Transcript Document 7910683

ProchymalTM
The dynamics of a new age
in medicine
Presented by Alla Danilkovitch, PhD
Senior Scientist, Prochymal
Potency Assay Development for
a Novel Cell Therapy Product:
ProchymalTM Adult
Mesenchymal Stem Cells
What is Prochymal?
PROCHYMAL is ex vivo cultured human mesenchymal stem cells
(hMSCs) derived from the bone marrow of healthy volunteer donors
Bone
Marrow
Aspirate
Adherence to
surface of cell
factory
Expansion
Passaged hMSCs
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
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
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 proinflammatory cytokines (TNF-a and IFN-g)
• Tissue repair
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
Potency Assay for Prochymal
• Suppression of immune response is the most distinguished
and desirable Prochymal characteristic for GVHD treatment
MSC dose-dependent effect on anti-CD3/CD28induced PBMC proliferation
200000
CPM
150000
100000
50000
0
0
156
312
625
1250
hMSC 75P5/well
2500
5000
10000
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
Potency Markers Selected for Screening
Marker
Justification for marker selection
Prostaglandin E2
(PGE2)
PGE2 suppresses immune response. MSCs produce PGE2, and
PGE2 mediates MSC-induced immunosuppressive and antiinflammatory effects in vitro.
Indoleamine 2,3dioxygenase
(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.
Potency Marker Screening
• TNFR I is the best potency marker for Prochymal
among screened candidates
Proliferation (CPM)
60000
180
160
140
120
100
80
60
40
20
0
50000
40000
30000
20000
10000
0
1
2
3
4
5
TNFR (pg/mL)
Correlation between TNFRI expression and MSC-mediated immunosuppression
Proliferation
TNFR, type I
6
1 – PBMC control; 2 – PBMC+MSC; 3, 4, 5 - PBMC+MSC treated by a 2 mM, 1 mM and 0.5 mM TNFRI antisense oligo respectively; 6 – PBMC+MSC treated by a 2 mM TNFRI sense (control) oligo
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: 7.8 - 500 pg/mL
Assay quantitaion range: 15.6 - 500 pg/mL
LLOQ: 15.6 pg/mL
LOD: 7.8 pg/mL
ULOQ: 500 pg/mL
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)
TNFRI Potency Marker Qualification
• Part 1: hMSC analysis from different donors
Experimental Design:
Frozen cells
at P5
(30 donors)
Thawing
and
counting
Cell lysis
TNFR detection in
lysates by ELISA
Plating into
96-well plates
with hPBMCs
5 days later
hPBMC proliferation
measurement
Experimental Results:
Parameter:
Mean+SD
Range
TNFRI expression
29+7 pg/ 106 MSCs
22- 36 pg/ 106 MSCs
Inhibition of hPBMC
proliferation
59+10%
49- 69%
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
5 days later
hPBMC proliferation
measurement
Cells lysis
TNFR detection in
lysates by ELISA
TNFRI Potency Marker Qualification
• Part 2: Potency cut-off point establishment
TNF RI (pg/mil cells)
60
75
71
70
50
40
49
39
38
30
28
20
10
11
0
0
1.25
2.5
5
TNF RI anti-sense oligonucleotide
concentration (mg/mL)
80
70
60
50
40
30
20
10
0
Inhibition of hPBMC
proliferation, % of control
Experimental Result: Potency cut off point is 13 pg/106 hMSCs (mean+SD)
TNF RI
Proliferation
Example of TNFRI Potency Assay Use
• Temperature tolerance study
Cell Viability
85
Cell storage at higher than -600C:
— Cell viability < 70%
% Viable cells
80
75
70
65
60
55
50
-80
— TNFRI < 13 pg/mil cells
-70
-60
-50
TNFRI Expression
— No inhibition of hPBMC
proliferation in vitro
TNFRI (pg/mil cells)
30
25
20
15
10
5
0
-80
-70
-60
-50
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