Comparison of a Novel Bisphosphonate Prodrug and Zoledronic Acid in the Induction of Cytotoxicity in Human Vγ2Vδ2 T Cells

Increasing attention has been paid to human gamma delta T cells expressing V gamma 2V delta 2 T cell receptor (also termed V gamma 9V delta 2) in the field of cancer immunotherapy. We have previously demonstrated that a novel bisphosphonate prodrug, tetrakis-pivaloyloxymethyl 2-(thiazole-2-ylamino)ethylidene-1,1-bisphosphonate (PTA), efficiently expands peripheral blood V gamma 2V delta 2 T cells to purities up to 95-99% in 10-11 days. In the present study, we first examined the effect of PTA on farnesyl diphosphate synthase (FDPS) using liquid chromatography mass spectrometry (LC-MS) to analyze the mechanism underlying the PTA-mediated expansion of V gamma 2V delta 2 T cells. We find that the prodrug induced the accumulation of both isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), direct upstream metabolites of FDPS. This indicates that not only IPP but also DMAPP plays an important role in PTA-mediated stimulation of V gamma 2V delta 2 T cells. We next analyzed TCR-independent cytotoxicity of V gamma 2V delta 2 T cells. When human lung cancer cell lines were challenged by V gamma 2V delta 2 T cells, no detectable cytotoxicity was observed in 40 min. The lung cancer cell lines were, however, significantly killed by V gamma 2V delta 2 T cells after 4-16 h in an effector-to-target ratio-dependent manner, demonstrating that V gamma 2V delta 2 T cell-based cell therapy required a large number of cells and longer time when tumor cells were not sensitized. By contrast, pulsing tumor cell lines with 10-30 nM of PTA induced significant lysis of tumor cells by V gamma 2V delta 2 T cells even in 40 min. Similar levels of cytotoxicity were elicited by ZOL at concentrations of 100-300 mu M, which were much higher than blood levels of ZOL after infusion (1-2 mu M), suggesting that standard 4 mg infusion of ZOL was not enough to sensitize lung cancer cells in clinical settings. In addition, V gamma 2V delta 2 T cells secreted interferon-gamma (IFN-gamma) when challenged by lung cancer cell lines pulsed with PTA in a dose-dependent manner. Taken together, PTA could be utilized for both expansion of V gamma 2V delta 2 T cellsex vivoand sensitization of tumor cellsin vivoin V gamma 2V delta 2 T cell-based cancer immunotherapy. For use in patients, further studies on drug delivery are essential because of the hydrophobic nature of the prodrug.