ProchymalTM

The dynamics of a new age in medicine

Presented by Alla Danilkovitch, PhDSenior Scientist, Prochymal

Potency Assay Development for a Novel Cell Therapy Product:

ProchymalTM Adult Mesenchymal Stem Cells

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

Passaged hMSCs

Bone Marrow Aspirate

Adherence to surface of cell factory

Expansion

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 pro-inflammatory cytokines (TNF- and IFN-

• 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

MSC dose-dependent effect on anti-CD3/CD28-induced PBMC proliferation

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hMSC 75P5/well

CP

M

• Suppression of immune response is the most distinguished and desirable Prochymal characteristic for GVHD treatment

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 anti-

inflammatory effects in vitro.

Indoleamine 2,3-dioxygenase (IDO) enzyme activity

IDO is an enzyme inducible by pro-inflammatory cytokines such as IFN- and TNF-. 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- ( TNF-)

TNF- is a pro-inflammatory cytokine playing an important role in GVHD. MSCs inhibit TNF-secretion by immune cells in vitro.

Interferon-

(IFN-)

IFN- is a cytokine secreted by Th1 cells that are involved in GVHD development. MSCs can inhibit secretion of IFN- that is beneficial for GVHD treatment

Tumor Necrosis Factor- Receptor (TNFR )

TNFR is expressed on MSCs. TNF is present in organs targeted by GVHD. TNF- 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

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Pro

life

rati

on

(C

PM

)

020406080100120140160180

TN

FR

(p

g/m

L)

Proliferation

TNFR, type I

1 – PBMC control; 2 – PBMC+MSC; 3, 4, 5 - PBMC+MSC treated by a 2 M, 1 M and 0.5 M TNFRI anti-sense oligo respectively; 6 – PBMC+MSC treated by a 2 M TNFRI sense (control) oligo

• TNFR I is the best potency marker for Prochymal among screened candidates

Correlation between TNFRI expression and MSC-mediated immunosuppression

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 ELISA is a Prochymal Endpoint Potency Assay

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

Frozen cellsat 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

• Part 1: hMSC analysis from different donors

Experimental Design:

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 cellsat P5(7 donors)

Thawing,Counting,Plating into 6-well plates

Cells lysisPlating into 96-well plates with hPBMCs

5 days later hPBMC proliferation measurement

TNFR detection in lysates by ELISA

Transfection with TNFRI oligos

TNFRI Potency Marker Qualification

Experimental Result: Potency cut off point is 13 pg/106 hMSCs (mean+SD)

• Part 2: Potency cut-off point establishment

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0 1.25 2.5 5

TNF RI anti-sense oligonucleotide concentration (g/mL)

TN

F R

I (p

g/m

il c

ells

)

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203040

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Inh

ibit

ion

of

hP

BM

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pro

life

rati

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, %

of

con

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l

TNF RI

Proliferation

49

38

28

11

75 71 70

39

Example of TNFRI Potency Assay Use• Temperature tolerance study Cell Viability

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% V

iab

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ells

-80 -70 -60 -50

TNFRI Expression

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Cell storage at higher than -600C:

— Cell viability < 70%

— TNFRI < 13 pg/mil cells

— No inhibition of hPBMC proliferation in vitro

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