1

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

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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