1. 1 DCGT research priorities: Led by Critical Path Challenges Virology Retroviruses, lentivirus, adeno, filovirus Immunology Anti-viral immunity, immunobiology of cell therapy and xenotransplantation Cell biology Control of differentiation in animal models, stem cell biology Cancer biology Antigen characterization, Molecular biomarkers, animal models Biotechnology - Genomics, flow cytometry, proteomics Tissue Safety Program

2. 2 Division of Cellular and Gene Therapies Raj Puri, Ph.D., M.D., Division Director Kimberly Benton, Ph.D., Deputy Director Division of Cellular and Gene Therapies Raj Puri, Ph.D., M.D., Division Director Kimberly Benton, Ph.D., Deputy Director Cellular and Tissue Therapy Branch Steven Bauer, Ph.D., Chief Tumor Vaccines and Biotechnology Branch Raj Puri, Ph.D., M.D., Chief Cell Therapies Branch Keith Wonnacott, Ph.D., Chief Gene Therapies Branch Daniel Takefman, Ph.D., Chief Gene Transfer and Immunogenicity Branch Andrew Byrnes, Ph.D., Chief

3. 3 Tumor Vaccines and Biotechnology Branch (TVBB) Raj K. Puri, M.D., Ph.D. Cancer Biology and Genomics Program Syed R. Husain, Ph.D., Staff Scientist Bharat H. Joshi, Ph.D., Staff Scientist Michail Alterman, Ph.D. Proteomics Program Shyh-Ching Lo, M.D., Ph.D. Tissue safety Program

4. 4 Scientific and Regulatory Challenges in cancer Vaccines and Immunotherapy – Puri lab  Complex biology of cancer  Identification of appropriate targets and specificity of target  Appropriate tests and biomarkers for identity and potency of cancer therapeutics/vaccines  Animal models – safety and efficacy  Immune biomarkers of response and disease monitoring

5. 5 Research programs addressing scientific and regulatory challenges Specific Programs:  Characterization of tumor associated cell surface proteins (antigens or receptors) for identity and potency and target these receptors for cancer therapy  Animal models of human cancer to assess safety and efficacy of tumor targeted agents including cancer vaccines, immunotoxins, cell and gene therapy products  Characterization of cancer vaccines and embryonic stem cells by genomics technology to identify cancer stem cells and biomarkers of identity and potency and response to cancer vaccines

6. 6 Characterization of tumor associated cell surface receptors and antigens Discovery of overexpression of Th-2 derived cytokine receptors in tumors  Interleukin-4 receptors  RCC, malignant glioma, AIDS-Kaposi’s sarcoma, colon cancer, breast carcinoma, NSCLC, prostate cancer, ovarian carcinoma, mesothelioma, hematological malignancies (CLL-B), PDA, biliary ductal cancer and SCCHN  Interleukin-13 receptors  RCC, malignant glioma, pediatric glioma, AIDS-Kaposi’s sarcoma, SCCHN, ovarian carcinoma, PDA, pheochromocytoma, brain stem glioma and prostate cancer

7. 7 Animal models of human cancer to assess safety, toxicity and efficacy of cancer targeted agents   Ovarian Cancer   Pancreatic cancer   Glioblastoma multiforme   SCCHN   Mesothelioma   Lung cancer   AIDS-Kaposi’s tumors   Breast cancer   Hodgkin’s lymphoma   Melanoma   Pheochromocytoma Pheochromocytoma

8. 8 Conclusions and Ongoing Projects  Certain human tumors express high levels of IL-4R and IL- 13R in vitro and in vivo; the structure and signal transduction through these receptors is different in cancer and normal cells.  IL-4R and IL-13R can be effectively targeted by immunotoxins by themselves or when combined with other agents for cancer therapy.  IL-13R  2 is involved in PDA invasion and metastasis and may be a prognostic biomarker of disease.  IL-13R  2 can signal through AP-1 pathway and regulated by Adrenomedullin in vitro and in vivo.  IL-13R  2 is a novel tumor rejection antigen - IL-13R  2 cDNA vaccination induces immune response in preventive and therapeutic murine cancer models.

