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

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-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

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 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.

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

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 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 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 Cells lysis 5 days later hPBMC proliferation measurement TNFR detection in lysates by ELISA

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

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

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