Development of a simple new flow cytometric antibody-dependent cellular cytotoxicity (ADCC) assay with excellent sensitivity

Antibody-based therapeutic strategies have become recognized as useful clinical options in several types of cancer, often with the expectation that such therapies will trigger target cell elimination via antibody-dependent cellar cytotoxicity (ADCC) by natural killer cells. The successful development of therapeutic monoclonal antibodies (mAbs) requires an assay system that permits a critical evaluation of their physicochemical and biological characteristics. At present a number of ADCC assay systems have been reported, however, there is still room for improvement in terms of usability, operability and sensitivity. Here we report a novel flow cytometric ADCC assay that uses a human natural killer cell line stably transfected with mouse FcγRIII, and Fc receptor common-γ chain (FcRγ) and a reporter gene as effector cells. This assay relies on discriminating effector and target cells by their differential immunofluorescence, which allows for clear-cut gating and accurate calculation of the number of surviving cells in a target population. This assay is easy and quick to perform and provides reliable data even for low frequency target cells in assay samples and with low concentrations of mAbs. Furthermore, our approach allows us to identify synergistic ADCC activity of mAbs with different epitope specificities on the same target antigen.