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Transcript of 04/10/2008©Novocell 2008 Developing a Safe hESC-Product for Diabetes FDA Advisory Committee Meeting...
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
Development of a SafehESC-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 aCell Product
Starting Material Cell Product
Definitive Endoderm
ForegutEndoderm
PancreaticEndodermhESC
InsulinProducing
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 1x1016
100 101 102 103 104
100
101
102
103
104
0.041 100
06.45e-3
Nanog
SS
EA
-4
100 101 102 103 104
100
101
102
103
104
0.015
99.80.19
4.79e-3
Oct3/4
TR
A-1
-81
% Oct3/4
% Nanog
% SSEA-4
% TRA-1-60
% TRA-1-81
CyT49 17 23 - 38 82 - >99 80 - >99 62 - >99 95 - >99 96 - >99
Cell Line
Percent of Cells Staining Over Background# of
cultures tested
Passage Range
In Vitro Characterization of Starting Material: 2) Markers
04/10/2008 ©Novocell 2008
In Vitro Characterization of Starting Material: 3) Karyotype
Cell Line Passage Range
CyT49 8-66
Normal Karyotype on Feeders
Cell Line
Passage #
at Thaw
Passage # at
Karyotype
CyT49 11 11 + 51
CyT49 9 9 + 57
CyT49 12 12 + 33
Normal Karyotype after Cryopreservation
Cell Line # Passages on Feeders
# Passages FF
Total # Passages
CyT49 10 28 47
CyT49 3 29 44
CyT49 32 39 82
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 Material Cell Product
Definitive Endoderm
ForegutEndoderm
Pancreaticendoderm
InsulinProducing
Cell
• Viability• Consistent Composition • Normal Karyotype• Ability to Differentiate
04/10/2008 ©Novocell 2008
In Vitro Characterization ofCell Product
Starting Material Cell Product
Enrichment of Cell Product &
In-Process Testing Predictive of Outcome
Definitive Endoderm
ForegutEndoderm
Pancreaticendoderm
Endocrine cellshESC
SOX17CER
FOXA2
OCT4NANOGSOX2ECAD
HNF1BHNF1AHNF4A
NKX6.1NGN3PAX4
NKX2.2
INSC-PEPPAX6GCG
GHRLSSTPP
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
Sen
siti
vity
04/10/2008 ©Novocell 2008
Detection of Rare ESCs Using Real-time PCR for OCT4
OCT4
0
1
2
3
4
5
6
7
HE
FH
EF
HE
FE
SC
ES
CE
SC
10%
10%
10% 1% 1% 1%
0.2%
0.2%
0.2%
0.04
%0.
04%
0.04
%0.
01%
0.01
%0.
01%
0.00
25%
0.00
25%
0.00
25%
2xH
EF
2xH
EF
FL
FL
AP
AP
pure pops HEFs+ESCs controls
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
OCT4
0
0.001
0.002
0.003
0.004
0.005
0.006
HE
FH
EF
HE
FE
SC
ES
CE
SC
10%
10%
10% 1% 1% 1%
0.2%
0.2%
0.2%
0.04
%0.
04%
0.04
%0.
01%
0.01
%0.
01%
0.00
25%
0.00
25%
0.00
25%
2xH
EF
2xH
EF
FL
FL
AP
AP
pure pops HEFs+ESCs controls
04/10/2008 ©Novocell 2008
0
200
400
600
800
10001.97 49.8
0
200
400
600
800
10002 5.6
0
200
400
600
800
1000
1.98 1.56
sid
e (9
0)
scat
ter
control Oct3/4
50%
5%
0.5%
hESC + HEF1.97
2.00
1.98 1.56
5.60
49.8
0
200
400
600
800
10001.93 97.2
control Oct3/4
sid
e (
90
) sc
atte
r
hESC only
1.93 97.2
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
Subcutaneous
Abdomen
Chest
CNS
Ris
k
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
Mouse Human
Morphological
Character
Rounded colonies
Flat colonies
Growth Requirements
LIF, BMP bFGF, activin
Marker
Expression
SSEA-1 SSEA-4
Spontaneous Trophoblast Differentiation
no yes
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
04/10/2008 Copyright 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