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

Steven R. Bauer, PhD, ChiefSteven R. Bauer, PhD, Chief Deborah Hursh, PhD, Senior Staff FellowDeborah Hursh, PhD, Senior Staff Fellow Gerald Marti, MD, PhD Senior Gerald Marti, MD, PhD Senior

InvestigatorInvestigator Brenton McCright, PhD, Senior Staff Brenton McCright, PhD, Senior Staff

FellowFellow Malcolm Moos, PhD, MD Senior Malcolm Moos, PhD, MD Senior

InvestigatorInvestigator John Terrig Thomas, PhD Visiting ScientistJohn Terrig Thomas, PhD Visiting Scientist

The ProblemsThe Problems Clinical benefit is highly variable, Clinical benefit is highly variable,

often hard to demonstrateoften hard to demonstrate In many cases, most cells In many cases, most cells

administered die immediatelyadministered die immediately Products may ‘misdifferentiate’Products may ‘misdifferentiate’ Inadequate supplyInadequate supply

Cell Therapy ChallengesCell Therapy Challenges Poor understanding of how cells Poor understanding of how cells

interact with their interact with their microenvironment microenvironment

Inadequate markers predictive of Inadequate markers predictive of cell state and cell fate cell state and cell fate

Poor survival of cells post Poor survival of cells post transplantationtransplantation

CTTB ApproachesCTTB Approaches Complementary SystemsComplementary Systems

Frogs, Flies, Mouse, and ManFrogs, Flies, Mouse, and Man Gene, Protein, Cell, Tissue InteractionsGene, Protein, Cell, Tissue Interactions

Normal Development and TumorigenicityNormal Development and Tumorigenicity Knowledge and manipulation of growth Knowledge and manipulation of growth

factor pathways factor pathways Knowledge and utilization of transformation Knowledge and utilization of transformation

pathwayspathways

Microenvironment in cell therapy: Microenvironment in cell therapy: manufacturing, patient - Bauermanufacturing, patient - Bauer

In vitro: reduced stromal dlkIn vitro: reduced stromal dlkNo differentiation or apoptosis upon removal of IL-7No changes in typical pre-B markers

In vivo effects: Dlk1-/- mouseIn vivo effects: Dlk1-/- mouseAlters B-cell development and function

Pre-BPre-B

Abnormal stroma=> Abnormal stroma=> Abnormal B cellsAbnormal B cells

Remove IL-7Remove IL-7

• Establish normal, Establish normal, neoplastic and neoplastic and preneoplastic pre-B cellspreneoplastic pre-B cells

•Mechanisms of Mechanisms of transformationtransformation

•Identify biomarkers of Identify biomarkers of transformation transformation

Systematic approach for identification Systematic approach for identification and qualification of tumorigenicity and qualification of tumorigenicity

biomarkers for cell therapy products- biomarkers for cell therapy products- BauerBauer

Impact for Cell Impact for Cell TherapyTherapy

Stroma can alter cell product in a way Stroma can alter cell product in a way that is not revealed in lot release teststhat is not revealed in lot release tests

Efficacy may be affected byEfficacy may be affected by Microenvironment during cell product Microenvironment during cell product

manufacturingmanufacturing Microenvironment in patientMicroenvironment in patient

Improved tumorigenicity assessmentsImproved tumorigenicity assessments

Approach:Approach: Genetically modify the mouse to the Genetically modify the mouse to the study function of proteins required for mammalian study function of proteins required for mammalian organ development in vivo.organ development in vivo.

Mammalian organogenesis as a model Mammalian organogenesis as a model for cellular and tissue engineered for cellular and tissue engineered

therapies – McCrighttherapies – 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

Notch2 expression and heart specific Notch2 expression and heart specific inactivation demonstrates a cell autonomous inactivation demonstrates a cell autonomous requirement for Notch2 during murine heart requirement for Notch2 during murine heart

developmentdevelopment

Notch2-Notch2-

*pa

Notch2+Notch2+

*

pa

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

Impact for Cell TherapyImpact for Cell Therapy Identification and analysis of molecules Identification and analysis of molecules

required for mammalian organogenesisrequired for mammalian organogenesis Notch2 as a biomarker for evaluating the Notch2 as a biomarker for evaluating the

developmental potential of cells used in developmental potential of cells used in cardiac repair. cardiac repair.

Notch1 or Notch2 activation may have Notch1 or Notch2 activation may have similar effects on cell products.similar effects on cell products.

