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BRMAC Jul-01/Hutchins1 RCA Assays and Clinical Data For a rAd-p53 in Cancer Patients Beth Hutchins, Ph.D. Director, Process Sciences Canji, Inc. (subsidiary of Schering Plough)

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BRMAC Jul-01/Hutchins2 RCA Assays and Clinical Data Overview of RCA Sources & Assays Details of the SPRI RCA Bioassay rAd-p53 Clinical Lot RCA Information Monitoring rAd-p53 Clinical Subjects for Shed RCA Summary Points

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BRMAC Jul-01/Hutchins3 Two Sources of RCA 1) Created during large fragment method of rAd construction where recombination to create construct occurs in production cell line (293 or PER.C6) Serial viral plaquing does not eliminate multiple viral constructs contained in initial plaque Use of newer E.coli plasmid construction methods where only final plasmid transfects production cell line eliminates this source of RCA

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BRMAC Jul-01/Hutchins4 RCA Sources Contd. 2) Created during production Recombination frequency is dependent on the amount of overlap between cell lines E1 gene cassette and rAd backbone Frequency of recombination must be estimated for each combination

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BRMAC Jul-01/Hutchins5 RCA Testing by Field Today Bioassay involving one or two cell lines Indicator cell line most commonly A549 cells (lung carcinoma) Detection via CPE, immunostaining, or PCR Positives confirmed if CPE is readout Set up and qualified to be sensitive to 1 pfu or IU of RCA at a certain confidence level +/- assay Results reported based on sample size tested

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BRMAC Jul-01/Hutchins6 RCA Bioassay is Quantal Quantification estimated through assays of different amounts of sample Example: Testing at 5 x 10 8, 1 x 10 9, 5 x 10 9, 1 x 10 10 vp Negative results at 5 x 10 8, 1 x 10 9 vp Positive results at 5 x 10 9, 1 x 10 10 vp Estimated amount of RCA is 1 RCA in 5 x 10 9 vp If clinical dose is 1 x 10 12 vp, then dose contains between 1,000 and 5,000 pfu RCA

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BRMAC Jul-01/Hutchins7 SCH 58500 rAd-p53 Construct Recombinant adenovirus achieves transient expression of p53 in target cells rAd backbone is E1a, E1b, E3 and protein IX deleted Deletions allow for p53 expression cassette insert pIX deletion decreases overlap of rAd-p53 backbone with 293 cell E1 region to 200 bp

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BRMAC Jul-01/Hutchins8 Phase I/II Clinical Trials rAd-p53

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BRMAC Jul-01/Hutchins9 Specifics of SP RCA Bioassay Quantal bioassay method with CPE readout 2 cell line (HeLa S3, then A549) 28 days long PCR confirmation of positive results sensitive to 1 pfu RCA Validated to detect 4 pfu of Ad5 WT spike with 95% confidence based on triplicate tests Only RCAs detected are dl327 backbone E1 region replaces p53 expression cassette

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BRMAC Jul-01/Hutchins10 Testing SCH 58500 rAd-p53 for RCA Specification for clinical product: < 40 pfu RCA in 7.5 x 10 10 vp rAd-p53 Each batch tested at 2 x 10 9 p in triplicate or tested at 3 different particle amounts 2 x 10 8 p, 2 x 10 9 p, and 2 x 10 10 p Based on assay confidence levels for triplicate test at 2 x 10 9 p: If # Positives = 0 or 1, then batch meets specification If # Positives = 2 or 3, then batch fails specification as amount of RCA > 40 pfu in 7.5 x 10 10 vp rAd-p53

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BRMAC Jul-01/Hutchins11 Confidence in Detecting RCA No. of Positives (Triplicate test @ 2 x 10 9 vp) Probability of