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Poster C Linical Nk Generation Ash 2009 Js

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Adoptive transfer of CD34+ derived NK cells in poor-prognosis AML patients ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Effective NK cell differentiation from umbilical cord blood (UCB) derived CD34+ cells using GBGM ® E x-vivo generated NK cells are devoid of T- and B cells and express specific NK cell antigens Stroma-free cell culture system for optimal NK cell generation ,[object Object],[object Object],[object Object],[object Object],[object Object],Clinical grade NK cell cultures provide sufficient numbers of functional NK cells which efficiently lyse various primary AML tumor cells Efficient CD34+ cell enrichment from cryopreserved UCB units using the CliniMACS system Efficient NK cell production from cryopreserved UCB units using various bioreactors CD56 content 99% ± 1% using GBGM ® 10 100 1,000 10,000 100,000 1 2 3 4 5 week Fold expansion total cells Medium1 (n=3) Medium 2 (n=3) GBGM ® (n=3) Percentage CD56+ cells A B * 0 20 40 60 80 100 3 4 5 week * Medium1 (n=3) Medium 2 (n=3) Cell expansion ~50,000 fold using GBGM ® expansion differentiation 1 • GBGM ® + human serum (HS) • SCF, Flt3L, IL-7, TPO • Clinical grade Heparin (GAGs) CD34+ cells Day 0 - 9 Day 9 - 14 Expansion medium: Differentiation medium 2: • SCF, IL-7, IL-2, IL-15 NK progenitors Mature NK cells STEP 1 STEP 2 differentiation 2 Day 14 - 35 Differentiation medium 1: • SCF, IL-7, Flt3L, IL-15 • Clinical grade Heparin (GAGs) Low - dose cytokine cocktail • Low - dose cytokine cocktail • Low - dose cytokine cocktail • • GBGM ® + HS • GBGM ® + HS Novel GMP grade Glycostem Basal Growth Medium (GBGM ® ) www.glycostem.nl Clinical scale generation of allogeneic NK cell products 0 500 1000 1500 2000 0 1 2 3 4 5 6 week CB0109 bag CB0209 bag CB0309 bag 0 20 40 60 80 100 0 1 2 3 4 5 6 week CB0109 plate CB0209 plate CB0309 plate 0 20 40 60 80 100 0 1 2 3 4 5 6 week CB0109 bag CB0209 bag CB0309 bag 0 2000 4000 6000 8000 0 1 2 3 4 5 6 week CB0109 plate CB0209 plate CB0309 plate 0 2000 4000 6000 0 1 2 3 4 5 6 week CB0709 wave CB0709 plate 0 20 40 60 80 100 0 1 2 3 4 5 6 week CB0709 wave CB0709 plate Fold expansion Fold expansion Fold expansion NK cells (%) NK cells (%) NK cells (%) 0 0 5 95 0 0 9 91 0 4 96 0 CD3 CD19 CD15 SSC CD14 CD38 CD56 CD45 0 20 40 60 80 100 AML1 AML2 AML3 AML4 AML5 K562 KG1a day2 day3 day1 specific lysis (%) NK cell products are analyzed for purity, showing no contamination with T- or B cells (A). The NK cell phenotype was determined by 10-color flowcytometry using the Gallios Flow Cytometer and the Kaluza software (B). A B The final NK cell products were analyzed for functionality in a CFSE based cytotoxicity assay for 18h with a 2:1 effector:target ratio. Control experiments in plates Experiments in static bags Experiments in bioreactors GBGM ® (n=3) NK cells per experiment CD34+ cells fold expansion CD56+ (%) NK cells experiment CB0109 1.7x10 6 1770 63 1.9x10 9 static bag CB0209 1.4x10 6 759 80 8.6x10 8 static bag CB0309 1.3x10 6 1291 70 1.2x10 9 static bag CB0709 0,81x10 6 2861 95 2.2x10 9 bioreactor

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