Exogenous Notch activation and the Exogenous Notch activation and the functional requirements for Notch2 can be functional requirements for Notch2 can be studied in most tissues studied in most tissues

A Genetic Model of Growth Factor A Genetic Model of Growth Factor Action to Develop Markers of Safety Action to Develop Markers of Safety and Efficacy of Cell-Based Products-and Efficacy of Cell-Based Products-

HurshHursh A system to study cell communication in intact A system to study cell communication in intact

tissuestissues Ability to alter gene expression within a Ability to alter gene expression within a

microenvironmentmicroenvironment High throughput screens identify critical High throughput screens identify critical

control pointscontrol points Markers predictive of pathway activityMarkers predictive of pathway activity

Analyze cell stress and viabilityAnalyze cell stress and viability Markers predictive of survivalMarkers predictive of survival

Genetic interaction Genetic interaction screens identify and place screens identify and place

genes in a functional genes in a functional pathwaypathway

Asks the organism to identify critical Asks the organism to identify critical control points.control points.

Avoid bias of abundance, Avoid bias of abundance, immunogenicity, modifications.immunogenicity, modifications.

Model organisms allow screens of Model organisms allow screens of sufficient size.sufficient size.

Knowledge of control points for cellular Knowledge of control points for cellular products is critical.products is critical.

identified > 20 genes that interact with identified > 20 genes that interact with BMP signaling.BMP signaling.

Loss of BMP causes cell Loss of BMP causes cell death, induces JNK death, induces JNK

pathway: a model of cell pathway: a model of cell competitioncompetition

WT,,caspaseMut, 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

Impact for Cell Therapy/Tissue Impact for Cell Therapy/Tissue EngineeringEngineering

Improve our ability to predict the Improve our ability to predict the survival of transplanted cells in their survival of transplanted cells in their new locationnew location

Cell interactions in tissue Cell interactions in tissue developmentdevelopment

Protein: Protein Interactions in Protein: Protein Interactions in Joint Development- MoosJoint Development- Moos

Proprotein Convertases and GDF5 colocalizeProprotein Convertases and GDF5 colocalize to establish normal joint structureto establish normal joint structure

Joint interzones

A novel BMP antagonist copurifiedcopurified and colocalizescolocalizes with

CDMP-1/GDF-5

Feedback and Crosstalk in Feedback and Crosstalk in Cell and Tissue Cell and Tissue SpecificationSpecification

Colocalization of several signals Colocalization of several signals is necessary to instruct formation is necessary to instruct formation of articular cartilageof articular cartilage

Developmental signals in Developmental signals in characterization of cell and characterization of cell and tissue engineering productstissue engineering products

Chronic Lymphocytic Leukemia Chronic Lymphocytic Leukemia in Mouse and Man- Martiin Mouse and Man- Marti

Molecular Lesion in CLLMolecular Lesion in CLL Precursor states: Monoclonal B Cell Lymphocytosis Precursor states: Monoclonal B Cell Lymphocytosis

(MBL)(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: ZAP70Biomarker: ZAP70 Quantitative Flow Cytometry (QFCM)Quantitative Flow Cytometry (QFCM)

Cell therapy product characterizationCell therapy product characterization Flow cytometry-based cell separationFlow cytometry-based cell separation

Standards Development: Standards Development: Fluorescence reference materialsFluorescence reference materials CLSI documentsCLSI documents

Mir16-1 mutations in Mouse and ManMir16-1 mutations in Mouse and Man

MicroRNA lesions in Human CLL and Mouse model of CLLNZB model of CLL reveals similar mutationNZB model of CLL reveals similar mutation

- Identification of critical lesion in CLL- Identification of critical lesion in CLL-Animal model for elucidation of mechanisms Animal model for elucidation of mechanisms of transformationof transformation- Improved diagnosis/treatment of CLLImproved diagnosis/treatment of CLL

ApplicabilityApplicability Early Detection: Early Detection:

Molecular lesions Molecular lesions Leukemogenesis Leukemogenesis InterventionIntervention

Product CharacterizationProduct Characterization In-processIn-process Lot ReleaseLot Release

Cellular and Gene Therapy ProductsCellular and Gene Therapy Products

Clinical Use of Flow Sorted Cells Clinical Use of Flow Sorted Cells Quantitative Flow Cytometry (QFCM) StandardsQuantitative Flow Cytometry (QFCM) Standards

Instrument/Assay PerformanceInstrument/Assay Performance Linearity of detectorsLinearity of detectors Compensation ControlsCompensation Controls

CTTB Research: Addressing CTTB Research: Addressing Cell Therapy Challenges Cell Therapy Challenges

Complementary approachesComplementary approaches Cell-Cell interactionsCell-Cell interactions Genetic interaction screensGenetic interaction screens Protein-Protein interactionsProtein-Protein interactions OrganogenesisOrganogenesis TumorigenesisTumorigenesis