Efficient in vitro expression of the human reverse transcriptase (hTERT) but impaired maturation in dendritic cells of lung cancer patients
13518 An innovative treatment strategy for patients with lung cancer is the targeting of tumor associated antigens with cellular effector cells. The telomerase catalytic subunit (hTERT) is an attractive target for cytotoxic T cells that is highly expressed in both NSCLC and SCLC cells. The aim of the study was the expression of hTERT in dendritic cells (DC) that are the most powerful antigen presenting cells for the induction of cellular immune responses. We used electrotransfection of DC with hTERT mRNA that enables an HLA independent whole antigen approach potentially targeting a wide range of hTERT epitopes. Immature DC were prepared from peripheral blood monocytes in serum-free growth medium, GM-CSF and IL-4. Subsequently the DC were electroporated with mRNA and matured in a cytokine cocktail consisting of IL-1 beta, IL-6, TNF-alpha and PGE2. Verification of hTERT mRNA transfection efficiency was performed by analyzing the induction of telomerase activity with the TRAP assay, the efficiency of GFP electrotransfection was determined by FACS. To optimize mRNA electrotransfection conditions the DCs of 3 healthy individuals were transfected with green fluorescent protein (GFP) mRNA. The percentage of electrotransfected DC was in between 18–89% (mean 46%). Subsequently we electrotransfected DCs in 3 lung cancer patients (1 SCLC, 2 NSCLC). In 1 patient GFP mRNA transfection resulted in 62% GFP positive DC. In the 2 other patients the telomerase activity in the DC 24 hrs after hTERT electrotransfection was equivalent to HL60 cells that biologically express high levels of hTERT. Importantly the DC of one patient did not mature after electrotransfection and incubation in the maturation cocktail. The same was true in another patient, in whom an electrotransfection was not done. In both patients the DC were expressing high levels of HLA-DR and no lineage markers, but CD83 as an indicator of maturation was found in only 1% and 9% respectively. Also the costimulatory molecules CD80 and CD86 were expressed at low levels only. These data show that strong hTERT expression can be achieved in DCs of lung cancer patients using mRNA electrotransfection but demontrate the need for other more potent maturation stimuli. No significant financial relationships to disclose.