:Identifyingcandidates
Qualification/Confirmation
ValidationPrototype assay
development
Technology
Product development
Shotgun mass spectrometry
Targeted MS MRM-LC-MS/MS
Prototype immunoassay
ImmunoassaymAb based/multiplexed
1000s
<100
5-25
<5
Number of analytes
Precision
Semi-quantitativeCV 30%
Quantitative
QuantitativeCV 5%
Quantitative CV 3%
Phase
Discovery:Identifyingcandidates
Qualification/Confirmation
ValidationPrototype assay
development
Technology
Product development
Proteome normalizationShotgun
immunization
Global mAB libraries to disease proteome
HTS ELISA screening
ImmunoassaymAb based/multiplexed
10000s
100
10
10
Number of analytes
Precision
QuantitativeCV < 10%
QuantitativeCV 5%
Quantitative CV 3%
Protein ID MS or peptide epitope
Assay developmentMultiplex
multivariate assay
Current, MS based workflow for translation of protein biomarker
discovery into clinical practice
May 22th 2008 Budapest 13
Antibody strategiesAntibody 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?
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
May 22th 2008 Budapest 14
Well chosen clinical
collection
Well chosen clinical
collection
Cntrl
Pos.
Enrichment of low
abundance proteins
Enrichment of low
abundance proteins
Prep. of a mAB panel
against virtually
all proteins
Prep. of a mAB panel
against virtually
all proteins
HTS(ELISA, etc.)
HTS(ELISA, etc.)
10,000mAbs
200Primary
Hits
10 Leads
In < 1 yr !
HT MS of biomarker candidate
s
HT MS of biomarker candidate
s
Validation on
clinical collection
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
Plasmacollection
“Normalized
antigen prep”
+ LC/MS
Hybridoma technology& Screening
Individual ELISAs,Protein ID, Data
integrationBSI- Patent pending
Dx development and test in clinical trial
Theranostics
BSI- Patent pending
Technology WorkflowTechnology Workflow
May 22th 2008 Budapest 15
Normalization of the plasma Normalization of the plasma proteome proteome (BSI patent pending)(BSI patent pending)
Relationship between aggregate number of peptides identified by MS/MS and measured concentration of 76 proteins by quantitative immunoassays is plotted.
David J States, Gilbert S Omenn, Thomas W Blackwell, Damian Fermin, J immy Eng, David W Speicher & Samir M Hanash Nature Biotechnology 24, 333 - 338 (2006)
10 100 1000
0
10
20
30
40
50
nu
mb
er
pe
ptid
es
ide
ntif
ied
by
MS
/MS
reported concentration of proteins in serum (µg/ml)
Agilent depleted High stringency Medium stringency Low stringency
Relationship between aggregate number of peptides identified by MS/MS and the reportedconcentration of the proteins identified in thenormalized samples
Plasmacollection
“Normalized
antigen prep”
+ LC/MS
Hybridoma technology& Screening
Individual ELISAs,Protein ID, Data
integrationBSI- Patent pending
Dx development and test in clinical trialBSI- Patent pending
May 22th 2008 Budapest 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
May 22th 2008 Budapest 17
0.0 0.5 1.0 1.5 2.0 2.5
0.0
0.5
1.0
1.5
2.0
2.5
Comparison of Average VmaxN values (and corresponding SD) between six experiments done with a single tracer, either on the same day or on different days
Average VmaxN 6 Experiments Same tracer Pool Different days(SD of the VmaxN from the 6 experiments)
Avera
ge V
maxN
6 E
xperi
ments Sam
e tra
cer
Pool Sam
e d
ay
(SD
of th
e Vm
axN
fro
m the
6 e
xper
imen
ts)
Scatter Plot
VmaxN Control NCI Y12 Tracer 270807-0.5 0 0.5 1 1.5 2
-0.5
0
0.5
1
1.5
2
Scatter Plot
VmaxN Control NCI Y12 Tracer 270807-0.5 0 0.5 1 1.5 2
-0.5
0
0.5
1
1.5
2
Sig
nal w
ith L
C t
race
r (V
max
, no
rm.)
Signal with control tracer (Vmax, norm.)
Screening of the libraries with Screening of the libraries with biotinylated pooled depleted plasma:biotinylated pooled depleted plasma:
LC tracer – pooled 20 patientsLC tracer – pooled 20 patientsControl tracer – pooled 20 matched controlsControl 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
Plasmacollection Plasma prep
Hybridoma technology& Screening
Protein I DI mmunoppt.
Peptide arrays
Anti-mouse I gG
Hybridoma supernatant
HRP-streptavidin
Biotinylated plasma protein tracer
Tracers: total plasma depleted plasma normalized plasma
May 22th 2008 Budapest 18
LC diagnostics candidates LC diagnostics candidates
ROC curve - E13NC926M6
1-Specificity
Se
nsi
tivity
0.0 0.2 0.4 0.6 0.8 1.0
0.0
0.2
0.4
0.6
0.8
1.0
LC/Ctrl
ROC curve - E13NC926M6
1-Specificity
Se
nsi
tivity
0.0 0.2 0.4 0.6 0.8 1.0
0.0
0.2
0.4
0.6
0.8
1.0
NSCLC/Ctrl
Plasmacollection Plasma prep
Hybridoma technology& Screening
Protein I DI mmunoppt.
Peptide arrays
0.6
0.8
1
1.2
1.4
1.6
1.8
2
2.2
AC Ctrl SCLC squamousCountMedianMeanStdDevMinMax
10 12 5 51.897 1.555 1.384 1.5131.846 1.497 1.285 1.3550.178 0.260 0.363 0.3201.513 1.059 0.713 0.8162.118 1.884 1.590 1.588
set(set(cancer subtype))
avg(V
max n
orm
ali
zed b
y P
C)
for
E12N
C70
E12N70
p < 0.01
AC Ctrl SCLC squamous
p < 0.005
p < 0.005
mAB2
May 22th 2008 Budapest 19
mAb multimarker panel can mAb multimarker panel can correctly classify squamous cell correctly classify squamous cell
carcinomascarcinomas
PCA 1-1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
AdenoSquamousSCLCControlPatient with
contradictory cytology/histology
A panel of 4 hybridomas can classify squamous cell carcinoma
Plasmacollection Plasma prep
Hybridoma technology& Screening
Protein I DI mmunoppt.
Peptide arrays
May 22th 2008 Budapest 20
72
55
40
33
24
17
170130
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
72554033
24
17
170130100
Ft:
Elution:
Plasmacollection
“Normalized antigen prep”
+ LC/ MS
Hybridoma technology& Screening
I ndividual ELI SAs,Protein ID, Data
integration
Dx development and test in clinical trial
BSI - Patent pending
72554033
24
17
170130100
Eluate
Protein ID-1Protein ID-1
May 22th 2008 Budapest 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)
Plasmacollection
“Normalized antigen prep”
+ LC/ MS
Hybridoma technology& Screening
I ndividual ELI SAs,Protein ID, Data
integration
Dx development and test in clinical trial
BSI - Patent pending
May 22th 2008 Budapest 22
Multiplex assaysMultiplex assaysLUMINEX PLATFORM
(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)
Plasmacollection
“Normalized antigen prep”
+ LC/MS
Hybridoma technology& Screening
Dx development and test in clinical trial
BSI - Patent pending
I ndividual ELISAs,Protein ID, Data
integration
May 22th 2008 Budapest 23
CD26 – dipeptidyl peptidase CD26 – dipeptidyl peptidase IVIV
o Mechanism of action was discovered via an unbiased mAB approach
o New generation type II diabetes drug
May 22th 2008 Budapest 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