Determination of functional EC50 values for atezolizumab, avelumab, durvalumab, nivolumab and pembrolizumab. PD-1 expressing reporter cells were stimulated for 24 h with TCS-CD86 and TCS-CD86/PD-L1 in presence of the indicated PD-1 and PD-L1 blocking antibodies at final concentrations ranging from 1000 to 0.98 ng/ml. Left: Flow cytometric measurement of eGFP expression on PD-1 reporter cells stimulated with TCS-CD86/PD-L1 in the absence (/) or presence of PD-1 blockers used at different concentrations. Data shown are representative of three independent experiments performed in duplicate. Middle: eGFP expression of PD-1 reporter cells stimulated with TCS-CD86/PD-L1 in presence of the indicated concentrations of PD-1 inhibitors. Data were normalized to the eGFP expression of PD-1 reporter cells stimulated in absence of PD-L1 (stimulation with TCS-CD86; dotted line). Right: Inhibition curves and half maximum effective concentrations (EC50) were calculated for the PD-1 and PD-L1 antibodies from normalized data using a 4-parameter logistic function. Results shown in the middle and right panels represent summarized data from three independent experiments performed in duplicate.