The correlation of trastuzumab and pertuzumab binding avidity-improvement and their antitumor activities. (A), antigen binding activities of trastuzumab and pertuzumab variants on SK-BR-3 cells were determined by FCM. (B), MTS assay examining the proliferation effects of 100 nmol/L of trastuzumab variants or pertuzumab variants in breast cancer cell BT-474 with the absence or presence of ErbB ligand (EGF or HRG). Results are shown as percentage of control cell proliferation. Error bars, SD. (C), in the absence of ligands, cell death induced by trastuzumab variants or pertuzumab variants were assessed by staining with SYTOX® Red and FCM. (D), In vitro ADCC analysis of trastuzumab and pertuzumab variants with binding avidity improvement. In the absence of ligands, PBMCs were added with BT-474 cells into 96-well plates containing trastuzumab or pertuzumab variants. Data are mean ± SD of at least three experiments.