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Absence of RCA Isolated from Patients Treated with INGN 201 (Ad5CMV-p53)

Absence of RCA Isolated from Patients Treated with INGN 201 (Ad5CMV-p53). Richard Sublett, Ph.D. Director Quality Systems, Introgen Therapeutics. Discussion of replication competent adenovirus (RCA) levels in INGN 201 semi- quantitative data

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Absence of RCA Isolated from Patients Treated with INGN 201 (Ad5CMV-p53)

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

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

  3. Origin of Replication Competent Adenovirus in INGN 201 • INGN 201 (Ad5CMV-p53) is an E1/partially E3 substituted adenovirus containing a p53 expression cassette • INGN 201 is produced in a HEK 293 cell which carries the adenovirus E1 region • Double 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. How does RCA arise ? Homologous overlap between 293-E1 and vector backbone Transgene cassette E1 in 293 genome ~ 450 bp ~ 1100 bp E1 from 293 genome

  5. Detection of RCA in Production Lots • Qualitative roller bottle CPE-type assay • Semi-quantitative plaque-like assay • Both assays used in a fashion that is near the limit of detection (LOD) for RCA (1 RCA in 3 X 1010 vp)

  6. Finding RCA

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

  8. RCA Results from Two Methods

  9. RCA Profile for 35 Lots Tested at 3 X 1010 vp # Lots RCA (Plaques per 3 X1010 vp) Mean = 1.3 RCA plaques per 3 X 1010 vp

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

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

  12. Estimated Patient Exposure to RCA • Maximum daily dose 3 X 1012 vp • Estimated RCA exposure 150 vp • Maximum total dose 4.6 X 1013 vp • Estimated RCA exposure 2400 vp

  13. Assay for RCA in Patients Treated with INGN 201 in Phase I Clinical Trials • Approximately 200 patients treated in Phase I clinical trials • Daily dosage ranged from 2.5 X 108 to 1 X 1012 vp • Primarily intratumoral injection • RCA 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 • No evidence of RCA in 3200 patient urine, plasma, and oral rinse samples

  14. Analysis of Samples from 190 Phase II Subjects • Samples included plasma, urine, feces, and oral rinses from patients treated by intratumoral injections • Included 400 samples from 39 household members • Testing 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. Study T201 • Multicenter Phase II clinical trial in subjects with recurrent or refractory SCCHN • Daily dosing ranged from 4 X 1011 to 2 X 1012 vp • Treatment cycles included treatment on days 1, 2, and 3 or on days 1, 3, 5, 8, 10 and 12 • Biodistribution samples collected pretreatment and on day 28 • 107 patients treated

  16. Study T202 • Multicenter Phase II clinical trial in subjects with recurrent or refractory SCCHN • Daily dosing ranged from 1 X 1010 to 4 X 1010 vp • Treatment cycles included treatment on days 1, 2, and 3 • Biodistribution samples collected pretreatment and on day 28 • 47 patients treated

  17. Study T207 • Multicenter Phase II clinical trial in subjects with recurrent or refractory SCCHN • Daily dosing 1 X 1012 vp • Treatment cycles included daily dosing on day 1 or on days 1, 3, 5, 8, 10, and 12 • Biodistribution samples collected for analysis pretreatment and every other day following last treatment through 15 days post- treatment • 36 patients treated • Household members also evaluated

  18. Incidence of CPE Positive Samples for T201, T202, and T207 *Both positives were serotype 11 adenovirus

  19. Results of Biodistribution Analysis • INGN 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 • Two CPE positives on A549 following treatment • Both were adenovirus serotype 11 • No evidence of horizontal transmission

  20. Immune Response of Patients Treated with INGN 201 Study T207

  21. Conclusions • INGN 201 contains RCA at levels of 1-2 RCA in 3 X 1010 vp • Near the limit of detection • Levels of RCA in INGN 201 are consistent from lot to lot • Patients treated with INGN 201 have been exposed to low levels of RCA • No INGN 201 derived RCA isolated from patients or household members • No evidence of adverse events related to RCA in patients treated with INGN 201 • Most patients treated with INGN 201 develop anti-adenovirus antibody • Anti-hexon and neutralizing titers

  22. Recommendations • Current RCA recommendation should be reconsidered • The following criteria should be taken into account when setting an RCA specification • Patient population • Product indication • Available safety data

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