Site Visit Update: Division of Therapeutic Proteins

1. Site Visit Update: Division of Therapeutic Proteins Amy Rosenberg, MD, Director

2. Amy Rosenberg, MD, Director Barry Cherney, Ph.D, Deputy Director Sandra Chung, Secretary Laboratory of Biochemistry Emily Shacter, Ph.D., Chief Jee Chung, Ph.D., IOTF Fellow Hiroshi Uehara, Ph.D., Biollgist Laboratory of Chemistry Gibbes Johnson, Ph.D., Chief Y-X Fan, Ph.D., Visiting Associate N. Spiridinov, Ph.D., Visiting Associate Lily Wong, Microbiologist Laboratory of Immunology Elizabeth Shores, Ph.D., Chief Baolin Zhang, Ph.D., Senior Staff Fellow Yaqin Zhang, MD Serge Beaucage, Ph.D., PI A Grajkowski, Ph.D., Visit Associate J. Cieslak, Ph.D., Visit Associate Michael Norcross, MD PI Jinhai Wang,MD Staff Scientist Gregory Rodriquez, Microbiologist Akhilesh Nagaich, Ph.D., Senior Staff Fellow Amy Rosenberg, MD PI Hugh McFarland, Ph.D, Staff Scientist Lai Xu, Ph.D., MD, Staff Fellow Karen Mason, Bio Lab Tech Full Time Review Staff Ingrid Markovic, Ph.D. Dov Pluznik, Ph.D.. Kathy Lee, MS Full Time Review Staff. Wei Guo, Ph.D. Kurt Stromberg, MD Emmanuela Lacana, Ph.D. • Edward Max, Ph.D., MD PI • Frederick Mills, Ph.D., Staff Scientist • Aruchandran, Ph.D. Microbiologist • Ralph Bernstein, Ph.D. Daniela Verthelyi, Ph.D., MD. Senior Staff Fellow J. Pedras-Vasconcelos, Visiting Associate M. Puig, Visiting Associate Vivian Wang, Microbiologist Raymond Donnelly, Ph.D., PI H. Dickensheets, Ph.D., Staff Scientist Faruk Sheikh, Ph.D. Visiting Associate Full Time Review Staff Susan Kirshner, Ph.D Ennan Guan, Ph.D., MD . The Division of Therapeutic Proteins

3. Laboratory of Immunology: Regulatory Responsibilities • Expert regulation of numerous products whose activity or origin is immune system based • Expert regulation of products regulated in other laboratories • Immunogenicity assessments on all licensed and IND products in DTP • Consultation to other FDA Centers regarding immunogenicity • Immunogenicity Guidance Documents: clinical concerns and assay guidance • Immunocomparability Guidance for follow-on proteins

4. Laboratory of Immunology: Regulatory Responsibilities - Products • Immunomodulators • Immunogenicity Assessments: all DTP products • (Wang, Bernstein, Mills, Norcross) • for autoimmune disease • (Mills, Bernstein, Norcross, Wang) • Innate immune system modulators • (Wang, Norcross) • Cytokines and Chemokines: • Interferons • (Bernstein, Wang, Norcross, Beaucage) • Interleukins (Norcross) Chemokines (Norcross)

5. Reseach Program Chemokine Biology HIV-1 Immunopathogenesis Cancer Biology Immunology and Autoimmunity Regulatory Program Regulation of chemokine products: potency assays, mechanism of action Regulation of protein therapeutics for HIV-1 treatment and prevention Cytokines and interferons for treatment and immune reconstitution Vaccine adjuvants Chemokine receptor inhibitors Regulation of protein therapeutics for treatment of malignant states Evaluation of immunogenicity of therapeutic protein products; TNF inhibitor products Research Program of Michael Norcross, MD

6. Michael Norcross, M.D. Accomplishments Since Site Visit Chemokines and Chemokine Receptors in HIV-1 infection and Immunology: Heterodimeric interactions between different types of chemokines and receptors have been studied. Expression vectors are being generated to dissect the interactions between CCR5, CXCR4, CD4 and CD26 using FRET techniques. Physical interactions between these cell surface proteins may control susceptibility to infection and sensitivity to receptor ligands and antagonists. Wang, J, Guan, E, and Norcross, MA. Heterodimer formation between CC and CXC chemokines (Manuscript in preparation) Immune modulation and HIV-1 pathogenesis: Studies are ongoing to examine the effects of HIV infection and HIV proteins on macrophage and dendritic cell function. HIV infection was found to induce partial dendritic cell activation that may contribute to chronic immune activation seen in HIV infected patients. Specific inhibitors of TLR receptors and scavenger receptors are being evaluated for effects on HIV mediated activation of monocytes and dendritic cells.

