Introduction to Protagen AG About Autoantibodies Technology Platform UNIarray ® Workflow

2. Agenda • Introduction to Protagen AG • About Autoantibodies • Technology Platform • UNIarray® Workflow • Protagen Dx Programs – Multiple Sclerosis • Protagen Dx Program Overview

3. Protagen AG – Facts and Location Established in 1997 Private Stock Corporation 36 Employees 1000 m² Modern Lab Space in the Technology Center Dortmund Otto-Hahn-Str.15 44227 Dortmund Germany

4. Protagen AG - Business Units Protein Services Diagnostics 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® 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

5. Agenda • Introduction to Protagen AG • About Autoantibodies • Technology Platform • UNIarray® Workflow • Protagen Dx Programs – Multiple Sclerosis • Protagen Dx Program Overview

6. 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 Autoantibodies as Diagnostics Non-disease Disease Arbuckle et al. 2003

7. Autoantibodies for Patient Stratification Disease Adverse effect Responder Non-Responder Colour Code:Disease DiseaseDiseaseResponder Non-Responder Adverse effect

8. Autoantibodies are Superior Markers • 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 Polyclonal AB-Pool

9. 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

10. We utilise… …a unique Resource for Discovery of Autoantibody Marker Panels • 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

11. Agenda • Introduction to Protagen AG • About Autoantibodies • Technology Platform • UNIarray® Workflow • Protagen Dx Programs – Multiple Sclerosis • Protagen Dx Program Overview

12. 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

13. High-Throughput Protein Expression & Purification • 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

14. Protein Characterization/QC kDa 130 72 55 43 34 26 17 • High-capacity 1D SDS-PAGE for purity control • Automated Capillary Electrophoresis for protein concentration determination using Agilent 5100 ALP

15. Biochip Production • Contact printing, regular arrays for optimal grid alignment during analysis • Highest flexibility with regard to sample conditions • Capacity: 2 Genetix QArray robots

16. Biochip Processing • High throughput automated chip processing • Highest reproducibility, temperature controlled • 2 Tecan stations HS 4800 Pro • Capacity: 2 x 24 biochips per run One HS 4800 Pro Station

17. Fluorescence Read-out Technology • ScanArray Express • Microscope slide formats • 4 lasers • High throughput scanning (automated processing of 20 slides) • Broad applicability in microarray scanning

18. Bioinformatic Analysis • Interdisciplinary team of biochemists, bioinformaticians, biologists, chemists, biotechnologists • Established algorithms for data normalization and analysis

19. Agenda • Introduction to Protagen AG • About Autoantibodies • Technology Platform • UNIarray® Workflow • Protagen Dx Programs – Prostate Cancer • Protagen Dx Program Overview

20. Functional Assay: Detection of Patient Autoantibodies AP AP AP Patient sample on MACROarray Clone ID:xxx_x14,address in microtiter plate 22 x 22 cm PVDF membrane Assay scheme: Fluorescent detection cascade (Anti-IgG and secondary labeled Ab) Serum dilution: 1:20 Polyclonal Autoantibodies (in Patient Serum) Recombinant protein/ E. coli lysate (MACROarray)

21. The UNIarray® -Workflow • 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)

22. Disease and control samples Indication-specific biomarker panel The UNIarray® -Workflow Affinity purification of putative biomarkers, printing protein biochips Screening of 37,000 human expression clones • Bioinformatical analysis: • Biostatistical ranking • Support vector machine • Neural nets • Treeboost algorithm • Threshold algorithm… Patient Sample Control Sample Diagnostic Protein Array

23. Agenda • Introduction to Protagen AG • About Autoantibodies • Technology Platform • UNIarray® Workflow • Protagen Dx Programs – Multiple Sclerosis • Protagen Dx Program Overview

24. Design of the Multiple Sclerosis Study Multiple Sclerosis Study Healthy controls Initial manifestation Secondary chronic progressive MS Primary chronic progressive MS Clinical partner: Dr. med. Sebastian Schimrigk(new affiliation:Director of NeurologyKlinikum Lüdenscheid) 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

25. Diagnostic Marker Candidates JIA 190 MS 312 59 147 RA 272 Non-disease 215 11 19 9 58 110 14 9 62 Indication-uniqueproteins 27 17 23 9 18 • Number of MACROarray-identified proteins • 20 individuals/indication DNA-Sequences BLASTed & clustered

26. Content of Clinical Screening Biochip Content: • Some 330 top-ranked MS biomarker candidateswere 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)

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

28. Quantitative Multiple Sclerosis Protein Biochip Non-diseased Multiple Sclerosis samples • Color Code: White: normal Blue: positive(cut-off: mean + 3 standard deviations, biomarker- specific) Biomarker

29. Multiple Sclerosis still difficult to diagnose…. Accurate , 85-95 % sensitivity in trained laboratories, Inconvenient procedure with , complications such as headache, numbness, infections, bleeding and brain herniation Current Testing for MS: Clinical Symptoms If suspicious MRI of brain and spinal cord¹ Lumbar Puncture (CSF²) Diagnosis • Goal: • Replace lumbar puncture • Idea: • Predictive, blood based in vitro diagnostic • better specificity of 90%, • higher sensitivity of 80% • robust Protagen MS-IVD ¹ MRI of value to exclude other diseases, EUR 400-500 / MRI ² Ceresbrospinal fluid

30. Results of MS Biochip Prototype • 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

31. Clinical Validation Study Design • 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

32. Agenda • Introduction to Protagen AG • About Autoantibodies • Technology Platform • UNIarray® Workflow • Protagen Dx Programs – Multiple Sclerosis • Protagen Dx Program Overview

33. UNIarray® Projects – Current Status è • Antigen Verification • Serum Screening Biochip • Multiple Sclerosis • Rheumatoid Arthritis • Juv. Idiop. Arthritis • Parkinson D. • Prostate C. • Pancreas C. • Alzheimer D. • SLE • Breast C. • Ovarian C

34. 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