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Absence of RCA Isolated fromAbsence of RCA Isolated fromPatients Treated with INGN 201 Patients Treated with INGN 201

(Ad5CMV-p53)(Ad5CMV-p53)

Richard Sublett, Ph.D.Richard Sublett, Ph.D.

Director Quality Systems, Director Quality Systems,

Introgen TherapeuticsIntrogen Therapeutics

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Presentation OutlinePresentation Outline

Discussion of replication competent adenovirus (RCA) levels Discussion of replication competent adenovirus (RCA) levels in INGN 201in INGN 201

semi-semi-quantitative dataquantitative data comparison of qualitative and semi-quantitative datacomparison of qualitative and semi-quantitative data

Patients treated with INGN 201 Patients treated with INGN 201 number of patientsnumber of patients number of dosesnumber of doses number of viral particlesnumber of viral particles estimated number of RCAestimated number of RCA

Attempts to detect RCA Attempts to detect RCA by by PCRPCR and and CPE assays in patient CPE assays in patient body fluidsbody fluids

Immune status of patientsImmune status of patients response to treatment (anti-Ad titers)response to treatment (anti-Ad titers)

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Origin of Replication Competent Origin of Replication Competent Adenovirus in INGN 201Adenovirus in INGN 201

INGN 201 (Ad5CMV-p53) is an E1/partially E3 INGN 201 (Ad5CMV-p53) is an E1/partially E3 substituted adenovirus containing a p53 expression substituted adenovirus containing a p53 expression cassettecassette

INGN 201 is produced in a HEK 293 cell which carries INGN 201 is produced in a HEK 293 cell which carries the adenovirus E1 regionthe adenovirus E1 region

Double homologous recombination between the E1 Double homologous recombination between the E1 region of the 293 host cell and homologous regions region of the 293 host cell and homologous regions flanking the p53 expression cassette results in a low flanking the p53 expression cassette results in a low level of RCAlevel of RCA

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How does RCA arise ?How does RCA arise ?

Homologous overlap between 293-E1 and vector Homologous overlap between 293-E1 and vector backbonebackbone

~ 450 bp

E1 in 293 genomeE1 in 293 genome

Transgene cassette

E1 from 293 genomeE1 from 293 genome

~ 1100 bp

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Detection of RCA in Production LotsDetection of RCA in Production Lots

Qualitative roller bottle CPE-type assayQualitative roller bottle CPE-type assay

Semi-quantitative plaque-like assaySemi-quantitative plaque-like assay

Both assays used in a fashion that is near the limit of Both assays used in a fashion that is near the limit of detection (LOD) for RCA (1 RCA in 3 X 10detection (LOD) for RCA (1 RCA in 3 X 101010 vp) vp)

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Finding RCAFinding RCA

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Consequence of Testing Near Consequence of Testing Near the Limit of Detectionthe Limit of Detection

Lots with the same RCA level may or may Lots with the same RCA level may or may not test as positivenot test as positive

Multiple tests of same lot may lead to Multiple tests of same lot may lead to inconsistent resultsinconsistent results

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RCA Results from Two MethodsRCA Results from Two Methods

INGN 201 Drug Lot Number

Semi-quantitative RCA (plaques)

Qualitative RCA (CPE positive)

US2B Sb A 0 Negative US4B Sb A 0 Negative US5B Sb A 1 Negative B0769701 0 Positive B1739602 3 Positive B2029701 7 Negative B2439602 0 Positive B2729701 1 Negative B0979701 0 Negative B2239701 1 Negative B3219701 2 Negative B2439601 2 Positive B1189801 0 Positive B1529801 2 Positive

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RCA Profile for 35 Lots RCA Profile for 35 Lots Tested at 3 X 10Tested at 3 X 101010 vp vp

0

2

4

6

8

10

12

14

16

0 1 2 3 4 5 6 7

RCA (Plaques per 3 X10RCA (Plaques per 3 X101010 vp) vp)

