1. INTROGEN Absence of RCA Isolated from Patients Treated with INGN 201 (Ad5CMV-p53) Richard Sublett, Ph.D. Director Quality Systems, Introgen Therapeutics

2. INTROGEN Presentation Outline qDiscussion of replication competent adenovirus (RCA) levels in INGN 201  semi-quantitative data  comparison of qualitative and semi-quantitative data qPatients treated with INGN 201  number of patients  number of doses  number of viral particles  estimated number of RCA qAttempts to detect RCA by PCR and CPE assays in patient body fluids qImmune status of patients  response to treatment (anti-Ad titers)

3. INTROGEN Origin of Replication Competent Adenovirus in INGN 201 qINGN 201 (Ad5CMV-p53) is an E1/partially E3 substituted adenovirus containing a p53 expression cassette qINGN 201 is produced in a HEK 293 cell which carries the adenovirus E1 region qDouble homologous recombination between the E1 region of the 293 host cell and homologous regions flanking the p53 expression cassette results in a low level of RCA

4. INTROGEN How does RCA arise ? Homologous overlap between 293-E1 and vector backbone ~ 450 bp E1 in 293 genome Transgene cassette E1 from 293 genome ~ 1100 bp

5. INTROGEN Detection of RCA in Production Lots qQualitative roller bottle CPE-type assay qSemi-quantitative plaque-like assay qBoth assays used in a fashion that is near the limit of detection (LOD) for RCA (1 RCA in 3 X 10 10 vp)

6. INTROGEN Finding RCA

7. INTROGEN Consequence of Testing Near the Limit of Detection qLots with the same RCA level may or may not test as positive qMultiple tests of same lot may lead to inconsistent results

8. INTROGEN RCA Results from Two Methods

9. INTROGEN RCA Profile for 35 Lots Tested at 3 X 10 10 vp RCA (Plaques per 3 X10 10 vp) # Lots Mean = 1.3 RCA plaques per 3 X 10 10 vp

10. INTROGEN Introgen’s Working Conclusion qAll INGN 201 lots contain some RCA qRCA level is consistently less than 0.1 parts per billion

11. INTROGEN Data Through June 2000 Data from Phase I and II trials in multiple indications

12. INTROGEN Estimated Patient Exposure to RCA qMaximum daily dose 3 X 10 12 vp  Estimated RCA exposure 150 vp qMaximum total dose 4.6 X 10 13 vp  Estimated RCA exposure 2400 vp

13. INTROGEN Assay for RCA in Patients Treated with INGN 201 in Phase I Clinical Trials qApproximately 200 patients treated in Phase I clinical trials qDaily dosage ranged from 2.5 X 10 8 to 1 X 10 12 vp qPrimarily intratumoral injection qRCA evaluated by virus replication on A549 cells (not permissive for E1 deleted adenovirus)  Acute and chronic testing  Assay sensitivity < 10 pfu/0.5 mL sample qNo evidence of RCA in 3200 patient urine, plasma, and oral rinse samples

14. INTROGEN Analysis of Samples from 190 Phase II Subjects qSamples included plasma, urine, feces, and oral rinses from patients treated by intratumoral injections qIncluded 400 samples from 39 household members qTesting included PCR for Hexon and p53 sequences and CPE  Plasma samples prescreened by PCR before CPE testing  Other samples tested by both PCR and CPE  CPE testing performed on 293 cells (permissive for E1 deleted adenovirus) followed by testing on A549 cells (not permissive for E1 deleted adenovirus)

15. INTROGEN Study T201 qMulticenter Phase II clinical trial in subjects with recurrent or refractory SCCHN qDaily dosing ranged from 4 X 10 11 to 2 X 10 12 vp qTreatment cycles included treatment on days 1, 2, and 3 or on days 1, 3, 5, 8, 10 and 12 qBiodistribution samples collected pretreatment and on day 28 q107 patients treated

16. INTROGEN Study T202 qMulticenter Phase II clinical trial in subjects with recurrent or refractory SCCHN qDaily dosing ranged from 1 X 10 10 to 4 X 10 10 vp qTreatment cycles included treatment on days 1, 2, and 3 qBiodistribution samples collected pretreatment and on day 28 q47 patients treated

17. INTROGEN Study T207 qMulticenter Phase II clinical trial in subjects with recurrent or refractory SCCHN qDaily dosing 1 X 10 12 vp qTreatment cycles included daily dosing on day 1 or on days 1, 3, 5, 8, 10, and 12 qBiodistribution samples collected for analysis pretreatment and every other day following last treatment through 15 days post- treatment q36 patients treated qHousehold members also evaluated

18. INTROGEN Incidence of CPE Positive Samples for T201, T202, and T207 Study # Samples Tested for CPE CPE Positive on 293 Cells CPE Positive on A549 Cells T20145980 T20217530 T207880182* *Both positives were serotype 11 adenovirus

19. INTROGEN Results of Biodistribution Analysis qINGN 201 or its DNA could be detected in body fluids up to four weeks post-treatment  Southern blot analysis of amplified virus isolates indicated no recombination events qTwo CPE positives on A549 following treatment  Both were adenovirus serotype 11 qNo evidence of horizontal transmission

20. INTROGEN Immune Response of Patients Treated with INGN 201 Pretreatment(n=29) Cycle 1 (n=22) Cycle 2 (n=9) ELISANeut.ELISANeut.ELISANeut.-15193411 +1246331 ++051302 +++011803 ++++2011452 Study T207

21. INTROGEN Conclusions qINGN 201 contains RCA at levels of 1-2 RCA in 3 X 10 10 vp  Near the limit of detection qLevels of RCA in INGN 201 are consistent from lot to lot qPatients treated with INGN 201 have been exposed to low levels of RCA qNo INGN 201 derived RCA isolated from patients or household members qNo evidence of adverse events related to RCA in patients treated with INGN 201 qMost patients treated with INGN 201 develop anti-adenovirus antibody  Anti-hexon and neutralizing titers

22. INTROGEN Recommendations qCurrent RCA recommendation should be reconsidered qThe following criteria should be taken into account when setting an RCA specification  Patient population  Product indication  Available safety data