1. Responses to FDA Gene Therapy Letter : Adenovirus Vector Titer Measurements and RCA Levels BRMAC July 13, 2001 Steven R. Bauer, Ph.D Division of Cellular and Gene Therapies CBER/FDA

3. Clinical IndicationRoute of AdministrationINDs CancerIntratumoral/Intralesional29 Ex vivo transduction13 Intravascular2 Intraperitoneal/pleural2 Oral1 Coronary / VascularMyocardial3 Intramuscular2 Intracoronary1 Genetic defectRespiratory tract3 Intravascular1 + NormalIntradermal1 58

4. Updates –Development of an adenovirus reference material –Change in recommendation on particle to infectious unit (IU) ratio –Change in recommendation on RCA limit Discussion –Application of RCA recommendation Purpose

5. RCA: replication competent, infectious particle IU: replication defective, infectious particle non-infectious particles Adenovirus Vector Final Product VP: all vector particles

6. Characterization of Particles in Adenovirus Vector Products VP: physical/chemical measurement –example : A 260 after lysis of VP IU: biological assay –examples: measure plaques on a lawn of permissive cells immunological detection of infected cells with fluorescent antibodies RCA: biological assay measure infection on cells that do not complement defective vector

7. Impact of VP, IU, RCA: Safety, Efficacy, Production Safety –replication competent virus –exposure to viral proteins Efficacy –non-transducing particles Production process

8. Development of Adenovirus Reference Material 1993: Adenoviral vectors used in CF Protocols CF Foundation recommended vector reference standard 1999: RAC Safety Symposium –RAC AdSAT calls for standards

9. What Will Be Accomplished by Reference Material Development? Produce more consistent, safer, adenoviral vectors –particle counts (10% inter-assay variability) –infectious units (over 30% inter-assay variability) Allow comparability between preclinical and clinical studies Develop regulatory policy –make recommendations based on “standardized” measurements

10. Reference Material Development Adenovirus Reference Material Working Group (ARMWG) Partnership: Government/Industry/Academia Williamsburg Bioprocessing Foundation –www.wilbio.com –www.fda.gov/cber/minutes/workshop-min.htm

11. Adenovirus Reference Material Production Scheme Ad5 wt virus Master Viral Seed Stock Production of purified formulated bulk virus Characterization, safety testing, provisional titer Vialing Master Cell Bank Particle and Infectivity Titer Determinations, Stability Repository and Distribution ARMWG Meetings March 22, 2001 May 31, 2001

12. Updates –Development of an adenovirus reference material –Change in recommendation on particle to infectious unit (IU) ratio –Change in recommendation on RCA limit Discussion –Application of RCA recommendation Purpose

13. Changes in Recommendations ALARA –A s L ow A s R easonably A chievable –New recommendations based on review of information submitted in March 6 letter responses –Applied to all Adenovirus GT products

14. Change in Recommendation on Particle to IU ratio Previous Recommendation –Based on review of production lot data < 100 vp/iu Current Recommendation –Based on review of production lot data from March 6 letter responses < 30 vp/iu (>3.3% iu)

15. Change in Recommendation on RCA Limit Previous recommendation – imprecise since based on infectious titer < 1RCA / 10 9 iu Current recommendation – based on particle number – better precision < 1RCA / 3 x 10 10 vp

16. Updates –Development of an adenovirus reference material –Change in particle to infectious unit (IU) ratio –Change in recommendation on RCA limit Discussion –Application of RCA recommendation Purpose

17. Application of RCA Recommendation < 1RCA / 3 x 10 10 vp –recommended for all adenovirus vector lots regardless of clinical use Exposure risk at highest current doses – dose of 3 x 10 13 vp – potential exposure up to 1000 RCA

18. ALARA vs Risk Based Shift from ALARA to Risk-Based recommendation? What information do we have? –Literature, clinical experience with wild- type ad infection/reactivation –Clinical experience with gene transfer studies –Some notable adverse events of unknown cause but indications of innate immuniity What information do we need?

19. Wild-type Adenovirus in Bone Marrow Transplantation Adenovirus, including types 2 and 5, is a significant cause of morbidity and mortality in BMT Important to consider recipient immune status –Infection/Reactivation

20.  Neonatal adenoviral pneumonia: Sporadic, severe, localized outbreaks  SCID population at high risk: Severe morbidity and mortality  DiGeoge syndrome: case reports of fatal hepatic necrosis  Solid organ transplant: ‒Infection (rejection?) of transplanted organ ‒Source: reactivation and donor  AIDS patients – Co-infection with other pathogens – Diversity of serotypes isolated Lessons Learned from Patients with Primary or other Secondary Immunodeficiencies

21. Topics for Discussion Should recommendations regarding acceptable levels of RCA in adenovirus gene transfer products be the same for all clinical uses? –ALARA vs risk-based recommendation for RCA exposure –severely immunosuppressed or immunocompromised patients –mildly immunosuppressed or immunocompromised patients –patients with genetic defects such as hemophilia, cystic fibrosis, or other. What data should be used to set acceptable limits for RCA exposure? Should RCA measurements be performed on ex-vivo transduced cells?