Gene Transfer and Immunogenicity Branch Site Visit March 2, 2007

1. Gene Transfer and Immunogenicity BranchSite Visit March 2, 2007 • Eda T. Bloom, PhD, Chief • Andrew P. Byrnes, PhD, Senior Staff Fellow • Suzanne L. Epstein, PhD, Senior Investigator • Nancy S. Markovitz PhD, Senior Staff Fellow • Carolyn A. Wilson, PhD, Senior Investigator • Takele Argaw, DVM, Staff Fellow

2. Problems in Development of Cell and Gene Therapy Products • Lack of preclinical models to predict performance of gene and cell therapy products in vivo • Potential for transmission of infectious agents to the patient and beyond • Products often intended for lengthy or permanent presence in the recipient • Complicated manufacture and structure of products • Challenging product characterization and testing

3. The Challenges for GTIB • Product safety is affected by virus-containing products • Unintended replicating viruses in viral vectors, xenotransplantation, and other products • Public health concern of spread beyond the patient • Viral vectors carry inherent risks (e.g., toxicity, tumorigenicity, off-target effects) • Immunogenicity impacts safety and efficacy • Immune responses to viruses and transgene products, xenotransplantation and cell therapy products • Immune activity of certain immunotherapy products

4. GTIB Addresses Challenges Through Critical Path Research • Multiple viral systems, e.g., adenovirus, filovirus, influenza virus, herpesvirus, retrovirus • Flexible systems to address immunogenicity and function of many OCTGT products • Predictability comes from understanding: • Fate and effect in vivo • Role of structure and function in safety • Interaction with immune system

5. Summary of Individual Programs Presented at the March 2 Site VisitandRegulatory Impact

6. Immunobiology of Cellular Therapy and Xenotransplantation Products: Immunogenicity Issues (Bloom) autologous xenotransplantation allotransplantation Autologous cellular therapy – cells themselves may be intended to be immunologically active, e.g., NK or T cell immunotherapy Primarily T cell, and sometimes NK cell immunity (e.g., hES cells & progeny), Ab • Immunity includes NK cells and T cells, and Ab • Regulatory T cells inhibit CD4+ T cell response

7. To understand role of Tregs in xenotransplantation in vivo: A preclinical model will be important Expanded baboon CD4+CD25+ T reg cells strongly suppress proliferation and cytokine production of baboon CD4+CD25- effector cells

8. Impact on Cell Therapy and Xenotransplantation Products • NK cells must be considered as immune mediators for rejection of both xenogeneic transplants and certain allogeneic cell therapy products • Efficacy may be affected by • Type(s) of immune response(s) of recipient • Microenvironment in patient • Studies of patient immune responses in vitro may predict immunity in vivo • Could serve as biomarkers of clinical benefit

9. Ad vectors are taken up by Kupffer cells, which then die Kupffer cells Control liver After i.v. injection of Ad vector Safety and biodistribution of adenovirus vectors (Byrnes) • >80 active INDs in US for adenovirus vectors • Poor pharmacokinetics after systemic injection • Rapid clearance of Ad by the liver • Inefficient therapy, toxicity

10. New assay for quantitating KC uptake of Ad Scavenger receptors are important for KC uptake of Ad How do Kupffer cells recognize adenovirus so well?

11. Critical Path issues in adenoviral gene therapy • Kupffer cells prevent Ad gene therapy from reaching its full potential • Goals: • Understand how Kupffer cells recognize adenovirus • Rationally develop strategies to block Kupffer cells in order to enhance gene delivery and decrease vector toxicity

12. Influenza, the public health problem (Epstein) High mortality from seasonal outbreaks, concern about pandemic (subtype new to humans). Vaccine supply delayed or inadequate. Work in this program: New vaccine technologies can cross-protect broadly against divergent influenza A subtypes. Historical data suggest possible cross-protection in humans during the pandemic of 1957. Producing vaccines Maintaining the cold chain

13. M2 can protect against a lethal H5N1 challenge Challenge with A/Thailand/SP83/04 17 days after boost DNA+rAd Morbidity also reduced (body weight)

14. Implications for public health and product regulation • Addresses DHHS and center-wide priorities on: • Control of epidemic and pandemic influenza. • Counter-bioterrorism: control of emerging infectious disease without having to know which strain is coming • Safety and efficacy impact in gene therapy: antibody and T cell responses to viral vectors can block efficacy, cause immunopathology.

15. Identity and Safety Studies of Herpes Simplex Viruses and Vectors(Markovitz) • Identity/Structure Replicating viruses used in cancer therapy studies contain unreported mutations. • Safety In contrast to what is frequently reported, HSVs similar to those in clinical trials doreplicate in normal brain cells in mice.

16. HSV-1(F) R3617 MW 45 [ UL3 WT ] UL3DC 31 1 2 Unexpected Mutations in Herpes Simplex Virus for Clinical Use • R3617 is the parent strain of virus G207, used in clinical trials. • Sequence analysis proved that the mobility shift was due to a single base substitution • Resulted in the truncation of the UL3 protein. Dambach et al. (2006) Molecular Therapy 13(5): 892-899.

17. High fatality rate; rapid disease progression. • No proven cure or vaccine • Infection suppresses both the innate and adaptive immune responses • Exacerbates pathogenic outcome • Prevents development of protective immune response DHHS high Priority Bioterrorism Agents Zaire Ivory Coast Sudan Reston Ebolavirus Filoviridae (Wilson) Marburg virus

18. Zaire Sudan VSV-G VSV-G Ivory Coast Anti-F88 MAbs Neutralize Three Ebolavirus Species* *Shown here with retroviral vector pseudotypes, now extended to WT Ebolavirus

19. Impact on Counter-Bioterrorism:Availability of Antibodies for Passive Transfer Targeting conserved epitopes: • Block Entry → Reduce virus burden • Critical for Entry → Avoid potential for immune escape mutants • Broad protection to Filovirus sp. Complements vaccine strategy: • Provides immediate protection in the event of an outbreak (natural or BT)

20. National Toxicology Program (NTP):Safety assessment of retroviral vectors for risk of tumorigenicity • Determine the sensitivity of a preclinical model for detecting retroviral vector-mediated insertional tumorigenesis • Impact of vector backbone, dose, and enhancer deletion on tumor frequency • Will assess retroviral vectors representing three different retroviral genera: • Gammaretrovirus (MLV) • Lentivirus (HIV) • Spumavirus (HFV)

21. Xenotransplantation: Identification of Porcine Endogenous Retrovirus Determinants Critical for Human Cell Tropism (Argaw)

22. Regulatory Impact of OCTGT Retrovirus Research • Addressing Risks Associated with Retroviral Vectors: • Identification of model to assess insertional mutagenesis from vector • Using model to assess relative risks, based on different vector types, dose, and structure • Porcine Xenotransplantation Products • Identification of residues required for transmission of porcine endogenous retrovirus may allow development of means to block infection and reduce risk of transmission

23. Summary - GTIB Research: Addressing Regulatory Challenges • Gene therapy vector safety • Xenotransplantation safety • Cellular therapy and xenotransplantation product efficacy • Center-wide and Departmental priorities • Pandemic influenza • Counter terrorism