7. JinHai Wang, MD: Promotion Action • Research focus: chemokine receptors as host factors in HIV-1 infection and cancer biology • 6 first author publications since 2001 • 1 patent filed • Non-CpG Immune Modulators • 1 publication • 1 patent filed • CMC Review Work: INDs and BLAs for HIV-1, HCV, cancer. Immunogenicity reviews for therapeutic enzymes

8. Scientific Accomplishments during Mar. 2007-Jul. 2007Jinhai Wang, M.D. Presentation • Jinhai Wang. Immune Suppression and Regulatory Oligodeoxynucleotides. CDER, DTP seminar series, June 8, 2007 Patent application • Jinhai Wang, Michael Norcross, and Gregory Roderiquez (FDA). Inhibiting immune response by regulatory orthophosphorothioate non-CpG oligodeoxynucleotides. U.S. Provisional Patent Application, filed July, 2006. International PCT application to be filed July, 2007. Publications • Jinhai Wang, Gregory Roderiquez, Taneishia Jones, Peter McPhie, and Michael A. Norcross. Control of In Vitro Immune Responses by Regulatory Oligodeoxynucleotides through Inhibition of pIII Promoter Directed Expression of MHC Class II Transactivator in Human Primary Monocytes. J Immunol 2007Jul 1,179, 45-52 • Jinhai Wang, Jay Lozier, Gibbes Johnson, Susan Kirshner, Daniela Verthelyi, Anne Pariser, Elizabeth Shores, and Amy Rosenberg.Neutralizing Antibodies to Therapeutic Enzymes: Prediction, Prevention, and Treatment (Review). (Submitted to Nature Biotechnology). • Jinhai Wang.Dimerization of chemokine receptors in living cells: key to receptor functions and novel targets for cancer therapy (Review). (Under internal review) Projects on going • Immune regulatory ODNs: ------Receptor, trafficking, and regulation of monocyte functions by pDCs. Rhodamine-labeled rODNs were taken up by monocytes within minutes w/o pDC (Confocal). Labeled rODN entry into monocytes was not inhibited by anti-CXCL16 (Confocal & FACS). rODN was colocalized with lysosome marker within one hour (Confocal). rODNs induced FSC and SSC changes and prolonged the survival of monocytes significantly. • Chemokine receptors and cancer biology ------CXCR4, cancer factor, migration, and function of immune cells: a soluble factor secreted by breast cancer cells upregulated HLA-DR, downmodulated CXCR4on monocytes, blocked by anti-GM-CSF.

9. Research Program Class switch recombination IgH Insulators: prediction, prevention, control of immune responses Regulatory Program Immunogenicity of protein therapeutics review Immunomodulatory product review products to activate B cells in setting of immune deficiency product qualities associated with Tind and Tdep B cell activation, ie aggregates Evaluation of biotech applications using insulators to stabilize and enhance transgene expression; replacement of defective genes, prevention of oncogene activation. Research Program of Edward Max, Ph.D., MD

10. Update: Dr. Max’s group Class Switch Recombination project (Arudchandran, Bernstein): Additional experiments completed to compare double- and single-strand DNA breaks in Ig Switch regions; only double stranded breaks are AID-dependent. Manuscript submitted to J Exp. Med. “Single-strand DNA breaks in immunoglobulin Class Switch Recombination that depend on UNG but not AID” ChIP experiments to assess APOBEC3 binding to Ig Switch regions; RTPCR for APOBEC3 expression in B cells IgH Insulator project (Bernstein, Mills): In vitro binding of candidate regulatory proteins USF1 and DB1 to insulator DNA has been characterized Barrier activity flanking insulator further mapped Manuscript draft: “A chromatin boundary element downstream of the human immunoglobulin heavy chain locus.”

