1. Gene Transfer and Immunogenicity Branch Site 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. 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. 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. 5 Summary of Individual Programs Presented at the March 2 Site Visit and Regulatory Impact

6. 6 Immunobiology of Cellular Therapy and Xenotransplantation Products: Immunogenicity Issues (Bloom) Immunity includes NK cells and T cells, and Ab Regulatory T cells inhibit CD4+ T cell response 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 xenotransplantation allotransplantation autologous

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

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

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

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

12. 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. Maintaining the cold chain Producing vaccines

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

14. 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. 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 do replicate in normal brain cells in mice.

16. 16 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. Unexpected Mutations in Herpes Simplex Virus for Clinical Use MW HSV-1(F) 45 31 R3617 1 2 UL3  C ] UL3 WT [ Dambach et al. (2006) Molecular Therapy 13(5): 892-899.

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

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

19. 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. 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. 21 Xenotransplantation: Identification of Porcine Endogenous Retrovirus Determinants Critical for Human Cell Tropism (Argaw)

22. 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 Gene therapy vector safety Xenotransplantation safety Xenotransplantation safety Cellular therapy and xenotransplantation product efficacy Cellular therapy and xenotransplantation product efficacy Center-wide and Departmental priorities Center-wide and Departmental priorities Pandemic influenza Pandemic influenza Counter terrorism Counter terrorism