19 September 2006

Gaithersburg, MD

David Peretz M.Sc., D.Sc.

Senior Scientist

CHIRON Submission To FDA TSE Advisory Committee Meeting

2 TSEAC 19 Sep 06

• Review vCJD assay development progress

• Review assay performance data

• Review proposed validation process

Presentation Outline

3 TSEAC 19 Sep 06

Issues and Implications…

Biology Unknown levels and prevalence of prions in vCJD donations

Disease models Questionable relevance of rodent models

Sample Availability < 30 human vCJD samples available, and small volumes

Confirmation Test None available in vitro

Sensitivity goal Nucleic acid test model not applicable

Incidence Declining incidence raises question about continued testing

Realism Atypical infectious disease test

Challenges in Assay Development

4 TSEAC 19 Sep 06

Development Assumptions and Rationale

Analyte Specificity• Assay was developed to detect human PrPSc presuming this to be both the cause

and biochemical marker of the disease(Prusiner et al. Science 1982, Bueler et al. Cell 1993, Legname et al. Science 2004)

Assay Sensitivity • Sensitivity requirements based on rodent studies, human transmission to primates

and transgenic mice, using the following criteria: (Bolton et al. Arch. Biochem. Biophes. 1987, Taguchi et al. Am. J. Patho. 2003, Brown et al. J. Lab. Clin. Med. 2001)

• 1LD50 = 0.1pg = 4.3 attomole Syrian hamster PrPSc

• 1LD50 = 0.1-10 nl 10% CJD brain homogenate

Initial Analytical Studies • Spiking experiments with animal and human tissues into plasma• Detection limit of endogenously animal infected blood

Clinical Validation Studies• Establish clinical sensitivity and specificity• Confirm with an assay which is as sensitive

5 TSEAC 19 Sep 06

Assay Format

Transfer Supernatant

Dissociation and Condition Capture PrPSc in plasma

ELISA - Capture ELISA - Detection

Level of Selection > 1000 fold

6 TSEAC 19 Sep 06

10% BH (nl)0.001 0.01 0.1 1 10 100 1000 10000

Sig

nal

(R

LU

)

1

10

100

1000

10000

Current 2006

March 2005

CHIRON Analytical Sensitivity for Capture and Detection of vCJD Human PrPSc Spiked in plasma

Human rPrP 23-231 (amol)

0.1 1 10 100 1000 10000

Sig

na

l (R

LU

)

1

10

100

1000

10000

Complete AssayrPrP ELISA alone

Performance Exceeds Benchmarks and Approaches the Limit of

Detection Required for Blood Screening

1pg PrP=43amol

7 TSEAC 19 Sep 06

0.0

1.0

2.0

3.0

4.0

5.0

1 2 3 4 5 6 7 8 910 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 PC

Negative Human Plasma Samples

S/CO

Initial Specificity of Prion Assay on Normal Human Plasma Samples

8 TSEAC 19 Sep 06

Proposed Pre-Clinical Confirmation Process

1. PSR-1 Selects PrPSc and Infectivity

Pull-down material is PK resistant, demonstrated

Pull-down material is infectious, ongoing study

2. Proof of Principle

Detection of PrPSc in sheep plasma, ongoing study

100 disease samples

100 Australian negatives

3. Biology of PrPSc in Blood

Time course in Syrian hamster , ongoing study

300 animals

9 TSEAC 19 Sep 06

Proposed Clinical Validation Process

1. Clinical Specificity a. Donor samples

• 5000 UK (high prevalence)• 5000 US (low prevalence)

b. Hospitalized patients• 100 non-neurodegenerative (non-ND) patients • 100 ND patients

c. Interfering substances• Bilirubin, Rheumatoid factor, Human anti-mouse antibodies, etc

2.  Clinical Sensitivity• All available vCJD plasma samples• >40 Symptomatic sCJD plasma samples

3.  Validation• Commercial validated assay with similar/better sensitivity• Research assay with similar/better sensitivity

10 TSEAC 19 Sep 06

Recommendation/Proposal

With currently available data, rodent infectivity studies appear unlikely to add value, given:

1. Persuasive evidence for transmission of vCJD by blood transfusion

2. Proven correlation between PrPSc and infectivity in multiple tissues

3. The variability of prion biology between species PrPSc levels vary (Brown et al. J. Lab. Clin. Med. 2001) Levels of infectivity vary (Brown et al. J. Lab. Clin. Med. 2001) Incubation period can exceed life span (Race et al. J. Virol. 2001)

4. An assay which reliably demonstrated PrPSc detection in disease progression models

5. An assay which was specific in separating vCJD positives from normal human PrP

6. An assay with surrogate specificity validated with a confirmatory test for PrPSc

Since assay validation will require defined standards:

1. Encourages the allocation of government funding to support generation of surrogate reference materials and a confirmatory assay

2. Surrogate reference material could be blood derived from a time-course in animals

3. Confirmatory assay could be WB or PMCA or cell culture or another technique

4. Intent is for manufacturers to calibrate their assays against the confirmatory assay

11 TSEAC 19 Sep 06

David Peretz, MSc, DSc, Senior Scientist, R&D

W. Andrew Heaton, MD, VP/Chief Medical Officer

Alisha J. McReynolds, Associate, Regulatory Affairs

Rainer Ziermann, PhD, Director, Scientific Affairs

The CHIRON Team