1. Gene Transfer and Immunogenicity Branch CTGTAC Meeting November 29, 2012 Andrew Byrnes, PhD, Senior Investigator, Branch Chief Carolyn Wilson, PhD, Senior Investigator, CBER ADR Suzanne Epstein, PhD, Senior Investigator, OCTGT ADR Graeme Price, PhD, Staff Fellow Jakob Reiser, PhD, Senior Staff Fellow Wu Ou, MD, Staff Fellow Cheng-Hong Wei, PhD, Visiting Scientist 1

2. 2 Overview of the Gene Transfer and Immunogenicity Branch Examples of regulated products Gene therapy products T cell products Stem cell-derived products Therapeutic vaccines Xenotransplantation products Mission relevance of research Improving product safety and efficacy Developing better preclinical models Characterizing complex products Addressing other FDA and HHS priorities: Pandemic influenza Counter-bioterrorism

3. 3 Overview of the Gene Transfer and Immunogenicity Branch Research topics Virology and gene therapy Gamma retroviruses Lentiviral vectors Adenoviral vectors Influenza virus Ebola virus Immunology Immune regulation Autoimmunity Immune responses to viral and plasmid vectors

4. 4 Viral safety studies in xenotransplantation Carolyn Wilson

5. Xenotransplantation raises public health concerns 5

6. 6 What are the viral and cellular determinants that allow PERV to infect human cells? Goals: 1) Identify regions of viral envelope important for human cell entry 2) Identify structural features of receptor for PERV-A required for viral infection

7. Major finding: Human cells express receptors for three classes of PERV IMPLICATIONS FOR XENOTRANSPLANTATION: Our data suggest that PERV-C envelopes may adapt to use the human PERV-C receptor through mutation of a single residue Argaw, et al, Journal of Virology, 2012 7

8. Identify structural features of receptor for PERV-A required for viral infection PERV-A receptor = Riboflavin Transporter 8

9. 9 Cross-linking suggests multimeric PERV-A receptor (huPAR2) SIRC SIRC-huPAR2 BS3: - - 5 10 mM Multimerization required for infection?

10. Determine structural and functional attributes related to infectivity and multimerization Library of huPAR-2 cDNAs with Cys-Ser mutations Express in non-permissive cells Initial Screen: Receptor expression Infectivity titer Potential Impact of Studies: Improved understanding of cellular factors that influence human cell infection If 1000-fold or greater decrease in titer: Determine multimerization 10

11. 11 Universal influenza vaccines Suzanne Epstein Graeme Price

12. Influenza, the public health problem  High mortality from seasonal outbreaks, concern about pandemics. Strain-matched vaccine can be delayed or insufficient Work in this program: Universal influenza vaccines  Cross-protection in animals by nucleoprotein (NP) and/or matrix 2 (M2) expressed by plasmid DNA or recombinant viral vectors.  Investigation of possible cross-protection in humans Relevance to CBER’s public health mission  Center-wide priority on control of epidemic and pandemic influenza  Gene therapy and tumor vaccines: Need to understand immune responses to viral and plasmid vectors; therapeutic or interfering.  Counter-bioterrorism: Control of emerging infections without knowing which strain is coming 12

13. Single dose mucosal emergency vaccine candidate protects mice against H5N1 Strong antibody and T cell responses induced, including locally in lungs. Protection against challenge with 10 LD 50 of A/VN1203 (H5N1) 10 months post-immunization Protection also seen as early as 2 weeks post-immunization 13

14. Cross-protection by NP+M2 vaccinations in animal models  NP and/or M2 universal vaccine candidates protect mice against divergent influenza virus strains, including H1N1, H3N2, and H5N1.  In mice, intranasal rAd induces strong mucosal responses (IgA, lung T cells), strong protection against challenge.  Prime-boost vaccination to A/NP+M2 protects ferrets against H5N1 challenge, with reduction in nasal virus shedding.  Mouse model of transmission demonstrates that NP+M2 vaccination reduces spread of infection to contacts. Suggests vaccination could protect both recipients and the community. 14

15. Human influenza surveillance study during the 2009 pandemic Cross-protection in humans? Did cross-reactive immune memory from past influenza exposures provide some protection against the 2009 pandemic strain? Baseline sera and PBMC collected, donors monitored during fall 2009 pandemic wave. Those with influenza-like illness tested for virus. Mild outbreak; few cases in cohort. With Jack Gorski, Blood Center of Wisconsin Analytical phase now in progress. T cells, cytokines, antibodies 15

