title: Comparative evaluation of techniques for the manufacturing of dendritic cell-based cancer vaccines.
abstract: Manufacturing procedures for cellular therapies are continuously improved with particular emphasis on product safety. We previously developed a dendritic cell (DC) cancer vaccine technology platform that uses clinical grade lipopolysaccharide (LPS) and interferon (IFN)-y for the maturation of monocyte derived DCs. DCs are frozen after 6 hrs exposure at a semi-mature stage (smDCs) retaining the capacity to secret interleukin (IL)-12 and thus support cytolytic T-cell responses, which is lost at full maturation. We compared closed systems for monocyte enrichment from leucocyte apheresis products from healthy individuals using plastic adherence, CD14 selection, or CD2/19 depletion with magnetic beads, or counter flow centrifugation (elutriation) using a clinical grade in comparison to a research grade culture medium for the following DC generation. We found that elutriation was superior compared to the other methods showing 36 +/- 4% recovery, which was approximately 5-fold higher as the most frequently used adherence protocol (8 +/- 1%), and a very good purity (92 +/- 5%) of smDCs. Immune phenotype and IL-12 secretion (adherence: 1.4 +/- 0.4; selection: 20 +/- 0.6; depletion: 1 +/-0.5; elutriation: 3.6 +/- 1.5 ng/ml) as well as the potency of all DCs to stimulate T cells in an allogeneic mixed leucocyte reaction did not show statistically significant differences. Research grade and clinical grade DC culture media were equally potent and freezing did not impair the functions of smDCs. Finally, we assessed the functional capacity of DC cancer vaccines manufactured for three patients using this optimized procedure thereby demonstrating the feasibility of manufacturing DC cancer vaccines that secret IL-12 (9.4 +/- 6.4 ng/ml). We conclude that significant steps were taken here towards clinical grade DC cancer vaccine manufacturing.
authors: Laboratory of Tumour Immunology, St Anna Children's Cancer Research Institute, Vienna, Austria.
authors: Alexander Michael
authors: AM
authors: Dohnal
authors: Sebastian
authors: S
authors: Graffi
authors: Volker
authors: V
authors: Witt
authors: Christina
authors: C
authors: Eichstill
authors: Dagmar
authors: D
authors: Wagner
authors: Sidrah
authors: S
authors: Ul-Haq
authors: Doris
authors: D
authors: Wimmer
authors: Thomas
authors: T
authors: Felzmann
mesh_terms: D001781:Blood Component Removal; D019496:Cancer Vaccines / Q000276:immunology*; D002469:Cell Separation / Q000379:methods*; D003713:Dendritic Cells / Q000166:cytology / Q000276:immunology*; D006801:Humans; D007371:Interferon-gamma / Q000276:immunology; D018664:Interleukin-12 / Q000276:immunology; D007962:Leukocytes / Q000166:cytology / Q000276:immunology; D008070:Lipopolysaccharides / Q000276:immunology; D009000:Monocytes / Q000166:cytology / Q000276:immunology; D009369:Neoplasms / Q000188:drug therapy / Q000276:immunology
chemical_list: D019496:Cancer Vaccines; D008070:Lipopolysaccharides; D018664:Interleukin-12; D007371:Interferon-gamma