Peter Schulz-Knappe

Autoantibodies for Patient Stratification –
Protein Arrays and beyond
EAPB, Berlin 2011
Dr. Peter Schulz-Knappe
Diagnostics
peter.schulz-knappe@protagen.de
Protagen AG
Otto-Hahn-Str. 15
44227 Dortmund
+49 231 9742 6300
www.protagen.de

Biomarker qualities
� Clinical Biomarkers
- Risk Assessment
- Screening
- Prognostic markers
- Disease Stratification
- Therapeutic monitoring
�� Biomarkers for drug discovery and development
- Mechanism (PoM)
- Toxicity
- Efficacy (PoC)
- Patient stratification (responder/non-responder)
- Surrogate for clinical endpoints
Diagnostic companies
Antibody-based?
Pharma companies
Fit-for purpose assays
� In each case, biomarkers may support the move to personalised medicine

Status of Pharmaceutical Industry 2011
Pharmaceutical R&D
• Duration
• Costs
• Risk
• 12-13 year average from first synthesis to market approval
• R&D cost per NME in 2001 estimated at €870 million
• 92% of NME’s tested in phase 1 will fail to reach market
• Only 30% of marketed medicines produce revenues that meet or
exceed average R&D cost
• Many drugs work in a small population of patients with the intended
disease indication
After M. Bottcher, Bayer Healthcare
Is molecular targeting and individual treatment the future?

Status of Pharmaceutical Industry 2011
Pharmaceutical Industry – Quo Vadis? Prof. Axel Kleemann, January 2011
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Status of Pharmaceutical Industry 2011
Pharmaceutical Industry – Quo Vadis? Prof. Axel Kleemann, January 2011
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Status of Pharmaceutical Industry 2011
Pharmaceutical Industry – Quo Vadis? Prof. Axel Kleemann, January 2011
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Status of Pharmaceutical Industry 2011
Pharmaceutical Industry – Quo Vadis? Prof. Axel Kleemann, January 2011
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Major Pharma M&A´s
Acquirer Target company Year Deal $ billion
Novartis (Ciba-Geigy) Sandoz 1996 26
Astra Zeneca 1999 35
Pfizer Warner Lambert 2000 90
GSK Smith Kline & French 2000 55
Pfizer Pharmacia 2003 57
Sanofi-Synthelabo Aventis (former Hoechst) 2004 62
Novartis Alcon 2009/10 39/28
Pfizer Wyeth 2009 68
Merck & Co Schering-Plough/Organon 2009 41
Bayer Schering AG 2006 20
Roche Genentech 2009 47
Sanofi-Aventis Genzyme ? 2011 ? ?
...to be continued...
The consequences are job lay-offs, plant (site) closures,
reduction of R&D projects, contract manufacturing deals in Asia,...!
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Status of Pharmaceutical Industry 2011
IMS Health ́s world pharma rankings
2006 Rank 2011 Rank
1 USA USA
2 Japan Japan
3 France China
4 Germany Germany
5 Italy France
6 Canada Italy
7 Spain Brasil
8 UK Canada
9 China Spain
10 Brasil UK
11 Mexico Russia
12 Australia India
13 South Korea South Korea
14 Russia Australia
15 India Mexico
16 Turkey Turkey
Pharmaceutical Industry – Quo Vadis? Prof. Axel Kleemann, January 2011
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Status of Pharmaceutical Industry 2011
Pharma layoffs
2009:
• Total layoffs: 61,109
thereof Pfizer (19,500), Merck & Co (16,000), J&J (8,900),
AstraZeneca (7,400), GSK (6,000), Eli Lilly (5,500)
2010:
• Total layoffs: 53,636
thereof AstraZeneca (8,550), Pfizer (8,480), GSK (5,201),
Roche (4,800), Bayer (4,500), Abbott (3,000), Sanofi‐Aventis
(2,500), Takeda (1,400), Novartis (1,400), Genzyme (1,280)
Pharmaceutical Industry – Quo Vadis? Prof. Axel Kleemann, January 2011
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Ok, so the pharmaceutical industry needs to change?

