1 biosystems international lung cancer biomarker test february 25th. 2011
TRANSCRIPT
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BioSystems InternationalBioSystems International
Lung Cancer Biomarker Lung Cancer Biomarker TestTest
February 25th. 2011February 25th. 2011
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BioSystems InternationalBioSystems International
o Founded in 2004 based on proteomics technology developed at Pfizer.
o Facilities in Evry, France and Debrecen, Hungary.
o 35 Employees (23 in France, 15 in Hungary).
o Recently merged with MicroBioChips, a French protein microarray company.
Evry, France Debrecen, Hungary
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Unique MAb Proteomics Unique MAb Proteomics Biomarker Discovery TechnologyBiomarker Discovery Technology
BSI has developed monoclonal antibody proteomics: a unique and superior technology for discovery of blood biomarkers:
o Targets native epitopes: nascent mAB libraries (10 000 mABs) cloned libraries (850 mABs)
o Unbiased: No knowledge of disease mechanism or potential biomarkers assumed or needed.
o High sensitivity: detects small changes in biomarker levels.o High-throughput: Biomarkers are confirmed by testing of hundreds of
patient samples.o Efficient translation: Avoids major bottleneck of mass spectrometry-
based biomarker discovery process: transition to clinical assay.o Diagnostic-grade MAbs against biomarkers are generated as part of
the biomarker identification process; allows a rapid transition to clinical validation and IVD product development
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Lung cancer Dx
mAB microarray
Colon and breast cc
Discovery
Validation
Market
Development
2008 2009 2010 2011 2012
Pipeline
5
Monoclonal antibody proteomics workflow
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
Mass spectrometry based workflow for translation of
protein biomarker discovery to clinical practice
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%
Translation bottleneck
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BSI Biomarker Discovery TechnologyBSI Biomarker Discovery Technology
o Disease-state plasma samples are pooled and processed to remove the most abundant proteins and to “normalize” the concentration of remaining proteins
o Mice are immunized with processed plasma sampleso Resulting hybridomas are screened to select MAbs which
discriminate disease state from control patient sampleso Hybridomas secreting antibodies to candidate biomarkers are
cloned.o Resulting MAbs used to identify biomarkero Libraries are searched for sandwich partnerso Translation of discovery to diagnostic candidate, clinical testing
Immunogenpreparation
Hybridomageneration
ScreeningCloning
Pooled (LC)
plasma
Depleted
plasma
Glycoprotein
rich plasma
Normalized
plasma
NascentmAb libraries
3848 hybridomas
IgG screen1051 hyb.
collection I collection II collection III collection IV
2 PooledplasmaLC/Ctrl
180 hyb.4 clones
45 hyb.16 clones
24 clones 13 clones
N Individual plasmas
Depletedbiotinylated
poolsDepleted biotinylated plasmas
32 271 3202
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Targets of Antibodieso Native proteins which
contain multiple epitopes
o Epitope: a single antigenic site on a protein to which an antibody reactso Linear or
Discontinuouso Peptideo Carbohydrates
o Antibodies detect PTRs
Putative glycanepitope
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More people die from lung cancer than any other type of cancer.Survival rate is ~16%
but
Why lung cancer / early diagnosis ?
55272Mountain 1987
58439Bluzebruck 1992
61.6288Shimizu 1993
63.5865Mountain 1989
64.6152Zhang 1993
65536Naruke 1988
68725Mountain 1987
71121Termach 1990
71461Williams 1981
72128Martini 1986
%NAuthor
55272Mountain 1987
58439Bluzebruck 1992
61.6288Shimizu 1993
63.5865Mountain 1989
64.6152Zhang 1993
65536Naruke 1988
68725Mountain 1987
71121Termach 1990
71461Williams 1981
72128Martini 1986
%NAuthor
Herbst et all ASCO Edutcational / 2000 Spring
Survival of NSCLC removed at Stage I
Average: 64.4Average is 64.4%
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Immunogenpreparation
Hybridomageneration
ScreeningCloning
Pooled (LC)plasma
Depletedplasma
Glycoproteinrich plasma
Normalizedplasma
NascentmAb libraries
3848 hybridomas
IgG screen1051 hyb.
collection I collection II collection III
2 PooledplasmaLC/Ctrl
180 hyb.4 clones
45 hyb.16 clones 24 clones
Individual plasmas
Depletedbiotinylated
pools
Depleted biotinylated plasmas
32 2712
An
tib
od
ych
arac
teri
zati
on
Immunogenpreparation
Hybridomageneration
ScreeningCloning
Pooled (LC)plasma
Depletedplasma
Glycoproteinrich plasma
Normalizedplasma
NascentmAb libraries
3848 hybridomas
IgG screen1051 hyb.
collection I collection II collection III
2 PooledplasmaLC/Ctrl
180 hyb.4 clones
45 hyb.16 clones 24 clones
Individual plasmas
Depletedbiotinylated
pools
Depleted biotinylated plasmas
32 2712
An
tib
od
ych
arac
teri
zati
on
Lung Cancer Discovery WorkflowLung Cancer Discovery Workflow
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-4 -3 -2 -1 0 1 2 3
0
5
10
15
20
Bsi0359
Bsi0352
Bsi0392
Bsi0272
Bsi0358
Bsi0077
-log10 p
(LC
vs C
trl)
log2 (Median LC/ Median Ctrl)
Bsi0071
-1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
2.5
No
rma
lize
dV
ma
x,
(re
l. u
.)
