# Presentation: 182285

## 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**

## Slide 2

**Notes:**
Active INDs
Complex products produced by biological systems
Briefing package:methods used to construct and then produce large amounts of these vectors
Vectors range in size from 3 kb up to 300 kb 
Major FDA concern: structure of the vector, also intermediates used  for such vector construction: examples are  plasmids for adenovirus and  retrovirus.   
Four major points for discussion later today will be 
How much structural characterization should be done
What kinds of structural characterization should be done
When in the course of product development it should be done.  
What should be done with sequencing information

Two major milestones during product development where this type of analysis is done
before initiation of a clinical trial
before licensure of a product

## Slide 3

| Clinical Indication | Route of Administration | INDs |
| --- | --- | --- |
| Cancer | Intratumoral/Intralesional | 29 |
|   | Ex vivo transduction | 13 |
|   | Intravascular | 2 |
|   | Intraperitoneal/pleural | 2 |
|   | Oral | 1 |
| Coronary / Vascular  | Myocardial | 3 |
|   | Intramuscular | 2 |
|   | Intracoronary | 1 |
| Genetic defect | Respiratory tract | 3 |
|   | Intravascular | 1 |
| +Normal | Intradermal | 1 |
|   |   | 58 |

## 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 

**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**

## Adenovirus Vector Final Product

**RCA: ****replication competent, infectious particle**

**IU: ****replication defective,**** ****infectious particle**

**non-infectious particles**

**Adenovirus Vector Final Product**

**VP: ****all vector particles**

## 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**

## Impact of VP, IU, RCA: Safety, Efficacy, Production

- Safety
- replication competent virus
- exposure to viral proteins
- Efficacy
- non-transducing particles
- Production process

**Notes:**
Replication competent viruses can be detected by assays that detect infectious recombinants

Other types of genetic alterations would not be detected by infectivity assays
Altered transgene:
 change in transgene function, pathogenic
 immunity/autoimmunity 
stop codons
Altered vector backbone:
	 inserted open reading frame
changed transcriptional control

## 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**

## 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**

## 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**

## 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**

**Notes:**
RFPs:   www.wilbio.com
1. Cell Bank Donation: Univ. of Alabama at Birmingham 
2. Viral Material Donation: Canji, Inc. 
3. Donation of Supplies or Other Services
4. Viral Bank Production and Testing Donation: Canji, Inc. 

5. Purified Formulated Bulk Virus Reference Material Production and Release Testing Donation: Introgen, Inc. 

6. Vialing and Freezing Donation: Introgen, Inc. 

7. Donation of Repository Services: ATCC

8. Participation in Assignment of Particle Concentration
9. Participation in Assignment of Infectious Titer
10. Participation in Characterization of Reference Material - Other Characterization
11. Short-term and Field Stability Studies
12. Long-term Stability Studies
13. Donation of Supplies for Characterization Phase

## 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 

**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**

## 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**

## 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)**

## 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**

## 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 

**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**

## 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**
**	****	****	**

## 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?**

## 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

## 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

**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 **

## 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?**