Understanding key assay parameters that affect measurements of trastuzumab-mediated ADCC against Her2 positive breast cancer cells

Use of the antibody trastuzumab to kill HER2(+) breast cancer cells is an attractive therapy because of its specificity and minimal adverse effects. However, a large fraction of HER2(+) positive patients are or will become resistant to this treatment. No other markers are used to determine sensitivity to trastuzumab other than HER2 status. Using the xCELLigence platform and flow cytometry, we have compared the ability of mononuclear cells (MNCs) from normal and breast cancer patients to kill different breast cancer cell lines in the presence (i.e., ADCC) or absence of trastuzumab. Image analysis and cell separation procedures were used to determine the differential contribution of immune cell subsets to ADCC activity. The assay demonstrated that ADCC activity is dependent on the presence of trastuzumab, the level of HER2 expression on the target, and the ratio of MNCs to tumor cells. There is a wide range of ADCC activity among normal individuals and breast cancer patients for high and low HER2-expressing tumor targets. Fresh MNCs display higher ADCC levels compared with cryopreserved cells. Natural killer cells display the highest ADCC followed by monocytes. T cells and B cells were ineffective in killing. A major mechanism of killing of tumor cells involves insertion of granzyme B and caspase enzymes via the antibody attached MNCs.