gms | German Medical Science

Immunotherapy with autologous dendritic cells (DC) loaded with tumour-antigen-encoding information (peptides, DNA, RNA) and prepared as a functional vaccine aiming specific immune response in humans requires clean DC of high quality and numbers.

Human peripheral blood mononuclear cells (PBMC) are harvested by leukapheresis in a closed system and enriched by density grading centrifugation. These PBMC are incubated in serum free media with a cell density of 1x10n8 cells per 35 ml in cell culture flaskes at 37°C/5%CO2 for one hour, than washed twice and human recombinant IL-4 (500U/ml) and GM-CSF (800U/ml) are added to the adherent cells for further incubation of a total of 11 days. The same cytokines are added again at day 3, 7 and 10 differentiating cells to immature DC. Additionally, TNFα (10ng/ml), IL-1β (2ng/ml) and IL-6 (100U/ml) are given for further maturation one day before collection. Thereafter, a phenotypic characterisation of cells with fluorochrome labelled monoclonal antibodies will be held by FACS to ensure the phenotype of mature DC. Subsequently, the transfection procedures will be performed. Finally, the generated vaccine is cryo-conserved in aliquots and undergoes a batch release test. This contains measurements of cell viability and microbial contamination. To achieve high quality test results and to release vaccine for patient treatment all steps including DC preparation and vaccine generation should performed in clean room under the guidelines of GMP and SOP.

The generation of myeloid, mature DC from PBMC and vaccine preparation procedures having regard to this protocol establishes the clinical application of the DC-based vaccination for anticancer immunotherapy beyond laboratory studies.