Theranostics and Personalised medicine
The Biomarker Promise
This will happen on the basis of Biomarkers

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UNIarray® Technology Platform
Autoantibodies
• are constitutive and dynamic components
of the immune system
• change specifically with the development
of diseases and treatment
• are used for diagnostic purposes
• are attractive for development of novel
serum based diagnostics
• make identification of indication specific
marker panels feasible with UNIarray
Non-disease Disease
SM Slide: 14

Autoantibodies for Patient Stratification
Disease
Responder Non-Responder Adverse effect
Colour Code: Disease Disease Disease
Responder Non-Responder Adverse effect
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The UNIarray ® -Workflow
Disease and
control samples
Patient
Sample
Control
Sample
Disease Non-disease
Screening of 40,000
human expression clones
Bioinformatical analysis:
• Biostatistical ranking
• Support vector
machine
• Neural nets
• Treeboost algorithm
• Threshold algorithm…
Affinity purification of
putative biomarkers,
printing protein biochips
Indication-specific
biomarker panel
Diagnostic Protein Array
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UNIarray ® Projects – Current Status
Indication-specific
Concept
Multiple Sclerosis
Rheumatoid Arthritis
SLE
Prostate Cancer
Pancreatic Cancer
Breast Cancer
Ovarian Cancer
Diabetes Typ I
Parkinson Disease
Alzheimer Disease
Thyroid Disease
Biomarker
Discovery
UNIarray ®
Antigen
Verification
Antigen
Concept ���� MACROarray ���� ����
Antigen
Verification ����
Serum Screening
Biochip Prototype
Clinical
Validation
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Multiple Sclerosis
Initial manifestation
Clinical partners:
Multiple Sclerosis Study
Secondary chronic
progressive MS
Primary chronic
progressive MS
1. Klinikum Lüdenscheid,Dr. Schimrigk, Germany
2. Polyclinic TU Munich, Prof. Hemmer, Germany
3. KTH Stockholm, Prof. Olsson, Sweden
4. Accelerated Cure Project, Waltham, MA, USA, with
collection sites at major US-centers
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Multiple Sclerosis still difficult to diagnose….
Clinical
Symptoms
Current Testing for MS:
MRI of
brain and
spinal
cord¹
Protagen
MS-IVD
Lumbar
Puncture
(CSF²)
¹ MRI of value to exclude other diseases, EUR 400-500 / MRI
² Cerebrospinal fluid
Accurate , 85-95 % sensitivity in
trained laboratories,
Inconvenient procedure with ,
complications such as
headache, numbness,
infections, bleeding and brain
herniation
Diagnosis
Goal: Replace lumbar puncture
Idea:
Predictive, blood based in vitro diagnostic
• better specificity of 90%,
• higher sensitivity of 80%
• robust
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Clinical Partners: Karolinska Sample Bank
Genome wide association study (GWAS) with the
international MS genetics consortium (IMSGC),
Illumina 1 million SNP chip
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Properties of MS TOP V3.0
l Nanoliter (10 -9 L) amounts of sample printed
l Low total incubation volumes (< 150 µl)
l 2,672 of quantitative data points/MS TOP V3.0
“multiplex immunoassays”
l 100 - 180 µm spot size,
analysis with microscopic resolution,
20 fmole/spot of 50 kDa protein = 1 ng/spot
Subarray
MS TOP V3.0
anti-RGS His
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Disease Mapping of MS TOP Chip Antigen Panel
Term No. of Genes
multiple sclerosis 21
breast cancer 14
Alzheimer's Disease 11
diabetes, type 1 10
colorectal cancer 10
schizophrenia 10
hepatitis C 8
Parkinson's disease 8
lung cancer 8
melanoma 7
tuberculosis 7
longevity 7
HIV 7
stomach cancer 7
bladder cancer 7
systemic lupus erythematosus 7
H. pylori infection 6
hepatitis C, chronic 6
arthritis 6
celiac disease 6
bone density 6
Database:
Genetic Association Database
Disease
(archive of human genetic association
studies of complex diseases and
disorders;
http://geneticassociationdb.nih.gov/)
