Responses to FDA Gene Therapy Letter : Adenovirus Vector Titer Measurements and RCA Levels

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

2
(No Transcript)
3
(No Transcript)
4
Purpose

• 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

5
Adenovirus Vector Final Product
non-infectious particles
IU replication defective, infectious particle
RCA replication competent, infectious particle
VP all vector particles
6
Characterization of Particles in Adenovirus
Vector Products

• VP physical/chemical measurement
• example A260 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
Master Cell Bank
ARMWG Meetings March 22, 2001 May 31, 2001
Ad5 wt virus
Master Viral Seed Stock
Production of purified formulated bulk virus
Characterization, safety testing, provisional
titer
Vialing
Particle and Infectivity Titer Determinations,
Stability
Repository and Distribution
12
Purpose

• 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

13
Changes in Recommendations

• ALARA
• As Low As Reasonably Achievable
• 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
• lt 100 vp/iu
• Current Recommendation
• Based on review of production lot data from March
  6 letter responses
• lt 30 vp/iu
• (gt3.3 iu)

15
Change in Recommendation on RCA Limit

• Previous recommendation
• imprecise since based on infectious titer
  lt 1RCA / 109 iu
• Current recommendation
• based on particle number
• better precision
• lt 1RCA / 3 x 1010 vp

16
Purpose

• 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

17
Application of RCA Recommendation

• lt 1RCA / 3 x 1010 vp
• recommended for all adenovirus vector lots
  regardless of clinical use
• Exposure risk at highest current doses
• dose of 3 x 1013 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
Lessons Learned from Patients with Primary or
other Secondary Immunodeficiencies

• 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

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?