Protagen AG Otto-Hahn-Str. 15 44227 Dortmund +49-231-9742 6300

Protagen AGOtto-Hahn-Str. 1544227 Dortmund+49-231-9742 6300

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1. Introduction to Protagen AG

2. About Autoantibodies

3. Technology Platform

4. UNIarray® Workflow

5. Protagen Dx Programs – Multiple Sclerosis

6. Protagen Dx Program Overview

Agenda

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1000 m² Modern Lab Space in the

Technology Center Dortmund

Otto-Hahn-Str.15

44227 Dortmund

Germany

Established in 1997

Private Stock Corporation

36 Employees

Protagen AG – Facts and Location

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Protagen AG - Business Units

Protein Services Diagnostics

UNIarray® Technology

High throughput protein expression &

production

Development of novel in-vitro diagnostics

based on autoantibody signatures

R&D focus on diagnostics for MS, RA,

and Prostate Cancer

Platform for companion diagnostics &

vaccine development

UNIchip® Protein Microarrays

Antibody specificity analysis

Antibody ranking

Antibody performance prediction

GMP-compliant Protein Analysis Complete protein characterization Glycans & PTM Impurities & HCP Bioassays

Customized method development

Biomarker & target discovery

Customized Bio-IT Solutions IT support for Protein analytics and Protein

Mass Spectrometry Array data analysis Statistical methods for biomarker

discovery Modiro® – The PTM Explorer ProteinScape®

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Agenda

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Arbuckle et al. 2003

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 further biomarker discoveries

Non-disease Disease

Autoantibodies as Diagnostics

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Disease

Responder Non-Responder Adverse effect

Colour Code: Disease Disease Disease Responder Non-Responder Adverse effect

Autoantibodies for Patient Stratification

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Autoantibodies are Superior Markers

Polyclonal AB-Pool

• Measuring autoantibodies in serum or plasma

• takes advantage of the specific polyclonal immune response

directed against immunogenic antigens which become presented

as part of a disease process

• has very high sensitivity of detection of a stable analyte (IgG)

• enables retrospective/prospective studies

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Autoantibodies are Superior Markers

Robust

• polyclonal antibodies are stable in serum for years

• antibody titers do not fluctuate during the day

Easy to obtain from Serum

• minimum invasive procedure

• established procedures in clinical use

Validated as Diagnostics

• used in routine in-vitro diagnostic procedures since decades

• surrogate & non-surrogate markers

• in part established for therapy or disease progression monitoring

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UNIclone® Protein Expression Technology

Largest diversity of recombinant human proteins

> 10,000 different human gene products represented in 38.000 E.coli expression clones

MACROarrays comprising the whole library for screening (E.Coli lysate)

High-throughput protein expression & purification workflow

Proprietary Protein Microarray Production Process

Protagen has exclusive worldwide license from

Max-Planck-Society (Hans Lehrach group), Germany

We utilise…

…a unique Resource for Discovery of Autoantibody Marker Panels

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Agenda

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Expression Library Management

Generation, characterization & maintenance of expression libraries

Automated liquid handling and rearraying of clones

Largest library on stock: > 10,000 different human recombinant proteins in > 38,000 expression clones

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High throughput expression of 100 – 1000 of proteins in E. coli/week

Optimized HTS-cultivation in microtiter plate formats

HIS-tag affinity purification, typical yield 150 µg/ml, scalable to low mg levels

Expression in human cell lines and yeast, low µg yields

High-Throughput Protein Expression & Purification

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High-capacity 1D SDS-PAGE for purity control

Automated Capillary Electrophoresis for protein concentration determination using Agilent 5100 ALP

72554334

26

17

130kDa

Protein Characterization/QC

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Contact printing, regular arrays for optimal grid alignment during analysis

Highest flexibility with regard to sample conditions

Capacity: 2 Genetix QArray robots

Biochip Production

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One HS 4800 Pro Station

High throughput automated chip processing

Highest reproducibility, temperature controlled

2 Tecan stations HS 4800 Pro

Capacity: 2 x 24 biochips per run

Biochip Processing

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Fluorescence Read-out Technology

ScanArray Express

Microscope slide formats

4 lasers

High throughput scanning (automated processing of 20 slides)

Broad applicability in microarray scanning

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Bioinformatic Analysis

Interdisciplinary team of biochemists, bioinformaticians, biologists, chemists, biotechnologists

Established algorithms for data normalization and analysis

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5. Protagen Dx Programs – Prostate Cancer


Agenda

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Functional Assay: Detection of Patient Autoantibodies

Patient sample on MACROarray

Clone ID:xxx_x14,address in microtiter plate

Polyclonal Autoantibodies (in Patient Serum)

Recombinant protein/ E. coli lysate (MACROarray)

Fluorescent detection cascade (Anti-IgG and secondary labeled Ab)

Serum dilution: 1:20

AP AP

APAssay scheme:

