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

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

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

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

5. Microenvironment in cell therapy: manufacturing, patient - Bauer 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 Pre-B Abnormal stroma=> Abnormal B cells Remove IL-7

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

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

8. Approach: Genetically modify the mouse to the study function of proteins required for mammalian organ development in vivo. Mammalian organogenesis as a model for cellular and tissue engineered therapies – McCright Genetic modification of the mouse by blastocyst injection Mouse models created: 1)Mice that allow us to either inactivate or over express Notch2 in a tissue specific manner 2)Mice that allow us to isolate stem cells based on their expression of Notch2 3)Mice to study a putative anti- oncogene, B56gamma

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

10. Impact for Cell Therapy Identification and analysis of molecules required for mammalian organogenesis Identification and analysis of molecules required for mammalian organogenesis Notch2 as a biomarker for evaluating the developmental potential of cells used in cardiac repair. 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. 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 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 A system to study cell communication in intact tissues Ability to alter gene expression within a microenvironment Ability to alter gene expression within a microenvironment High throughput screens identify critical control points High throughput screens identify critical control points Markers predictive of pathway activity Markers predictive of pathway activity Analyze cell stress and viability Analyze cell stress and viability Markers predictive of survival Markers predictive of survival

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

13. Loss of BMP causes cell death, induces JNK pathway: a model of cell competition WT,,caspase Mut, caspase,JNK activity 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

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

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

16. Joint interzones copurified colocalizes A novel BMP antagonist copurified and colocalizes with CDMP-1/GDF-5

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

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

19. Mir16-1 mutations in Mouse and Man MicroRNA lesions in Human CLL and Mouse model of CLL 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: Early Detection: Molecular lesions Molecular lesions Leukemogenesis Leukemogenesis Intervention Intervention Product Characterization Product Characterization In-process In-process Lot Release Lot Release Cellular and Gene Therapy Products Cellular and Gene Therapy Products Clinical Use of Flow Sorted Cells Clinical Use of Flow Sorted Cells Quantitative Flow Cytometry (QFCM) Standards Quantitative Flow Cytometry (QFCM) Standards Instrument/Assay Performance Instrument/Assay Performance Linearity of detectors Linearity of detectors Compensation Controls Compensation Controls

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