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Disease-specific
Concept
Biomarker
Discovery
MACRO/Microarray Screening:
plasma samples of 20+ patients
plasma samples of 20+ controls
Antigen Verification:
plasma samples of 50-100 patients
plasma samples of 50-100 controls
UNIarray ®
Antigen
Verification
Marker Panel Validation:
Serum Screening
Biochip Prototype
plasma samples of 200+ patients
plasma samples of 200+ controls
Multicentric
Retrospective/prospective
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Development of Protein Biomarker Candidates
?
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Need to switch from
High content/low throughput technology
to
Medium/low content/high throughput technology,
Ideally with
good/excellent single data point quality
High installed instrument base
FDA-clearance...
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Principle of bead based arrays (Luminex)
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Generation of BBAs
Bead
count
384 Proteins
Coupling
Coupling Control
Signal
intensities
NEU
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BBAs for Antigen/Autoantibody Interaction Assays
Intra-Plate:
Average CV Values of
reference serum
Inter-Plate:
Average CV Values of
reference serum
Plate Mean CV
# 1 5 %
# 2 2 %
# 3 2 %
# 4 2 %
# 5 3 %
# 6 2 %
Mean 3 %
Plate Mean CV
# 1 -6 10 %
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Intraassay Performance
MFI
45000
40000
35000
30000
25000
20000
15000
10000
5000
Sera (min. 10 microliter, 1:100)
0
# 91
B07_E2
# 92
E02_E1
# 93
D12_E2
# 94
C03_E1
# 95
G09_E2
# 96
F11_E2
# 97
F09_E2
# 98
F07_E2
# 99
B11_E2
# 100
B08_E2
MS-00030_10_1
MS-00030_10_2
MS-00030_10_3
MS-00030_10_4
MS-00030_g_10_1
MS-00030_15_1
MS-00030_15_2
MS-00030_15_3
MS-00030_15_4
MS-00030_20_1
MS-00030_20_2
MS-00030_20_3
MS-00030_20_4
MS-00030_25_1
MS-00030_25_2
MS-00030_25_3
MS-00030_25_4
MS-00030_30_1
MS-00030_30_2
MS-00030_30_3
MS-00030_30_4
MS-00030_40_1
MS-00030_40_2
MS-00030_40_3
MS-00030_40_4
MS-00030_50_1
MS-00030_50_2
MS-00030_50_3
MS-00030_50_4
MS-00030_250_1
MS-00030_250_2
MS-00030_250_3
MS-00030_250_4
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20-plex Test with 20 Samples
Each Sample was measured in Quadruplicates. Table displays CV
• Mean CV of antigens is 6%
• Mean CV of Samples is 6%
• FDA recommends CV < 15%
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UNIarray ® -Workflow
Screening Cohort:
Disease/Control Samples
Study Cohort:
Disease/Control Samples
• Screening of antigen/autoantibody interactions
Planar Array
Content: 3000 Proteins
• Validation of Biomarker Panels
Bead Based Array
Content: < 500 Proteins
Data Analysis: Selection
of Identified Antigens
Biostatistical Analysis
Diagnostic Protein Panel
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Development of Protein Biomarkers
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Autoantibodies as Companion Diagnostics
Protagen sees a strong rationale to utilise autoantibodies as companion
diagnostics in autoimmune disease and oncology.
Partnership with
• Bayer (Endometriosis)
• BiogenIdec (Multiple Sclerosis)
• Suppremol AG (phase II trial for their novel Lupus Erythematodes
development candidate)

Autoantibodies as Companion Diagnostics
• Benlysta (Human Genome Sciences/Glaxo) was introduced in 4/2011
as the first novel Lupus Erythematodes treatment in 50 years.
• During clinical development, Benlysta failed to reach clinical endpoints
in phase II trials
• Patient stratification against certain Autoantibodies identified the
seropositive patients as responders
• Phase III trials mandating seropositive patients were successful
• Benlysta is expected to reach 1 bn $ sales within 4 years after market
introduction

Explore Terra Incognita

Contact
Protagen AG
Otto-Hahn-Str. 15
44227 Dortmund
Germany
T + 49 231 9742 6300
F + 49 231 9742 6301
info@protagen.de
www.protagen.de
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