11. Ralph Bernstein, Ph.D., Conversion to Staff Scientist • Research focus: Insulator elements 3’ of the Murine 3’ IgH LCR. • 3 papers since 2001 • 3 manuscripts in preparation • CMC work: • primary reviewer of multiple therapeutic INDs; • on 4 committees for recently licensed DTP BLAs; • consult reviewer for CDRH and CDER licensed and IND protein products (not regulated by DTP) • Workshop presentations at multiple joint FDA/Industry meetings. • Co Moderator at 3 consecutive WCBP meetings • Techniques used to characterize tertiary structure: Tool Time for Tertiary Structure 2005 • Comparability: Same Pieces + Same Blueprint = Same Product. 2006. • Challenges associated with the Development of Product Specific Assays for Cleaning validation. 2007. • Presentations: • Expectations & Case Reports for Biological Product Quality, at the Biological Therapeutic Products Workshop. April-06 • Core Team Biologics TrainingOctober 21, 2004, ELISA and PCR assays • Multiple presentations of scientific and regulatory topics for FDA staff

12. Ralph Bernstein, Ph.D., Conversion to Staff Scientist • Inspection Coordinationfor DTP Products: • coordination of biennial inspections (with OC); • identification of issues for inspection and briefing of inspectors, • advice for field inspectors on scientific issues; • performance of multiple inspections • IT Coordinator for DTP • Approval of computer hard and software for regulators and researchers • Email and data base access for reviewers

13. Update: Ralph Bernstein IgH Insulator project: • Long-lasting barrier activity localized to a 0.5 kb DNA segment upstream of insulator • Study of DNase I hypersensitivity site upstream of murine insulator • Identification of insulator DNA fragments binding to DB1 and MAR-binding protein SATB2. • Preparation of a library of DNA fragments for an enhancer-blocking trap screen • Characterization of DNA methylation status of CpG sites in murine insulator • Critical contributions to two manuscripts

14. Laboratory of Chemistry: Regulatory Responsibilities - Products • Therapeutic Enzymes • Oncology Indications • (Beaucage) • Enzyme Replacement Therapies. • (Mills, Beaucage, Wang, Max) • Infectious Disease • (Norcross) • Counter Terrorism Enzyme • (Norcross) • Cystic Fibrosis • Pancreatic Insufficiency

15. Research Program Optimization of methods for production of diagnostic and therapeutic oligonucleotides Expertise in Chemistry Regulatory Program Co-author Oligonucleotide guidance document; Review of therapeutic oligonucleotides, siRNA INDs Review of BLA-PCR Based Diagnostic (CBER) Regulation of pegylated products: reaction kinetics, stability Regulation of therapeutic enzymes: enzyme activity, stability. Serge Beaucage, Ph.D., Progress Update

16. Scientific Accomplishments of Serge L. Beaucage Since the Site Visit Publications: 1. Grajkowski, A., Cieślak, J., and Beaucage, S. L. Functionalization of Long Chain Alkylamine Controlled-Pore Glass for Thermolytic Release of DNA Oligonucleotides and Their Conjugates. To besubmitted for publication in Bioconjugate Chemistry. 2. Ausín, C. and Beaucage, S. L. Synthesis of Deoxyribonucleoside Phosphoramidites Functionalized with Lipophilic and Hydrophobic Groups at P(III) for Incorporation into CpG-Oligonucleotide Produgs. To besubmitted for publication in the Journal of Organic Chemistry. 3. Cieślak, J., Grajkowski, A., and Beaucage, S. L. The 4-(N-dichloroacetyl-N-methylaminobenzyloxy)methyl group for 2’-Hydroxyl protection in the Solid-Phase Synthesis of Oligoribonucleotides. To besubmitted for publication in the Journal of Organic Chemistry. 4. Beaucage, S. L. Solid-Phase Synthesis of siRNA Oligonucleotides. To besubmitted for publication in Current Opinion in Drug Discovery & Development.

17. Scientific Accomplishments of Serge L. Beaucage Since the Site Visit • Invited Presentations at Symposia: • Assessment of Novel Phosphate/Thiophosphate Thermosensitive Protecting Groups in the Design of Thermolytic Oligonucleotide Prodrugs. EuroTIDES, Hotel Palace Berlin, Berlin, Germany, December 4-5, 2007. • Progress in the Synthesis of Thermolytic DNA Oligonucleotide Prodrugs and Oligoribonucleotides for Potential Therapeutic Applications. 41st IUPAC and CHEM-BIOTECH 2007 Congress in the Synthesis of Natural Products, Torino, Italy, August 5-11, 2007. • 4-Nitrogenated Benzyloxymethyl Groups for 2’-Hydroxyl Protection in the Solid-Phase Synthesis of siRNA Oligonucleotides. TIDES 2007 Oligonucleotides and Peptides Technology and Product Development, Red Rock Casino, Resort and Spa, Las Vegas, Nevada, May 20-23, 2007. • Thermosensitive Phosphate/thiophosphate Protecting Groups and the Development of Thermolytic Olifonucleotide Prodrugs. 17th International Conference on Phosphorus Chemistry, Xiamen, China, April 15-19, 2007.