04/10/2008©Novocell 2008 Developing a Safe hESC-Product for Diabetes FDA Advisory Committee Meeting April 10, 2008 Melissa Carpenter, Ph.D.

04/10/2008©Novocell 2008 Cell Therapy for Diabetes: The Edmonton Protocol Adapted from J. Shapiro

04/10/2008©Novocell 2008 Development of a Safe hESC-Derived Product Characterize the Cell Product In Vitro Evaluate Cell Product In Vivo in appropriate animal model –Efficacy –Stability –Toxicity –Tumorigenicity

04/10/2008©Novocell 2008 Steps in Characterizing a Cell Product Starting Material Cell Product Definitive Endoderm Foregut Endoderm Pancreatic Endoderm hESC Insulin Producing Cell Starting cellular material –hESCs Intermediate populations Final cellular product –Insulin producing cell population

04/10/2008©Novocell 2008 Established on GMP-compliant human fibroblast feeders PTC testing completed and passed All processing reagents compliant for clinical manufacturing Initial characterization of cell line complete (surface markers, karyotype, differentiation) MCB & WCB established PTC testing complete Starting Material: Derivation Under Clinical Manufacturing Conditions CyT49

04/10/2008©Novocell 2008 Assays for In Vitro Characterization of Starting Material (hESCs) 1.Genetic identity 2.Markers Flow cytometry Q-PCR Immunocytochemistry 3.Karyotype 4.Stability 5.Differentiation

04/10/2008©Novocell 2008 In Vitro Characterization of Starting Material: 1) Genetic Identity Cell line identity by STR genotyping PowerPlex® 1.1 Plus 2.1 System (14 STR loci) Power of 1 in 1x10 16

Nanog SSEA-4 Oct3/4 TRA-1-81 In Vitro Characterization of Starting Material: 2) Markers

04/10/2008©Novocell 2008 In Vitro Characterization of Starting Material: 3) Karyotype Cell LinePassage Range CyT Normal Karyotype on Feeders Cell Line Passage # at Thaw Passage # at Karyotype CyT CyT CyT Normal Karyotype after Cryopreservation Cell Line# Passages on Feeders # Passages FF Total # Passages CyT CyT CyT Normal Karyotype Feeder-Free CyT49 p25

04/10/2008©Novocell 2008 Cytogenetic Analysis G-Banding –Allows detection of numerical abnormalities, inter- chromosomal abnormalities, intra-chromosomal abnormalities –Performed in cytogenetics lab –20 cells or more examined –Clinically correlated Spectral Karyotype (SKY) analysis –Allows detection of unknown rearrangements Correlation between aneuploidy and adverse outcome currently unclear

04/10/2008©Novocell 2008 In Vitro Characterization of Starting Material: 4) Stability Key Requirements for Stability –Stable over long term culture Expansion of cells prior to differentiation for cell product –Stable over long term cryopreservation

04/10/2008©Novocell 2008 Expansion of hESCs hESC Starting MaterialCell Product Definitive Endoderm Foregut Endoderm Pancreatic endoderm Insulin Producing Cell Viability Consistent Composition Normal Karyotype Ability to Differentiate

04/10/2008©Novocell 2008 In Vitro Characterization of Cell Product Starting Material Cell Product Enrichment of Cell Product & In-Process Testing Predictive of Outcome Definitive Endoderm Foregut Endoderm Pancreatic endoderm Endocrine cells hESC SOX17 CER FOXA2 OCT4 NANOG SOX2 ECAD HNF1B HNF1A HNF4A NKX6.1 NGN3 PAX4 NKX2.2 INS C-PEP PAX6 GCG GHRL SST PP

04/10/2008©Novocell 2008 Recommended Characterization of Cell Based Product Code of Federal Regulation for Food and Drugs (21 CFR 600 – Biologics) –Sterility –Purity –Potency –Identity –Stability –Safety –Efficacy “Active component/cell”, accessory cells, carrier solution, inappropriate components

04/10/2008©Novocell 2008 Identity Analysis Includes Assessment of Different Populations in Product Cell Product might be a heterogeneous population Cell Product assessment will include: –“Functional” cell Cell population capable of producing insulin –Accessory cells Other endocrine cells –Inappropriate cells Undifferentiated cells Cytotoxic cells –“Bystander” cells

04/10/2008©Novocell 2008 In Vitro Assays for Identity Analysis hESC- insulin QPCR Flow Cytometry Immunocytochemistry Sensitivity

04/10/2008©Novocell 2008 Detection of Rare ESCs Using Real- time PCR for OCT4 0.04% ESCs detected = 8 ESCs in 20,000 HEFs results in signal detectable above HEF expression Decreasing numbers of ESCs mixed in with 20,000 HEFs

04/10/2008©Novocell 2008 side (90  ) scatter controlOct3/4 50% 5% 0.5% hESC + HEF controlOct3/4 side (90  ) scatter hESC only Flow Cytometric Analysis for Detection of hESCs

04/10/2008©Novocell 2008 Summary of In Vitro Characterization of Cell Product Assessment of Starting Material Identity and stability of cell product Assay validation required Sensitivity of assays needs to be balanced with consumption of product for QC Predictive of clinical outcome –Safety –Efficacy

04/10/2008©Novocell 2008 Safety/Tumorigenicity for Cellular Product Dosing / Toxicity Biodistribution Where do the cells go? Maintain identity if found in other tissues? Stability Glycemic control De-differentiated cells? Tumorigenicity In vivo In vitro

04/10/2008©Novocell 2008 What is a Relevant Animal Model? Many cell based products are species-specific Will large animal studies be meaningful? –Is there a suitable large animal model? ??

04/10/2008©Novocell 2008 Teratoma vs Teratocarcinoma Teratoma = benign tumor Teratocarcinoma = malignant tumor Risk of teratoma formation will be balanced with patient population and implant site

04/10/2008©Novocell 2008 Tumorigenicity: What is the appropriate assay? How many ES cells does it take to make a teratoma? –Is there an absolute number of cells required? –Is there a frequency required (percentage of cells)? –Needs to be measured for each cell line, each product? What is the effect of implant site on teratoma formation? –Are some sites more permissive? –Do the neighboring cells (from graft or from implant site) influence teratoma formation? Are other cell types tumorigenic? Does the immune status of the recipient affect teratoma formation? What does a negative result mean?

04/10/2008©Novocell 2008 All ES Cells are NOT Equal: Origin May Influence Tumorigenicity Human does not equal mouse –Single cell cloning –Requirements for self-renewal are different MouseHuman Morphological Character Rounded colonies Flat colonies Growth Requirements LIF, BMPbFGF, activin Marker Expression SSEA-1SSEA-4 Spontaneous Trophoblast Differentiation noyes

04/10/2008©Novocell 2008 Developing a Safe hESC-derived Product: Summary Characterize the cell product Demonstrate safety and efficacy of cell product Sensitive and specific assays required Assays which are predictive of clinical outcome required

04/10/2008Copyright Novocell 2008 Developing a Cell Product for Diabetes Edmonton protocol and others successfully implanted autologous islets under immunosuppression hESCs provide a cell source for Diabetic therapies Novocell has developed encapsulation technology –Preliminary clinical evaluation