# L

ots

# L

ots

Mean = 1.3 RCA plaques per 3 X 10Mean = 1.3 RCA plaques per 3 X 101010 vp vp

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Introgen’s Working ConclusionIntrogen’s Working Conclusion

All INGN 201 lots contain some RCAAll INGN 201 lots contain some RCA

RCA level is consistently less than 0.1 parts RCA level is consistently less than 0.1 parts

per billionper billion

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Data Through June 2000Data Through June 2000

Number of Patients

Number of Treatment

Days

Total vp Administered

Estimated vp RCA

Administered

406 2816 3 X 1015 2 X 105

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

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Estimated Patient Exposure to RCAEstimated Patient Exposure to RCA

Maximum daily dose 3 X 10Maximum daily dose 3 X 101212 vp vp

Estimated RCA exposure 150 vpEstimated RCA exposure 150 vp

Maximum total dose 4.6 X 10Maximum total dose 4.6 X 101313 vp vp

Estimated RCA exposure 2400 vpEstimated RCA exposure 2400 vp

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Assay for RCA in Patients Treated with Assay for RCA in Patients Treated with INGN 201 in Phase I Clinical TrialsINGN 201 in Phase I Clinical Trials

Approximately 200 patients treated in Phase I clinical trialsApproximately 200 patients treated in Phase I clinical trials

Daily dosage ranged from 2.5 X 10Daily dosage ranged from 2.5 X 1088 to 1 X 10 to 1 X 101212 vp vp

Primarily intratumoral injectionPrimarily intratumoral injection

RCA evaluated by virus replication on A549 cells (not RCA evaluated by virus replication on A549 cells (not permissive for E1 deleted adenovirus)permissive for E1 deleted adenovirus)

Acute and chronic testing Acute and chronic testing

Assay sensitivity Assay sensitivity << 10 pfu/0.5 mL sample 10 pfu/0.5 mL sample

No evidence of RCA in 3200 patient urine, plasma, and oral rinse No evidence of RCA in 3200 patient urine, plasma, and oral rinse samplessamples

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Analysis of Samples from 190 Phase II SubjectsAnalysis of Samples from 190 Phase II Subjects

Samples included plasma, urine, feces, and oral rinses from Samples included plasma, urine, feces, and oral rinses from patients treated by intratumoral injectionspatients treated by intratumoral injections

Included 400 samples from 39 household membersIncluded 400 samples from 39 household members

Testing included PCR for Hexon and p53 sequences and CPETesting included PCR for Hexon and p53 sequences and CPE

Plasma samples prescreened by PCR before CPE testingPlasma samples prescreened by PCR before CPE testing

Other samples tested by both PCR and CPEOther samples tested by both PCR and CPE

CPE testing performed on 293 cells (permissive for E1 CPE testing performed on 293 cells (permissive for E1 deleted adenovirus) followed by testing on A549 cells deleted adenovirus) followed by testing on A549 cells (not permissive for E1 deleted adenovirus)(not permissive for E1 deleted adenovirus)

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Study T201Study T201

Multicenter Phase II clinical trial in subjects with Multicenter Phase II clinical trial in subjects with recurrent or refractory SCCHNrecurrent or refractory SCCHN

Daily dosing ranged from 4 X 10Daily dosing ranged from 4 X 101111 to 2 X 10 to 2 X 101212 vp vp

Treatment cycles included treatment on days 1, 2, Treatment cycles included treatment on days 1, 2, and 3 or on days 1, 3, 5, 8, 10 and 12and 3 or on days 1, 3, 5, 8, 10 and 12

Biodistribution samples collected pretreatment and Biodistribution samples collected pretreatment and on day 28on day 28

107 patients treated107 patients treated

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Study T202Study T202

Multicenter Phase II clinical trial in subjects with Multicenter Phase II clinical trial in subjects with recurrent or refractory SCCHNrecurrent or refractory SCCHN

Daily dosing ranged from 1 X 10Daily dosing ranged from 1 X 101010 to 4 X 10 to 4 X 101010 vp vp

