To establish a method for manufacturing mature myeloid dendritic cells for a Phase I clinical trial in immunotherapy of CML, we validated scaling up and translation of the standard laboratory method. We compared DC prepared from three normal mononuclear cell apheresis products (AP) with DC prepared from three AP from CML patients. By the use of CD14-specific MicroBeads and a CliniMACS magnetic separator (both Miltenyi Biotec, Auburn, CA), we enriched normal CD14+ cells from 15.0+2.1 % in AP to 98.1±1.5% and the CML cells from 13.5±5.0% in AP to 92.6111.8% (P=0.5 for enriched cells). Enrichment in CD14+-cells increased the level of Philadelphia-chromosome-positive cells from 72.6±20.7% to 97.5±2.2%. To obtain mature DC, we incubated CD14+ cells in X-V1VO 15 medium supplemented with GM-CSF and IL-4 for seven days and with additional TNF-a, IL-1 β, 1L6 and PGE2 for three more days. The final DC yield from CD14 cells was 30.3±6.4% for normal cells and 32.8± 12.3% for CML cells; the corresponding yields from from total nucleated cells were 2.2±0.7% and 2.5%, respectively. DC were highly mature (84.5+1.5% CD83 ) and functional as assessed by stimulation of allogeneic MLR. The extent of maturity and function wassimilartoDC prepared from the same AP by the standard laboratory method. Mature DC could be stored by cryopreservation; this mode of storage did not affect DC phenotype and yielded cells 81.0+9.5% viable. We received an IND by the PDA for this DC preparation method and have started enrollment into the Phase 1 trial. We gratefully acknowledge the generous support by Miltenyi Biotec GmbH.
|Original language||English (US)|
|Issue number||11 PART II|
|State||Published - Dec 1 2000|
ASJC Scopus subject areas
- Cell Biology