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 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