Fig. 5.

Inhibition of antibody internalization results in increased ADCC. a (Above) ADCC experimental design schema. (Below) Average (± SD) neutrophil-mediated ADCC against Lan1GFP cells (% of control) measured by Incucyte 6 h after addition of neutrophils (E:T of 10:1) with dinutuximab (5 µg/ml) and GM-CSF (100 ng/ml). Prior to addition, cells were pretreated with EIPA, chlorpromazine, MBCD, or media control for 30 min (4 replicate wells each over 3 individual experiments). b Mean (± SEM) normalized 24 h AUC of 14G2a internalization for Lan1 cells pretreated with twofold dilutions of MBCD as noted or media only (mean of 3 individual replicates). c Average (± SEM) neutrophil-mediated ADCC against Lan1 cells (% of control) measured 6 h after addition of neutrophils (E:T of 10:1), dinutuximab (5 ug/ml) and GM-CSF (100 ng/ml) following the design schema shown in (A). Prior to addition, cells were pretreated with MBCD at twofold dilutions as noted for 30 min. (mean of 3 individual replicates). d Mean (± SD) 24-h AUC (expressed as % of control) of 14G2a internalization for Lan1 cells following a 4-h pre-treatment period with indicated endocytosis inhibitors or media only followed by addition of 14G2a-pHrodo (10 ug/ml) in the continued presence of inhibitor (solid bars) or immediately following drug removal (striped bars). e A modified ADCC experimental design schema is on top. Below, Average (± SD) neutrophil-mediated ADCC against LAN2, CHLA90, and CHLA136 neuroblastoma cells (expressed as % of control) measured 6 h after addition of neutrophils (E:T of 10:1), dinutuximab (1 ug/ml) and GM-CSF (100 ng/ml) is shown. Prior to addition, cells were pretreated with EIPA, chlorpromazine, MBCD or media for 30 min followed by addition of dinutuximab in the continued presence of drug or media for 4 h and then washing the cells 3x to remove drug and dinutuximab. This allowed to evaluate ADCC with remaining cell-bound dinutuximab and evaluate effect of inhibitors. (**p < 0.01, ns = non-significant)