1. The dynamics of a new age in medicine
ProchymalTM
The dynamics of a new age in medicine
Presented by Alla Danilkovitch, PhD
Senior Scientist, Prochymal

2. Potency Assay Development for a Novel Cell Therapy Product:
ProchymalTM Adult Mesenchymal Stem Cells

3. What is Prochymal?
PROCHYMAL is ex vivo cultured human mesenchymal stem cells (hMSCs) derived from the bone marrow of healthy volunteer donors
Passaged hMSCs
Bone
Marrow
Aspirate
Adherence to
surface of cell factory
Expansion

4. How is Prochymal supplied?
100 million cells in a bag
15 ml
Plasma-Lyte A, 5% HSA, 10% DMSO
Homogenous cell population
Storage < – 140 degrees C,
LN2 vapor
Stability > 2 years

5. Prochymal Indication Acute Graft versus Host Disease (GVHD)
Occurs in about 50% of bone marrow transplants
New immune system (graft) starts attacking the patient (host)
Similar to organ rejection
Can involve the skin, liver, and GI system
Severe acute GVHD is fatal in 50-80% of cases

6. Prochymal Mechanisms
Prochymal functional properties beneficial for GVHD treatment
Homing to sites of injury/inflammation
Immunomodulation: suppression of T-lymphocytes at injury/inflammation sites
Anti-inflammatory activity: inhibition of pro-inflammatory cytokines (TNF-a and IFN-g)
Tissue repair

7. Potency Assay for Prochymal
Potency assay must guarantee that each lot of the product performing acceptably will have the desired clinical effect or characteristics for disease treatment
Desirable Prochymal characteristics for successful treatment of GVHD are:
suppression of immune response and/or
inhibition of inflammation and/or
healing of damaged tissues

8. Potency Assay for Prochymal
Suppression of immune response is the most distinguished
and desirable Prochymal characteristic for GVHD treatment

9. Potency Assay Development Strategy
Select potency markers that are linked to MSC immunomodulative activity using
Literature data
Data accumulated at Osiris
Screen selected markers for correlations to MSC ability to suppress lymphocyte proliferation in vitro
Potency assay method validation and potency marker qualification

10. Potency Markers Selected for Screening
Justification for marker selection
Prostaglandin E2 (PGE2)
PGE2 suppresses immune response. MSCs produce PGE2, and PGE2 mediates MSC-induced immunosuppressive and anti-inflammatory effects in vitro.
Indoleamine 2,3-dioxygenase (IDO) enzyme activity
IDO is an enzyme inducible by pro-inflammatory cytokines such as IFN-g and TNF-a. IDO inhibits immune response via depletion of tryptophan, an amino acid that is essential for immune cell activation. IDO enzyme mediates MSC-induced immunosuppression in vitro.
Tumor Necrosis Factor-a ( TNF-a)
TNF-a is a pro-inflammatory cytokine playing an important role in GVHD. MSCs inhibit TNF-a secretion by immune cells in vitro.
Interferon-g
(IFN-g)
IFN-g is a cytokine secreted by Th1 cells that are involved in GVHD development. MSCs can inhibit secretion of IFN-g that is beneficial for GVHD treatment
Tumor Necrosis Factor-a Receptor (TNFR )
TNFR is expressed on MSCs. TNFa is present in organs targeted by GVHD. TNF-a via TNFR up-regulates secretion of PGE2, induces expression of IDO and stimulates MSC migration in vitro. TNFR is a mediator of MSC biological activities.

11. Potency Marker Screening
TNFR I is the best potency marker for Prochymal
among screened candidates
Correlation between TNFRI expression and MSC-mediated immunosuppression
10000
20000
30000
40000
50000
60000 1 2 3 4 5 6
Proliferation (CPM) 20 40 60 80
100
120
140
160
180
TNFR (pg/mL)
Proliferation
TNFR, type I
1 – PBMC control; 2 – PBMC+MSC; 3, 4, 5 - PBMC+MSC treated by a 2 mM, 1 mM and 0.5 mM TNFRI anti-sense oligo respectively; 6 – PBMC+MSC treated by a 2 mM TNFRI sense (control) oligo

12. Prochymal Endpoint Potency Assay
TNFRI ELISA is a Prochymal Endpoint Potency Assay
Commercially available kit assay (R&D Systems)
Quantitative/Sensitive/Short duration
TNFRI ELISA parameters
Calibration standard range: pg/mL
Assay quantitaion range: pg/mL
LLOQ: 15.6 pg/mL
LOD: 7.8 pg/mL
ULOQ: 500 pg/mL

13. TNFRI Potency Marker Qualification
Part 1:
hMSC analysis for TNFR expression and its ability
to inhibit hPBMC proliferation in vitro
Part 2:
Potency cut-off point establishment – the level of TNFRI
expression correlating with less than 50% inhibition of
hPBMC proliferation (a TNFRI anti-sense oligonucleotide
was used for generation of non-potent hMSCs)

14. TNFRI Potency Marker Qualification
Part 1: hMSC analysis from different donors
Experimental Design:
Frozen cells
at P5
(30 donors)
Thawing
and
counting
Cell lysis
Plating into
96-well plates
with hPBMCs
5 days later
hPBMC proliferation
measurement
TNFR detection in
lysates by ELISA
Experimental Results:
Parameter:
Mean+SD
Range
TNFRI expression
29+7 pg/ 106 MSCs
pg/ 106 MSCs
Inhibition of hPBMC proliferation
59+10%
49- 69%

15. TNFRI Potency Marker Qualification
Part 2: Potency cut-off point establishment
Experimental Design:
Frozen
cells
at P5
(7 donors)
Thawing,
Counting,
Plating into
6-well plates
Transfection
with TNFRI
oligos
Plating into
96-well plates
with hPBMCs
Cells lysis
5 days later
hPBMC proliferation
measurement
TNFR detection in
lysates by ELISA

16. TNFRI Potency Marker Qualification
Part 2: Potency cut-off point establishment
Experimental Result: Potency cut off point is 13 pg/106 hMSCs (mean+SD) 49 38 28 11 75 71 70 39

17. Example of TNFRI Potency Assay Use
Temperature tolerance study
-80
-70
-60
-50
Cell storage at higher than -600C:
Cell viability < 70%
TNFRI < 13 pg/mil cells
No inhibition of hPBMC
proliferation in vitro
-80
-70
-60
-50

18. Summary Prochymal Potency Assay measures cellular TNFRI by ELISA
TNFRI is a marker linked to MSC immunosuppression, which is a desirable MSC biological activity for GVHD treatment
TNFRI expression level linked to MSC functionality: an ability to identify poor quality product lots
The endpoint assay: quantitative sandwich ELISA
Osiris experience shows that an indication-specific marker selection is a useful strategy for development of potency assays for cell therapy products