Diagnostics

Alan S. Rudolph PhD MBAChief Executive Officer

Adlyfe Inc.Rockville, MD

arudolph@adlyfe.com301-424-8344

Innovation is in our blood

Diagnostics

DiagnosticsBackground and Perspective

• Aggregate nature of misfolded prion (PrPsc) protein presents fundamental challenges in detection and sample preparation for wide variety of risk materials including blood

• Adlyfe has developed specific and sensitive ligands that exploit prion folding behavior for direct detection of PrPSC and are developing high throughput kits for diagnostics and screening.

• We have shown presymptomatic antemortem detection of PrPSC in blood and tissue using Adlyfe proprietary peptide Pronucleon ligands

• The lack of controlled studies and matched samples to correlate etiology of potential disease from risk materials in animals (over expected timecourse of disease) limits progress

• Standardized source materials for testing would accelerate development of needed tests

DiagnosticsAdlyfe Pronucleon Ligands MimicAdlyfe Pronucleon Ligands MimicFolding and Detect PrPFolding and Detect PrPscsc Directly Directly

PrP

Adlyfe Pronucleon Ligands

-helix

-sheet

PrPsc

(TSE)

Sequence specific peptide ligands with conformationally sensitive fluorescent labels detect PrPSC directly

Folding and Aggregation of PrPSC

Infectivity and Disease

-helix -sheet

Diagnostics

Dendrimer Target:Amplification of signal and propagation of conformational change ,

without aggregation.

• Adlyfe ligands Adlyfe ligands associate with PrPassociate with PrPSCSC and transduce signaland transduce signal

•Ligands seed Ligands seed nucleation reaction nucleation reaction amplifying signalamplifying signal

Unparalleled Sensitivity through Novel Amplification

DiagnosticsAdlyfe Ligands Enable

Blood Test for BSE

BSE detection in blood plasma (red) samples were also confirmed positive by western blot in matched brain tissue

Negatives (blue) from blood plasma were confirmed negative by western blot of matched brain tissue). Two false positives (pink) were later confirmed positive by western

MPD Assay of BSE Plasma (Western: Positive &

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DiagnosticsDetection in Different Animals

Under Different Modes of Infection

Species infected control mode of animals animals inoculation Tissues Hamster - study 1 36 20 i.c. Brain, spleen and plasma -study 2 27 15 i.c. Brain, spleen and plasma

-study 3 25 15 oral gavage Brain, spleen and plasma -study 4 30 8 oral toxicity In progress Sheep 27 2 endemic Plasma or serum Bovine 45 45 endemic Brain and plasma Tot al: 190 105

DiagnosticsThreshold of Detection

• Based upon calculations by Dr. Paul Brown of blood levels of prion in disease we estimate we are detecting in the fM range or 0.1-1 ng PrP-Sc approaching the equivalent of one IU

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Concentration of prion protein (pM)

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Advanceddiagnostics

Conventionaldiagnostics

Infectivity: 1 IU = 3 fM = 200,000 PrP

Diagnostics TSE Test Kit

• 96-well polypropylene plate pre-seeded with target peptide

• Sequence specific ligands would create blood screen and diagnostic kits for BSE, vCJD and other neurodegerative protein misfolding diseases

• We are preparing to produce kits for validation and approval with USDA, FDA, EFSA, MHRA

Diagnostics Summary and Plans

• More sensitive detection of PrPSC will enable surveillance of risk materials and reduce risk of transmission

• Adlyfe ligands can detect misfolded PrPSC in risk materials including brain tissue and blood and demonstrate presymptomatic, ante mortem detection of disease

• We are in discussions with strategic partners to develop a high throughput diagnostic and screening test for detection and removal applications for risk materials

• Future R&D will further exploit detection of protein folding in neurodegenerative diseases