Normalized Vmax, (rel. u.)
LC
Nascent hybridoma library: initial hitsNascent hybridoma library: initial hits
Anti-mouse I gG
Hybridoma supernatant
HRP-streptavidin
Biotinylated plasma protein tracer
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Best candidates: cloned mABsBest candidates: cloned mABs
0.0 0.2 0.4 0.6 0.8 1.0
0.0
0.2
0.4
0.6
0.8
1.0
Sen
siti
vity
1-specificity
Ctrl LC-200
0200400600800
100012001400160018002000
Co
nc.
Ctrl LC
0
500
1000
1500
2000
Co
nc
.
Ctrl LC
0
50
100
150
200
Co
nc.
Ctrl LC
0
200
400
600
800
1000
1200C
on
c.
Ctrl LC0
200
400
600
800
1000
Co
nc.
coll. III coll. IV
0.85 0.77
0.80 0.90
0.80 0.89
0.74 0.78
AUC
0.6 --
0.8 0.89
0.74 0.78
Anti-mouse I gG
Hybridoma supernatant
HRP-streptavidin
Biotinylated plasma protein tracer
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Cognate antigen identificationCognate antigen identification
Bsi
0071
Bsi
0077
Bsi
0271
Bsi
0272
Bsi
0352
Bsi
0358
Bsi
0392
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
ELI
SA
with
cyt
C c
apt.
LRG
1
A
Bsi0071 Bsi0077 Bsi0270 Bsi0271 Bsi0272 Bsi0358 Bsi03590.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
mAbs
CFHApoB100ApoA1C3HSAHptIgC9FrnACTATAMG
Vm
axN
(rel
.u)
Bsi0071 Bsi0077 Bsi0270 Bsi0271 Bsi0272 Bsi0358 Bsi03590.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
mAbs
CFHApoB100ApoA1C3HSAHptIgC9FrnACTATAMG
Vm
axN
(rel
.u)
Hpt CFH C9 CFH C9 ACT Cntr LRG1
Direct ELISA with purified natural proteins Recombinant LRG1
Protein ID: immunoprecipitation mass spectrometry verification
“redundancy”
BM 4
BM1 BM2 BM3 BM2 BM3 BM5
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Immunohistology: cancer tissueImmunohistology: cancer tissue
Bsi0033(Hpt)
Bsi0077(CFH)
Bsi0272(C9)
Bsi0358(ACT)
Negative control
BM1 BM2 BM3 BM5
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BM5: MAb-1 Recognizes Lung BM5: MAb-1 Recognizes Lung Cancer-Specific EpitopeCancer-Specific Epitope
Figure 2: Antigen search for Bsi 0358 monoclonal antibody and contol/lung cancer specific plasma comparison via Western Blot after SDS-polyacrilamide gelectrophoresis. Left: membrane after ECL developing. Right:
membrane after amido-black staining. Band with arrow is specific for the antibody.
Ana
lyte
spec
ific
mA
B-
2
Control
LC
Analyte specific mAB - 1Ana
lyte
spec
ific
mA
B-
2
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Translation: Discovery Method to Translation: Discovery Method to Sandwich ELISA AssaysSandwich ELISA Assays
ACT Hpt CFH
LRG-1 C9
Disease specific isoforms?
BM 1 BM 2 BM 3
BM 4 BM 5
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Lung Cancer Risk Indicator PanelLung Cancer Risk Indicator Panel
o Results of 5 biomarkers were combined using support vector machine (SVM) algorithm.
o Lung cancer risk indicator is the composite linear function of individual biomarker concentrations.
o Gives improved sensitivity/specificity compared to any single biomarker.
