Generierung hochreiner Fusionszellen aus Tumorund Antigen-präsentierenden Zellen

Abstract

Background: The aim of tumor therapy is to induce a potent CTL response against autologous tumors.

One of the different approaches is the fusion of antigen presenting cells (APCs) and tumor cells. These hybrid cells combine antigenicity of tumor cells and immunstimulation of APCs. An enhancement of the immunstimulatory capacity can be achieved by an increased proportion of pure fusion cells. The aim of our study was to gain highly pure fusion cells. Material and methods: As fusion partners we used a sporadic-microsatellite-instable (MSI-H, wt APC, wt K-Ras) colorectal tumor cell line and autologous B-LCLs (EBV immortalised pheriphere lymphocytes). Tumor cells and B-LCLs were stained to monitor all steps by FACS analysis. Results: First, the optimal conditions for standard polyethylene-glycol (PEG) fusion, the handling and the analysis were established. Finally, the optimized fusion protocol resulted in reproducible fusion efficiencies of 19.8 % (n = 16, range 8.9–33.3 %). In a second step, the B-LCLs were incubated with a mouse antibody against MHC class I (clone W6/32) followed by goat-anti mouse IgG magnetic beads (Miltenyi). The tumor cells were labeled with CFSE prior to fusion. Subsequent to these steps, standard fusion was performed. Unfused tumor cells which were not labeled with microbeads could easily be eliminated from the cell mixture by magnetic separation (about 90 %). For depletion of unfused B-LCLs the remaining mixture was transferred into culture flask. Fusion cells adhere to plastic and the B-LCLs could be removed by washing. We were able to achieve a purity of 83.9 % (n = 8, 69.7–95.4 %). Conclusion: Using a combination of magnetic separation and selective adhesion we were able to increase the purity of fusion cells high significant from 19.8 % to 83.9 % (p < 0.001). In functional T cell analyses the purified fusion cells demonstrated their immustimulatory capacity. The presented results are a cornerstone for advanced preclinical analyses with fusion vaccines. This study was substantially supported by «Else Kröner Fresenius Stiftung».