22 x 22 cm PVDF membrane

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• Screening of patient sera on MACROarrays (20 patients)

• Selection of all proteins (Hits) which pass a predefined frequency (i.e. 20% of patient samples express autoantibodies against a certain protein)

• Mixing of all proteins (Hits) with already identified autoantigens from other indications in a customized microarray (current size: 2700x proteins, 3200x design ready)

• Analysis of larger cohorts (n= 50 samples/group), appropriate controls, active controls on 2700x microarrays

• Selection and ranking of best protein marker panels according to statistical significance

• Production of an indication-specific microarray (10-100 proteins)

The UNIarray® -Workflow

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Disease and control samples

Screening of 37,000 human expression clones

Affinity purification of putative biomarkers,

printing protein biochips

Bioinformatical analysis:• Biostatistical ranking• Support vector

machine• Neural nets• Treeboost algorithm• Threshold algorithm…

Patient Sample

Control Sample

Indication-specificbiomarker panel

Diagnostic Protein Array

Disease Non-disease

The UNIarray® -Workflow

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Agenda

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Design of the Multiple Sclerosis Study

Multiple Sclerosis Study

Healthy controls

Primary chronic progressive MSInitial manifestation

Secondary chronic progressive MS

Dr. med. Sebastian Schimrigk(new affiliation:Director of NeurologyKlinikum Lüdenscheid)

Clinical partner:

Clinical patient classification according to:

Polman et al., 2005. Diagnostic Criteria for Multiple Sclerosis: 2005 Revisions to the “McDonald Criteria”. Ann Neurol 2005;58:840–846

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Diagnostic Marker Candidates

DNA-Sequences BLASTed & clustered

Number of MACROarray-identified proteins 20 individuals/indication

14

62

9

11

147

110

17 27

58

59

9 23

18

MS

312

9

JIA

190

19

Non-disease

215

RA

272

Indication-uniqueproteins

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Content of Clinical Screening Biochip

Content:

Some 330 top-ranked MS biomarker candidates

were purified and printed in proprietary UNIchip® layout

Analysed samples:

13 non-diseased(7 female, 6 male, mean age: 36.3 y +/- 9.2 y)

30 MS patients (initial manifestation & sec. progr. MS)

(20 female, 10 male, mean age: 39.8 y +/- 8.1 y)

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Signals in relative units,White: normal Blue: positive (cut-off: [mean non-diseased samples + 3 SD; biomarker-specific]) Multiple Sclerosis samples Non-diseased samples

Bio

mar

ker

Quantitative Multiple Sclerosis Protein Biochip

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Color Code: White: normal Blue: positive (cut-off: mean + 3 standard

deviations, biomarker- specific)

Bio

mar

ker

Quantitative Multiple Sclerosis Protein Biochip

Multiple Sclerosis samples Non-diseased

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If suspicious

Protagen MS-IVD

MRI of brain and

spinal cord¹

Lumbar Puncture (CSF²)

Diagnosis

Current Testing for MS:

¹ MRI of value to exclude other diseases, EUR 400-500 / MRI

² Ceresbrospinal fluid

Accurate , 85-95 % sensitivity in trained laboratories,

Inconvenient procedure with , complications such as headache, numbness, infections, bleeding and brain herniation

Clinical Symptoms

Goal:

Replace lumbar puncture

Idea:

Predictive, blood based in vitro diagnostic• better specificity of 90%, • higher sensitivity of 80%

• robust

Multiple Sclerosis still difficult to diagnose….

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SVM analysis using 10-fold cross validation of the currently analysed

samples a high diagnostic sensitivity & specificity is achieved Analysis of 96 CIS patient versus 96 healthy persons (blood donors)

71 % Sensitivity

61 % Specificity

Results of MS Biochip Prototype

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Pre-Product Development Phase Definition of TOP Markers Raw Material Development Assay Development and Optimization

Pre-Validation and Validation Phase Appointment of Clinical Partner(s) - Polyclinic TU Munich, Prof. Hemmer, Germany - Accelerated Cure Project, MA, USA, with collection sites at:

Assay Performance Test on MS Dx

Clinical Validation Study Design

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Agenda

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UNIarray®

Indication-specific

Concept

MACROarray

Screening

Antigen

Verification

Serum Screening

Biochip Prototype

•Concept •MACROarray

UNIarray® Projects – Current Status

•Alzheimer D.

•Prostate C.

•Parkinson D.

•Juv. Idiop. Arthritis

•Rheumatoid Arthritis

•Multiple Sclerosis

•Serum Screening

Biochip

•Antigen Verification

• Pancreas C.

•SLE• Breast C.•Ovarian C

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Protagen AG

Otto-Hahn-Str. 15

44227 Dortmund

Germany

T + 49 231 9742 6300

F + 49 231 9742 6301

[email protected]

www.protagen.de

Contact

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Protagen AG Otto-Hahn-Str. 15 44227 Dortmund +49-231-9742 6300