16. Public health implications: Broad cross-protection for control of influenza Broadly protective influenza vaccines could be used off the shelf early in an outbreak, when matched vaccines are not yet available, and could perhaps someday be used routinely. Reduce illness, death, viral titers, spread of infection With recovery from mild or asymptomatic infection, people would make antibodies to the strain circulating locally 16

17. 17 Immunoregulation and T cell tolerance induction Cheng-Hong Wei NOD

18. 18 Focus of research In vivo expansion of regulatory T cells (Tregs) prevents type 1 diabetes in mice Assays to measure the immunosuppressive activity of human MSCs

19. 19 Regulatory T cells (Treg cells) Foxp3+CD4+CD25+ regulatory T (Treg) cells are essential for the control of autoimmunity. Treg cells can suppress the proliferation and function of effector T cells. Therefore, Tregs are viewed as promising therapeutics for prevention/treatment of autoimmune diseases, transplant rejection

20. Major Findings (I) 1.A new combined regimen can significantly expand CD4+CD25+ regulatory T cells in vivo. Peptide + IL2 antibody complex + rapamycin 2.The expanded Tregs express a classical CD4+CD25+ Treg phenotype and are functionally suppressive both in vitro and in vivo. 3.Most importantly, the combined regimen significantly protects NOD mice against both spontaneous and induced type 1 diabetes. 20

21. MSC = Multipotent Stromal Cells (or Mesenchymal Stem Cells) 21

22. Major Findings (II) 1.Currently, there is a need for potency assays that measure the immunosuppressive activity of human MSCs. 2.In this work, we find that human MSCs can alter multiple aspects of clonal murine T cell activation, including: Proliferation Surface activation markers Cytokine production Transcription factors 3.Therefore, clonal murine T cells can be used to measure the immunosuppressive activity of human MSCs. This is a promising approach to measure product potency and to elucidate the mechanisms for immunosuppression 22

23. 23 Lentiviral vector safety and targeting Jakob Reiser Wu Ou

24. 24 Background The goal of the Reiser lab is to develop safer HIV-1-based lentiviral vectors by: Directing vector integration to sites in the human genome that are devoid of proto- oncogene/tumor suppressor sequences (“safe harbor sites”) Narrowing the vector’s tissue tropism through targeted transduction

25. 25 Targeted lentiviral vector integration Ongoing approach: Use of integrase-defective lentiviral vectors bearing long homology arms to mediate homologous recombination (HR) at genomic “safe harbor” sites (such as the AAVS1 site) Future plans: –To improve HR frequencies using zinc finger nickases –To use zinc finger recombinases to mediate site-specific transgene integration

26. 26 Fusion protein Cell-specific protein ligand (e.g. IL-13) Cell-specific RNA aptamer (e.g. anti-IL-13R  2 aptamer) Receptor (e.g. IL-13R  2) Targeted lentiviral vector transduction: General outline

27. 27 Targeted lentiviral vector transduction Future plans: –To further pursue IL-13R  2-positive tumor cells as a model for targeted vector delivery in vivo –To design high-affinity RNA aptamers to allow high-efficiency targeting of IL-13R  2- positive cells in vivo

28. 28 Adenovirus Vector Biodistribution and Toxicity Andrew Byrnes

29. 29 Adenovirus: A popular vector in clinical trials A large number of active adenovirus INDs: ~ 50 Ad gene therapies and oncolytic Ad vectors ~ 30 Ad-based vaccines Two approved adenoviruses in China Gendicine: p53-expressing Ad vector H101: replication-selective oncolytic Ad

30. 30 Ad clearance from the circulation Our focus is on systemic gene therapy with adenovirus vectors in vivo We study Non-replicating adenovirus vectors (Ad5) Administered intravenously to rodents The potential Gene delivery to any organ or tumor The reality Poor pharmacokinetics Acute toxicity due to innate immune activation

31. 31 Research topics Adenovirus biodistribution Opsonization by plasma proteins Transduction of hepatocytes Clearance of vector by Kupffer cells Safety of adenovirus gene therapy Complement activation Shock MAP kinases

32. 32 Plasma proteins that opsonize Ad Adenovirus IgM Complement C1q Complement C3b Coagulation Factor X Drawn to scale

33. Ad and the innate immune system Overall goals: Understand how innate defense mechanisms affect safety and efficacy Use this information to develop better vectors 33

34. Questions? Carolyn Wilson Porcine endogenous retroviruses Suzanne Epstein and Graeme Price Universal influenza vaccines Cheng-Hong Wei Immunosuppression by Tregs and MSCs Autoimmunity / tolerance in diabetes Jakob Reiser and Wu Ou Lentiviral vector safety and targeting Andrew Byrnes Safety and efficacy of adenovirus vectors 34