# Presentation: 688890

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

**Richard Sublett, Ph.D.**
**Director Quality Systems, **
**Introgen Therapeutics**

## Presentation Outline

**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)**

## 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**

## 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**

## 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 10****10**** vp)**

## Finding RCA

## Consequence of Testing Near the Limit of Detection

**Lots with the same RCA level may or may not test as positive**
**Multiple tests of same lot may lead to inconsistent results**

## RCA Results from Two Methods

## RCA Profile for 35 Lots Tested at 3 X 1010 vp

**RCA (Plaques per 3 X10****10**** vp)**

**# Lots**

**Mean = 1.3 RCA plaques per 3 X 10****10**** vp**

## Introgen’s Working Conclusion

**All INGN 201 lots contain some RCA**
**RCA level is consistently less than 0.1 parts per billion**

## Data Through June 2000

**Data from Phase I and II trials in multiple indications**

## Estimated Patient Exposure to RCA

**Maximum daily dose 3 X 10****12**** vp**
**Estimated RCA exposure 150 vp**
**Maximum total dose 4.6 X 10****13**** vp**
**Estimated RCA exposure 2400 vp**

## 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 10****8**** to 1 X 10****12**** 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**

## 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)**

## Study T201

**Multicenter Phase II clinical trial in subjects with recurrent or refractory SCCHN**
**Daily dosing ranged from 4 X 10****11**** to 2 X 10****12**** 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**

## Study T202

**Multicenter Phase II clinical trial in subjects with recurrent or refractory SCCHN**
**Daily dosing ranged from 1 X 10****10**** to 4 X 10****10**** vp**
**Treatment cycles included treatment on days 1, 2, and 3**
**Biodistribution samples collected pretreatment and on day 28**
**47 patients treated**

## Study T207

**Multicenter Phase II clinical trial in subjects with recurrent or refractory SCCHN**
**Daily dosing 1 X 10****12**** 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 **

## 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 |
| --- | --- | --- | --- |
| T201 | 459 | 8 | 0 |
| T202 | 175 | 3 | 0 |
| T207 | 880 | 18 | 2* |

***Both positives were serotype 11 adenovirus**

## 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**

## Immune Response of Patients Treated with INGN 201

| Pretreatment
(n=29) | Cycle 1
(n=22) | Cycle 2
(n=9) |
| --- | --- | --- |

|  | ELISA | Neut. | ELISA | Neut. | ELISA | Neut. |
| --- | --- | --- | --- | --- | --- | --- |
| - | 15 | 19 | 3 | 4 | 1 | 1 |
| + | 12 | 4 | 6 | 3 | 3 | 1 |
| ++ | 0 | 5 | 1 | 3 | 0 | 2 |
| +++ | 0 | 1 | 1 | 8 | 0 | 3 |
| ++++ | 2 | 0 | 11 | 4 | 5 | 2 |

**Study T207**

## Conclusions

**INGN 201 contains RCA at levels of 1-2 RCA in 3 ****X 10****10**** 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**

## 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**