9. 9 Analytical proteomics for the characterization of biologic products and discovery of biomarkers Michail Alterman, Ph.D. Senior Investigator, Tumor Vaccines and Biotechnology Branch, Division of Cellular and Gene Therapies Laboratory Goal: Development and application of mass spectrometry- based proteomic tools for qualitative and quantitative measurement of cellular products, cell substrates and vaccines and discovery of potential biomarkers of cancer Development and application of mass spectrometry- based proteomic tools for qualitative and quantitative measurement of cellular products, cell substrates and vaccines and discovery of potential biomarkers of cancer

10. 10 Mission relevant questions to be impacted by application of proteomics tools   Implementation of new better, clearer and more defined means for regulatory decision making in evaluation and review of cell substrates, vaccine products and other biologics   Development of valid protein biomarkers for examining drug safety and efficacy through prediction of therapeutic effect, selection of appropriate dose, and identification of risk of toxicity.   Development of improved strategies for biotechnology product physicochemical comparability assessments   Development of effective tools to be used in biologics and protein therapeutics manufacturing process controls and release testing

11. 11 Specific aims and achievements since Site Visit in April 2010 Specific Aim # 1. Development of mass spectrometry-based analytical tools for testing of biological product quality and identity. Manuscript published in Analytical Biochemistry Specific Aim # 2. Identification of proteomics-based cellular molecular signatures that may be tested as predictors of therapeutic success and be developed as biomarkers of safety and effectiveness in preclinical animal models. Invited talk at an international meeting, one poster presented at an international meeting and one at a national meeting, two manuscripts in preparation Specific Aim #3. Proteomics-based analysis of influenza virus and vaccines. One invited seminar, manuscript in preparation

12. 12 Conclusions  A new label free quantitation technique was developed that allows determination of a ratio of co-migrating in gel electrophoresis proteins  Two prospective protein groups were identified that could be used as biomarkers for characterization of cell substrates  MS-based proteomics methods can be used to identify/distinguish different influenza variants and clades and quantify proteins in flu vaccines and virus preparations  A comprehensive proteome map of human MSC is under development; a number of prospective biomarkers of cell sub-culturing was found among >4000 identified proteins

13. 13 Ongoing projects 1. Development of a new potency/stability assay for flu vaccine. 2. MSC project: i. identify protein signatures characteristic for MSC from multiple donors ii. identify molecular signatures that could be used to discriminate product that produced a desirable clinical outcome

14. Tissue Microbiology Laboratory for Safety of Human Tissues Intended for Transplantation Shyh-Ching Lo, Ph.D., M.D. SV Report (2010) for The Tissue Laboratory Program of Tumor Vaccines and Biotechnology Branch

15. 15 Public Health, Regulatory Challenges and Critical Path Scientific Research: To Better Understand The Scientific Trends and Further Strengthen The Dialogue with The Stakeholders of Human Tissue  Increasing number of tissues are recovered and processed as grafts by the industry and used by the medical practice each year.  The OCTGT within CBER regulates human cells, tissues, and cellular and tissue-based products (HCT/Ps) in order to prevent the spread or transmission of communicable diseases.  The scientific capability of the new program needs to cover a wide spectrum of expertise with very different microbial pathogens, including bacteria, fungi, viruses and protozoa parasites.

16. 16 Research Programs of Tissue Laboratory Specific Aims:  To establish the required scientific capabilities to directly support regulatory needs for tissue safety  To adopt new technologies for rapid detection of infectious pathogens with high sensitivity in human tissues being processed for transplantation  To evaluate innovative methods for effective inactivation of various pathogens while preserving tissue quality for clinical needs  To explore new scientific approaches for detection and characterization of previously unknown or newly emerging infectious pathogens that would likely threaten the safety of human tissue grafts

17. 17 Laboratory research projects initiated to accomplish the specific aims:  Establishment of the laboratory capability of clinical microbiology for detection and characterization of microbes with tissue safety concerns -- Investigation of incidents and validation of industrial sterilization methods  Development of rapid, highly sensitive technology for detection of pathogens in tissues intended for transplantation -- Real-time qPCR arrays of the targeted high-risk bacteria and RCDAD viruses  Development of high-throughput genomic sequencing capability for detection and characterization of previously unknown infectious pathogens in tissue or tissue-based products -- Readiness of scientific capability against potential newly emerging threats of public health  Identification of “biomarkers” associated with the injury mechanisms in cell/tissue and development of the injury- associated microarray -- Assessment of the degree of tissue injury in pathogen inactivation using various chemical treatments

18. 18 Conclusions and Ongoing Projects  A new tissue microbiology laboratory has been established under the DCGT and the DHT to enhance both the safety and the availability of high quality human tissue intended for transplantation.  Development of New Technologies and evaluation of innovative methods is being implemented to guard the safety and quality of tissue intended for transplantation

19. 19 Thank you