Pronucleon TM Amplified Misfolded Protein Diagnostic Assay (AMP-D) Kenton L. Lohman, Ph.D. VP Assay Development Adlyfe Inc., Rockville, MD

Proprietary2 Amplified Misfolded Protein Diagnostic (AMP-D) Assay Exploits the basis of the misfolded protein disease protein conformational change A unique peptide based mimic of mechanism of disease –Specificity through amino acid sequence homology and conformational recognition of target –Sensitivity through fluorescent amplification of reaction Alpha helix to Beta sheet

Proprietary3 Association of Conformational Change with Fluorescence Conformational change in the peptide is associated with a shift in fluorescence intensity from excimer emission The ratio of relative excimer fluorescence to monomer relative fluorescence emission provides a measurement of the presence of  -sheet rich PrP TSE target substrate.

Proprietary4 Development Issues: PrP TSE Blood Tests Pronucleon™ AMP-D positive assay controls. Physical state of target Time course of target appearance in the etiology of disease Prognostic correlation of measurement to infectivity Association with other proteins and/or amyloids

Proprietary5 Development of Synthetic Assay Controls Synthetic Peptide Aggregate Control for TSE Controlled conditions for creating target substrates. Stable production and storage conditions. Amplification kinetics defined for the Adlyfe AMP-D assay.

Proprietary6 Assessment of “Natural” Controls Caughey/Silveira Laboratory of Persistent Viral Diseases (LPVD/NIAID) FlFFF size fractionated, purified PrP Sc oligomers and fibrils from Infected Hamster brains. Healthy animal brains were fractionated similarly and tested in parallel. Pronucleon™ AMP-D assay outcome on these purified fractions: Assay sensitivity is linked to: –Infectivity and PrPc-converting activity in individual fractions. –Conformation not concentration of target substrate.

Proprietary7 AMP-D TSE Assay Results with Blood Experimental disease –Clinical Hamster Squirrel monkey Murine –Preclinical Hamster Endemic disease –Clinical Sheep Bovine Human –Preclinical Sheep

Proprietary8 Defining a Model System for TSE Diagnostics Animal model bioassays can be diverse and costly. Standardization of samples and controls for confirmation of assay is a challenge. Should we pursue other options for bioassay such as cell culture? Will regulatory agencies make sample sets available with compliance standards?

Proprietary9 Performance Requirements : Steps toward TSE Ante-mortem tests Regulatory guidelines are needed for Sensitivity and Specificity in human TSE blood screening assays. Specifications for confirming TSE assay performance using animal endemic and/or controlled disease are needed. Correlation of findings with multiple tests –Cell culture-based infectivity, animal models or antibody based tests could be useful alternatives. Third party validation and site testing using a standardized battery of samples provided by regulatory authorities would be helpful.

Proprietary10 Summary The Pronucleon™ AMP-D Assay exploits the basis of the misfolded protein disease, or protein conformational change through Pronucleon™ ligands –Proof-of-principle with TSE in endemic disease states in rodents, sheep, bovine and humans –Technology concept being extended to Aβ Continued AMP-D Assay development is underway Regulatory guidelines are needed for controls and confirmation. CE marking guidelines are needed and could help formulate guidelines in other regulatory environments.

Proprietary11 Acknowledgements Adlyfe Inc. Alan Rudolph, CEO Cindy Orser, Ph.D. Renee Wegrzyn, Ph.D. Shankarama Shivaprasad, Ph.D. Jasmeet Sethi, PhD Peter Andreotti, PhD Craig Nelsen Miatta Morgan Georgetown University Anne Grosset, PhD Eugene Davidson, PhD Olga Tcherkasskaya, PhD Funding Sources DARPA, Defense Science Office NIH - NHLBI UK DEFRA Human/Murine Samples Paul Brown, MD, NIH (retired) Larisa Cervenakova, PhD MD, ARC Inga Zerr, MD, Gottingen Maurizio Pocchiari, MD, Rome VA Brain Blood Bank, LA Byron Caughey, NIAID, LPVD Monkey sCJD Thomas Kreil, PhD, Baxter Bioscience Univ. of Southern Alabama Bovine BSE Samples VLA in Weybridge UK Sheep Scrapie Samples Katherine O’Rourke, PhD (USDA) Michelle Crocheck, DVM (USDA) Moredun Institute Linda Detwiler, DVM (retired)