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Prochymal TM

Prochymal TM. 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: Prochymal TM Adult Mesenchymal Stem Cells. Bone Marrow Aspirate. Adherence to

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Prochymal TM

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

  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

  11. 60000 180 160 50000 140 40000 120 Proliferation 100 Proliferation (CPM) TNFR (pg/mL) 30000 80 TNFR, type I 20000 60 40 10000 20 0 0 1 2 3 4 5 6 Potency Marker Screening • TNFR I is the best potency marker for Prochymal • among screened candidates Correlation between TNFRI expression and MSC-mediated immunosuppression 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 • 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

  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. TNFR detection in lysates by ELISA Cell lysis Frozen cells at P5 (30 donors) Thawing and counting Plating into 96-well plates with hPBMCs 5 days later hPBMC proliferation measurement TNFRI Potency Marker Qualification • Part 1: hMSC analysis from different donors Experimental Design: Experimental Results:

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

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

  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

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