Antibody-Dependent Cellular Cytotoxicity (ADCC) Assay

ADCC assays were performed using the HER2-overexpressing BT-474 cells as target cells and fresh peripheral blood NK cells as effector cells. NK cells and cancer cells were mixed at E:T ratio 10:1 (0.2 × 10⁶ NK cells: 0.02 × 10⁶ target cells) in RPMI GlutaMAX™ (Thermo Fischer Scientific) containing 2% FCS (Thermo Fischer Scientific). They were incubated in 96-well round bottom plates for 4 h (Thermo Fischer Scientific) in the presence of serially diluted anti-HER2 antibody variants or isotype control antibodies (at concentrations ranging from 64 pg/mL to 5 µg/mL). Cellular cytotoxicity was assessed using the Pierce LDH Cytotoxicity Assay Kit (Thermo Fischer Scientific), according to the manufacturer’s instructions. Absorbance read out was measured at 490 and 680 nm using Fluostar^® Omega Spectrophotometer (BMG Labtech) and % cytotoxicity was calculated as per manufacturer’s instructions using the following formula:

Half maximal effective concentration (EC50) values at which different anti-HER2 Fc variant antibodies induced ADCC were calculated using the GraphPad Prism software (version 6, GraphPad Software Inc., USA). The data were normalized to a maximal (detergent induced) and minimal (isotype control) cell lysis and analyzed using a non-linear regression model.