Treatment cycles included treatment on days 1, 2, Treatment cycles included treatment on days 1, 2, and 3and 3

Biodistribution samples collected pretreatment and Biodistribution samples collected pretreatment and on day 28on day 28

47 patients treated47 patients treated

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Study T207Study T207

Multicenter Phase II clinical trial in subjects with recurrent or Multicenter Phase II clinical trial in subjects with recurrent or refractory SCCHNrefractory SCCHN

Daily dosing 1 X 10Daily dosing 1 X 101212 vp vp

Treatment cycles included daily dosing on day 1 or on days 1, 3, Treatment cycles included daily dosing on day 1 or on days 1, 3, 5, 8, 10, and 125, 8, 10, and 12

Biodistribution samples collected for analysis pretreatment and Biodistribution samples collected for analysis pretreatment and every other day following last treatment through 15 days post- every other day following last treatment through 15 days post- treatmenttreatment

36 patients treated36 patients treated

Household members also evaluated Household members also evaluated

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Incidence of CPE Positive Samples for Incidence of CPE Positive Samples for T201, T202, and T207T201, T202, and T207

StudyStudy # Samples # Samples Tested for Tested for

CPECPE

CPE CPE Positive Positive on 293 on 293 CellsCells

CPE CPE Positive Positive on A549 on A549

CellsCells

T201T201 459459 88 00

T202T202 175175 33 00

T207T207 880880 1818 2*2*

*Both positives were serotype 11 adenovirus*Both positives were serotype 11 adenovirus

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Results of Biodistribution AnalysisResults of Biodistribution Analysis

INGN 201 or its DNA could be detected in body fluids up to four INGN 201 or its DNA could be detected in body fluids up to four weeks post-treatmentweeks post-treatment

Southern blot analysis of amplified virus isolates Southern blot analysis of amplified virus isolates indicated no recombination eventsindicated no recombination events

Two CPE positives on A549 following treatmentTwo CPE positives on A549 following treatment

Both were adenovirus serotype 11Both were adenovirus serotype 11

No evidence of horizontal transmissionNo evidence of horizontal transmission

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Immune Response of Patients Treated Immune Response of Patients Treated with INGN 201with INGN 201

PretreatmentPretreatment

(n=29)(n=29)

Cycle 1Cycle 1

(n=22)(n=22)

Cycle 2Cycle 2

(n=9)(n=9)

ELISAELISA Neut.Neut. ELISAELISA Neut.Neut. ELISAELISA Neut.Neut.

-- 1515 1919 33 44 11 11

++ 1212 44 66 33 33 11

++++ 00 55 11 33 00 22

++++++ 00 11 11 88 00 33

++++++++ 22 00 1111 44 55 22

Study T207

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ConclusionsConclusions

INGN 201 contains RCA at levels of 1-2 RCA in 3 INGN 201 contains RCA at levels of 1-2 RCA in 3 X 10X 101010 vp vp Near the limit of detectionNear the limit of detection

Levels of RCA in INGN 201 are consistent from lot to lotLevels of RCA in INGN 201 are consistent from lot to lot Patients treated with INGN 201 have been exposed to low levels of Patients treated with INGN 201 have been exposed to low levels of

RCARCA No INGN 201 derived RCA isolated from patients or household No INGN 201 derived RCA isolated from patients or household

membersmembers No evidence of adverse events related to RCA in patients treated No evidence of adverse events related to RCA in patients treated

with INGN 201with INGN 201 Most patients treated with INGN 201 develop anti-adenovirus Most patients treated with INGN 201 develop anti-adenovirus

antibodyantibody Anti-hexon and neutralizing titersAnti-hexon and neutralizing titers

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RecommendationsRecommendations

Current RCA recommendation should be Current RCA recommendation should be reconsideredreconsidered

The following criteria should be taken into account The following criteria should be taken into account when setting an RCA specificationwhen setting an RCA specification

Patient populationPatient population

Product indicationProduct indication

Available safety dataAvailable safety data