In vitro effects of trastuzumab and T-DM1. A. Growth inhibitory effects of trastuzumab and T-DM1 on HER2 positive breast cancer cell lines in vitro. T-DM1 (1 μg/ml, hatched column) significantly inhibited the growth of all HER2 positive breast cancer cell lines in comparison to trastuzumab (10 μg/ml, black column) (*, P < 0.05). Neither trastuzumab nor T-DM1 had any growth inhibitory effect on MCF-7 cells with low trastuzumab binding capacity. Note that trastuzumab was used in a 10-times higher dose than T-DM1. B. T-DM1 inhibited the growth of HER2 positive breast cancer cells in a dose-dependent manner. The effect of T-DM1 was tested at a concentration of 0.001, 0.01, 0.1 and 1 μg/ml. Its dose-dependent growth inhibitory effect was seen on SKBR-3 (black circle), BT-474 (white circle) and MDA-453 (black triangle) HER2 positive breast cancer cell lines. No inhibition of HER2 negative MCF-7 (white triangle) breast cancer cells was seen. C. Trastuzumab and T-DM1 evoke similar efficient ADCC on HER2 positive breast cancer cells in vitro. Target tumour cells (SKBR-3, JIMT-1, MCF-7) were mixed with peripheral blood mononuclear cells (PBMCs) freshly isolated from peripheral blood at effector/target ratios of 5:1, 10:1, 20:1, 40:1, and 80:1. ADCC was analyzed by measuring the lactate dehydrogenase (LDH) released from the cancer cells as a result of ADCC activity of PBMCs in the presence of trastuzumab, T-DM1 or control antibody (rituximab) (20 μg/ml). The percentage of killed cells was calculated as described in Materials and methods. Killing of SKBR-3 cells was significantly higher in the presence of both trastuzumab (blue) and T-DM1 (pink) in comparison to that in the presence of control antibody (yellow) when the effector/target cell ratio was 5 or above (blue and pink stars, P < 0.05). Similarly, significantly higher killing of JIMT-1 cells was detected in the presence of both trastuzumab (gray) and T-DM1 (brown) compared to that in the presence of control antibody (pale blue) when the effector/target cell ratio was 20 or above (gray and brown stars, P < 0.05). Trastuzumab and T-DM1 evoked similar tumour cell killing on the HER2 positive breast cancer cell lines (SKBR-3 and JIMT-1). MCF-7 cells with weak trastuzumab binding capacity showed low levels of both trastuzumab- and T-DM1-evoked ADCC-mediated killing (red and green, respectively).