PronucleonTM Amplified Misfolded Protein Diagnostic Assay (AMP-D)

Kenton L. Lohman, Ph.D.VP Assay Development

Adlyfe Inc., Rockville, MD

Proprietary 2

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

Proprietary 3

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 PrPTSE

target substrate.

Proprietary 4

Development Issues: PrPTSE 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

Proprietary 5

Development of Synthetic Assay ControlsSynthetic 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.

0

5

10

15

20

T = 0hrs

T = 1hr

T = 2hrs

T = 3hrs

T = 4hrs

time

Exi

cmer

:Mon

omer

Rat

io

Synthetic PeptideControl

TSE diPyr

Proprietary 6

Assessment of “Natural” Controls • Caughey/Silveira Laboratory of Persistent Viral Diseases (LPVD/NIAID)

FlFFF size fractionated, purified PrPSc 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.

Proprietary 7

AMP-D TSE Assay Results with Blood

• Experimental disease– Clinical

• Hamster• Squirrel monkey• Murine

– Preclinical• Hamster

• Endemic disease– Clinical

• Sheep• Bovine• Human

– Preclinical• Sheep

Proprietary 8

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?

Proprietary 9

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.

Proprietary 10

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.

Proprietary 11

Acknowledgements

• Adlyfe Inc.Alan Rudolph, CEOCindy Orser, Ph.D.Renee Wegrzyn, Ph.D.Shankarama Shivaprasad, Ph.D.Jasmeet Sethi, PhDPeter Andreotti, PhDCraig NelsenMiatta Morgan

• Georgetown UniversityAnne Grosset, PhDEugene Davidson, PhDOlga Tcherkasskaya, PhD

• Funding SourcesDARPA, Defense Science OfficeNIH - NHLBIUK DEFRA

• Human/Murine SamplesPaul Brown, MD, NIH (retired)Larisa Cervenakova, PhD MD, ARCInga Zerr, MD, GottingenMaurizio Pocchiari, MD, RomeVA Brain Blood Bank, LAByron 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)