Bioprocess Development for the Generation of Monocyte-derived Dendritic Cells

Abstract

Monocyte-derived dendritic cells (DCs) are currently under extensive evaluation as cell vaccines for cancer treatment. The requirement for large-scale cell products demands optimized and standardized protocols. This study demonstrates that highly viable DCs (93.4 ± 5.9%) can be produced from CD14+ monocytes enriched via immunomagnetic beads in a high yield (66.3 ± 13.6%) and purity (95.4 ± 2.1%) with X-VIVO 15, 400 U/mL GM-CSF and 2000 U/mL IL-4 without serum and feeding. Furthermore, analysis of apoptosis of the starting population of monocytes demonstrated that 37.8 ± 11.1% (n=8) of freshly isolated monocytes from buffy coats of healthy donors underwent programmed cell death. The yield of viable matured dendritic cells from non-apoptotic monocytes was calculated to be 90.2 ± 16.7%. These results indicate that the yield of dendritic cells is mainly influenced by the percentage of apoptotic cells in the inoculum, and impact on DC generation for clinical application.<br /> For cancer immunotherapy the loading of DCs with whole tumor cell lysate preparations represents a simple and promising approach to utilize all potential known and unknown tumor-associated antigens (TAAs) and to circumvent the disadvantages like HLA-mismatching and the requirements for known TAAs. For this reason it is important whether lysate-pulsed DCs efficiently cross-prime cytotoxic T cells (CTLs) and induce a strong Th1 cell response. Additionally, this was compared to FLU M1 and Melan-A / Mart-1 peptide pulsed DCs. As a model system breast carcinoma cell lysate from either MCF-7 or MDA-MB-231 cells expressing the TAA MUC1 were chosen. The epithelial mucin MUC1 is a large molecular weight O-glycosylated protein, which is overexpressed in the majority of breast, ovarian and other epithelial malignancies and represents a promising target in cancer immunotherapy. A simple lysate preparation method was developed to solubilize all cell proteins without degradation. For loading of monocyte-derived dendritic cells, 100 µg/mL of breast carcinoma cell lysate was used, accompanied by an adjuvant consisting of tumor necrosis factor-a and Prostaglandin-E2. T cells were co-cultivated with lysate or peptide pulsed DCs and were restimulated weekly. Before cultivation, and after the 3rd stimulation, tetramer frequencies for the MUC1 epitopes F7 and M1.2 as well as for the FLU M1 and Melan-A / Mart-1 epitopes were determined. After stimulation with lysate, higher frequencies for M1.2 specific T cells were observed compared with the F7 epitope. Furthermore, the expansion factor for M1.2 specific T cells that had been stimulated with MCF-7 lysate-pulsed DCs were found to be up to 19-fold. The analysis of typical Th1 / Th2 cytokines (IFN-γ, TNF-α, IL-12p70, IL-2, IL-4, IL-5, IL-10) revealed a strong Th1 response: up to 50,000 pg/mL IFN-γ were determined in the supernatants of the stimulations with either lysate or peptide pulsed DCs. These results provide evidence for a strong Th1 polarization and cross-priming of MUC1 specific CTLs and demonstrate the feasibility of using lysate-pulsed dendritic cells in breast cancer immunotherapy.