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Comparison of Individual Biomarkers Comparison of Individual Biomarkers to Panelto Panel
Biomarker or Panel Sensitivity* AUC
BM1 62.3 0.85
BM2 43.9 0.80
BM3 60.9 0.81
BM4 32.1 0.74
Panel BM1-3 63.7 0.85
Panel BM1-5 72.2 0.90
* Sensitivity at 95% Specificity
Biomarker or Panel Sensitivity* AUC
BM1 62.3 0.85
BM2 43.9 0.80
BM3 60.9 0.81
BM4 32.1 0.74
Panel BM1-3 63.7 0.85
Panel BM1-5 72.2 0.90
* Sensitivity at 95% Specificity
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Risk Indicator Panel: performanceRisk Indicator Panel: performance
0.0 0.2 0.4 0.6 0.8 1.0
0.0
0.2
0.4
0.6
0.8
1.0
Sen
siti
vity
1-specificity
0.0 0.2 0.4 0.6 0.8 1.0
0.0
0.2
0.4
0.6
0.8
1.0
Sen
siti
vity
1-specificity0.0 0.2 0.4 0.6 0.8 1.0
0.0
0.2
0.4
0.6
0.8
1.0
Sen
siti
vity
1-specificity
BSI panel
All LC Stage I LC
Stage II, III and IV LC BSI panel: LRG1, ACT, Hpt, C9, CFH
AUC: 09Sens*:72%n**: 166/212
AUC: 0.86Sens*: :77%n**: 166/121
AUC: 0.94Sens*: :87%n**: 166/75
* Sensitivity @ 95% specificity** n:control / lung cancer
BM 1-5
19Febr. 23. 2011 CONFIDENTIAL
Potential confounding effectsPotential confounding effects
Lung cancer
Control
Pneumonia
COPD
Fibrosis
Sarcoidosis
IndexIndex-4 -3 -2 -1 0 1 2 3 4 5
Additional factors tested (capture assay)
-Gender-Smoking habit-“Other” (non lung) cancer-Lung metastasis of “other” cancers
SW ELISA: Cohort IV
20Febr. 23. 2011 CONFIDENTIAL
Combination with CYFRA : LC stagesCombination with CYFRA : LC stages
CONFI DENTI AL
BSI panel + CYFRABSI panel
All LC Stage I LC
Stage II, III and IV LC
0.0 0.2 0.4 0.6 0.8 1.0
0.0
0.2
0.4
0.6
0.8
1.0
Sen
siti
vity
1-specificity
0.0 0.2 0.4 0.6 0.8 1.0
0.0
0.2
0.4
0.6
0.8
1.0
Sen
siti
vity
1-specificity0.0 0.2 0.4 0.6 0.8 1.0
0.0
0.2
0.4
0.6
0.8
1.0
Sen
siti
vity
1-specificity
0.0 0.2 0.4 0.6 0.8 1.0
0.0
0.2
0.4
0.6
0.8
1.0
Sen
siti
vity
1-specificity
0.0 0.2 0.4 0.6 0.8 1.0
0.0
0.2
0.4
0.6
0.8
1.0
Sen
siti
vity
1-specificity0.0 0.2 0.4 0.6 0.8 1.0
0.0
0.2
0.4
0.6
0.8
1.0
Sen
siti
vity
1-specificity
BSI panel
BSI panel: LRG1, ACT, Hpt, C9
AUC*: 0.94Sens.**: 84%n***: 158/203
*BSI panel + CYFRA, ** Sensitivity @ 95% specifcity, *** n: ctrl/LC
AUC*: 0.93Sens.**: 82.6%n***: 158/115
AUC*: 0.97Sens.**: 90.3%n***: 158/72
BM 1-4
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Independent validationIndependent validation
0.0 0.2 0.4 0.6 0.8 1.0
0.0
0.2
0.4
0.6
0.8
1.0
Se
ns
itiv
ity
1-specificity
0.0 0.2 0.4 0.6 0.8 1.0
0.0
0.2
0.4
0.6
0.8
1.0
Se
ns
itiv
ity
1-specificity
Independent cohorts: protocol, geography, anticoagulant differences
Panel: LRG1, ACT, C9, Hpt, CFH Panel: LRG1, ACT, C9, Hpt, CYFRA
Training Testing
BM 1-5 BM 1- 4 + CYFRA
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Conclusions, perspectivesConclusions, perspectives
o BSI’s MAb proteomics technology identifies novel disease state biomarkers
o The new biomarkers combined with CYFRA have higher specificity and sensitivity for early LC than any previously reported biomarker panel
o Clinical use potential:o Diagnostic aid (current development focus)o Early diagnosis (partnering)o Management, monitoring (v2 panel is in the
pipeline)
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AcknowledgmentsMariana Kuras Ph.DIstván Kurucz Ph.D.
Nadège Tardieu M.S.William Hempel Ph.DJozsef Lazar Ph.D.Yann Kiefert M.S.János Kádas Ph.D.Carole Malderez-Bloes M.S.Anne Jullien M.S.András Guttman Ph.D.
Balazs Dezso M.D.
Eszter Csanky M.D.
Barry L. Karger Ph.D.
.
Mariana Kuras Ph.D.
Biosystems International SAS, Evry, FranceBiosystems International Kft., Debrecen, Hungary
Department of Pathology, University of Debrecen
Department of Pulmonology, University of Debrecen
Northeastern University Barnett Institute, USA
Director of Research Biosystems Intl. SAS, France
OSEO