1. 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)

2. 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

3. 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

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

5. 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

6. 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

7. 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

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

9. 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

10. 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

11. BRMAC Jul-01/Hutchins11 Confidence in Detecting RCA No. of Positives (Triplicate test @ 2 x 10 9 vp) Probability of <4 pfu RCA in 7.5 x 10 10 vp Probability of <40 pfu RCA in 7.5 x 10 10 vp pfu RCA Detection Limit @ 95% CL in 7.5 x 10 10 vp 00.270.96~40 10.030.73~75 20.000.28~155

12. BRMAC Jul-01/Hutchins12 Amount of RCA in Clinical Materials % Clinical Batches RCA Level in 7.5 x 10 10 vp (Specification Level) RCA Level in 7.5 x 10 13 vp (Highest Clinical Dose) ~45%< 4 RCA pfu< 4,000 RCA pfu ~45%< 40 RCA pfu< 40,000 RCA pfu ~10% Fail Specifications < 400 RCA pfu< 400,000 RCA pfu (< 4 x 10 5 RCA)

13. BRMAC Jul-01/Hutchins13 Analytical Testing Methods for Adenoviral Shedding  ELISA (Enzyme-linked Immunosorbent Assay) –Detects hexon antigen –Does not distinguish intact virus from protein  FACS (“Fluorescent Activated Cell Sorter”) – 293 Cells –Detects infectious Ad w/o distinguishing type  FACS (“Fluorescent Activated Cell Sorter”) – A549 Cells –Detects infectious WT Ad or RCA; not product  PCR ( Polymerase Chain Reaction) –Detects DNA; does not distinguish intact virus –Specific for RCA and WT Ad

14. BRMAC Jul-01/Hutchins14 SCH 58500 Clinical Subjects Positive for Adenoviral Shedding StudyELISAFACS-IFACS-R (RCA) PCR Skin – IT0/60/2 Head/Neck – IT0/140/2 Ph I NSCLC – IT2/15NA Ph II NSCLC – IT1/251/24 a 0/170/1 Colorectal – IHA1/530/310/221/1 b Ovarian – IP0/530/380/200/6 TOTAL4/1661/970/631/12

15. BRMAC Jul-01/Hutchins15 SCH 58500 Clinical Subject Population  Cancer patients  All subjects were initially selected to be anti-adenovirus positive prior to entry onto studies reported  Neutralizing anti-adenovirus titers varied prior to SCH 58500 administration and increased with SCH 58500 administration  For subjects where Ad shedding was detected, no adverse clinical sequelae were identified

16. BRMAC Jul-01/Hutchins16 Serological Monitoring Serum Anti-Adenovirus Titer

17. BRMAC Jul-01/Hutchins17 Summary Points  Quantal bioassays allow sensitive & reliable detection of RCA  More precise quantitation impractical using bioassay due to the amount of testing required –Quantitative PCR (real-time PCR) is option  No viral shedding of replication competent adenovirus from clinical subjects detected –No PCR + for RCA (only SCH 58500), No FACS-R +  No adverse sequelae identified in clinical subjects where Ad shedding was detected –Very low viral shedding of infectious SCH 58500 –1.0% by FACS-I assay

18. BRMAC Jul-01/Hutchins18 Backup Slides

19. BRMAC Jul-01/Hutchins19 SCH 58500 rAd-p53 Vector

20. BRMAC Jul-01/Hutchins20 How the PCR Assay Discriminates between SCH58500-Encoded p53 and WT p53 SCH 58500 PCR Primers PCR Amplification NO PCR Amplification Human Cell

21. BRMAC Jul-01/Hutchins21 ELISA Enzyme-linked Immunosorbent Assay  Purpose rapid on-site assay used for patient discharge from hospital qualified for urine, stool, sputum and additional body fluids as requested  Characteristics colorimetric assay detects hexon coat protein of adenovirus does not discriminate between hexon on intact virus and free hexon from dissociated virus

22. BRMAC Jul-01/Hutchins22 FACS-Infectivity “Fluorescent Activated Cell Sorter” infectivity assay  Purpose measures infectious adenovirus qualified for urine, stool, sputum and additional body fluids as requested  Characteristics detects hexon coat protein of adenovirus that is made as a result of the infection of permissive cells (HEK 293 cell line) Single replication cycle (2 day) LOD 10 3 -10 4 particles/mL (matrix dependent) does not discriminate infectious SCH 58500 from wild- type adenovirus or RCA

23. BRMAC Jul-01/Hutchins23 FACS-RCA “Fluorescent Activated Cell Sorter” RCA assay  Purpose measures infectious RCA qualified for urine, stool, sputum and additional body fluids as requested  Characteristics detects hexon coat protein of adenovirus that is made as a result of the infection of non-permissive cells (A549 cell line) Multi-replication cycles (7-day) discriminates between infectious wild-type & RCA adenovirus and clinical product

24. BRMAC Jul-01/Hutchins24 PCR for RCA  Purpose highly specific for SCH 58500 encoded p53 DNA vs. wild-type or homologous recombinant (RCA) qualified for urine, stool, sputum and additional body fluids as requested  Characteristics highly specific for identity high sensitivity (matrix dependent) 3 assays: TPL (SCH 58500), E1a, and E3 does not discriminate between intact virus and free viral DNA

25. BRMAC Jul-01/Hutchins25 Possible Sequence Analysis Results for Plaque-Purified Isolates from RCA Testing dl327 E1a E1b E2 E4 SCH58500 CMV/TPL/p53 E2 E4 Ad5 E1a E1b E2 E3 E4 ??? E2 E3?? E4 Other Recombinants All RCA to date

26. BRMAC Jul-01/Hutchins26 Expected Results from PCR Assays