Large-scale generation of mature monocyte-derived dendritic cells for clinical application in cell factories

Dendritic cells (DC) are increasingly used for the immunotherapy of cancer. Both the induction of tumor-specific T cells and some clinical regressions have been observed in early phase I/II trials by using either DC isolated from blood, DC generated from CD34+ precursors ex vivo, and most frequently, by employing monocyte-derived DC. As DC vaccination is now awaiting phase II/III trials with larger patient collectives, it becomes increasingly important to overcome prior limitations such as the repetitive, labor-intensive generation of DC in a large number of open culture vessels. We describe here as a result of several years of optimization, in detail, a procedure that uses the so-called Nunc cell factories to process a whole apheresis product, labor- and cost-effectively in a quasi-closed system to reproducibly generate (by using GM-CSF+IL-4 followed by a maturation cocktail composed of IL-1beta+IL-6+TNF-alpha +PGE(2)) large numbers (8.32+/-3.8% of input peripheral blood mononuclear cells (PBMC)) of mature (>85% CD83+), monocyte-derived DC that can be successfully cryopreserved. Our report is based on the processing of >100 aphereses including 52 unselected aphereses in advanced melanoma patients. This allows us also to suggest meaningful quality and validation criteria. The DC generation method appears particularly promising as respective DC vaccination proved to be immunogenic in cancer patients and cell factories can readily be converted to a fully closed system by using appropriate valves, tubings, and bags.