Avelumab

Human NSCLC cell lines were obtained by the American Type Culture Collection (ATCC, Manassas, Virginia, USA) and were cultured in RPMI-1640 (Sigma-Aldrich) medium which was supplemented with 10% foetal bovine serum (FBS; Life Technologies, Gaithersburg, Maryland, USA) in a humidified atmosphere with 5% CO₂. The identity of cell lines was confirmed by Short tandem repeat profiling (Promega). Cetuximab, atezolizumab and panitumumab were purchased from Selleck Chemicals (Munich, Germany). Avelumab, was provided by Merck (Darmstadt, Germany), as part of a Cooperative Research and Development Agreement. Peripheral blood mononuclear cells (PBMCs) from healthy donors (HD) or from NSCLC patients were isolated by Ficoll-Paque Plus (GE Healthcare). NK cells were obtained through magnetic separation by using the NK CELL isolation kit, HUMAN (Miltenyi Biotech 130-092-657) according to the manufacturer’s protocol.

96-well plates were coated with the indicated proteins at 2 μg/ml overnight, washed three times with PBS-T, and blocked with 4 % nonfat dried milk for 1 h at room temperature (RT). Twofold serial dilution of Avelumab (Merck) or trastuzumab (Roche) from 6 μg/ml and incubated for 1 h at RT. The wells were washed three times and HRP-conjugated goat anti-human IgG Fc Secondary Antibody (1:6000) was added and incubated for 30min at RT. TMB substrate (Invitrogen) was used for detection at 450 nm.