Cellular and Tissue Therapies Branch (CTTB) Site Visit November 3, 2006

1. Cellular and Tissue Therapies Branch (CTTB)Site Visit November 3, 2006 • Steven R. Bauer, PhD, Chief • Deborah Hursh, PhD, Senior Staff Fellow • Gerald Marti, MD, PhD Senior Investigator • Brenton McCright, PhD, Senior Staff Fellow • Malcolm Moos, PhD, MD Senior Investigator • John Terrig Thomas, PhD Visiting Scientist

2. The Problems • Clinical benefit is highly variable, often hard to demonstrate • In many cases, most cells administered die immediately • Products may ‘misdifferentiate’ • Inadequate supply

3. Cell Therapy Challenges • Poor understanding of how cells interact with their microenvironment • Inadequate markers predictive of cell state and cell fate • Poor survival of cells post transplantation

4. CTTB Approaches • Complementary Systems • Frogs, Flies, Mouse, and Man • Gene, Protein, Cell, Tissue Interactions • Normal Development and Tumorigenicity • Knowledge and manipulation of growth factor pathways • Knowledge and utilization of transformation pathways

5. Microenvironment in cell therapy: manufacturing, patient - Bauer Pre-B Remove IL-7 In vitro: reduced stromal dlk No differentiation or apoptosis upon removal of IL-7 No changes in typical pre-B markers In vivo effects: Dlk1-/- mouse Alters B-cell development and function Abnormal stroma=> Abnormal B cells

6. Systematic approach for identification and qualification of tumorigenicity biomarkers for cell therapy products- Bauer • Establish normal, neoplastic and preneoplastic pre-B cells • Mechanisms of transformation • Identify biomarkers of transformation

7. Impact for Cell Therapy • Stroma can alter cell product in a way that is not revealed in lot release tests • Efficacy may be affected by • Microenvironment during cell product manufacturing • Microenvironment in patient • Improved tumorigenicity assessments

8. Mammalian organogenesis as a model for cellular and tissue engineered therapies – McCright Approach:Genetically modify the mouse to the study function of proteins required for mammalian organ development in vivo. • Mouse models created: • Mice that allow us to either inactivate or over express Notch2 in a tissue specific manner • Mice that allow us to isolate stem cells based on their expression of Notch2 • Mice to study a putative anti-oncogene, B56gamma Genetic modification of the mouse by blastocyst injection

9. Notch2 expression and heart specific inactivation demonstrates a cell autonomous requirement for Notch2 during murine heart development Notch2 inactivation results in developmental defects in the outflow tract and the right ventricle Notch2 is highly expressed in the embryonic heart Notch2- Notch2+ pa pa * * Hearts from newborn mice, 2/3 of the mice with Notch2 heart specific inactivation die perinatally Notch2-bgal expressing cells stain blue with Xgal in the E17 heart

10. Impact for Cell Therapy • Identification and analysis of molecules required for mammalian organogenesis • Notch2 as a biomarker for evaluating the developmental potential of cells used in cardiac repair. • Notch1 or Notch2 activation may have similar effects on cell products. • Exogenous Notch activation and the functional requirements for Notch2 can be studied in most tissues

11. A Genetic Model of Growth Factor Action to Develop Markers of Safety and Efficacy of Cell-Based Products-Hursh • A system to study cell communication in intact tissues • Ability to alter gene expression within a microenvironment • High throughput screens identify critical control points • Markers predictive of pathway activity • Analyze cell stress and viability • Markers predictive of survival

12. Genetic interaction screens identify and place genes in a functional pathway • Asks the organism to identify critical control points. • Avoid bias of abundance, immunogenicity, modifications. • Model organisms allow screens of sufficient size. • Knowledge of control points for cellular products is critical. • identified > 20 genes that interact with BMP signaling.

13. Loss of BMP causes cell death, induces JNK pathway: a model of cell competition • TGF-ß/BMP, insulin, EGF serve • as cell survival factors. • Loss of growth factor causes • inability of cells to “compete” • with normal neighbors, can cause • apoptosis, and overgrowth Mut, caspase ,JNK activity WT,,caspase

14. Impact for Cell Therapy/Tissue Engineering • Improve our ability to predict the survival of transplanted cells in their new location • Cell interactions in tissue development

15. Protein: Protein Interactions in Joint Development- Moos Proprotein Convertases and GDF5 colocalize to establish normal joint structure

16. A novel BMP antagonistcopurifiedandcolocalizeswithCDMP-1/GDF-5 Joint interzones

17. Feedback and Crosstalk in Cell and Tissue Specification • Colocalization of several signals is necessary to instruct formation of articular cartilage • Developmental signals in characterization of cell and tissue engineering products

18. Chronic Lymphocytic Leukemia in Mouse and Man- Marti • Molecular Lesion in CLL • Precursor states: Monoclonal B Cell Lymphocytosis (MBL) • Familial Chronic Lymphocytic Leukemia (CLL) • NZB mouse model of CLL • Shared micro RNA lesion in human and mouse CLL • Biomarker: ZAP70 • Quantitative Flow Cytometry (QFCM) • Cell therapy product characterization • Flow cytometry-based cell separation • Standards Development: • Fluorescence reference materials • CLSI documents

19. MicroRNA lesions in Human CLL and Mouse model of CLL Mir16-1 mutations in Mouse and Man • NZB model of CLL reveals similar mutation • - Identification of critical lesion in CLL • Animal model for elucidation of mechanisms of transformation • Improved diagnosis/treatment of CLL

20. Applicability • Early Detection: • Molecular lesions • Leukemogenesis • Intervention • Product Characterization • In-process • Lot Release • Cellular and Gene Therapy Products • Clinical Use of Flow Sorted Cells • Quantitative Flow Cytometry (QFCM) Standards • Instrument/Assay Performance • Linearity of detectors • Compensation Controls

21. CTTB Research: Addressing Cell Therapy Challenges • Complementary approaches • Cell-Cell interactions • Genetic interaction screens • Protein-Protein interactions • Organogenesis • Tumorigenesis