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

1
Absence of RCA Isolated fromPatients Treated
with INGN 201 (Ad5CMV-p53)

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

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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)

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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

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How does RCA arise ?
Transgene cassette
E1 in 293 genome
450 bp
1100 bp
E1 from 293 genome

• Homologous overlap between 293-E1 and vector
  backbone

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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)

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Finding RCA
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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

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RCA Results from Two Methods
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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
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Introgens Working Conclusion

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

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

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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 lt 10 pfu/0.5 mL sample
• No evidence of RCA in 3200 patient urine, plasma,
  and oral rinse samples

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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)

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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

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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

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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

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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
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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

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Immune Response of Patients Treated with INGN 201
Pretreatment (n29) Cycle 1 (n22) Cycle 2 (n9)
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
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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

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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