Nk supplement

ADCC of BCA101 was assessed using TGFβ pretreated NK cells and A431 (EGFR^high) or MCF7 (EGFR^low) cell lines, procured from ATCC. NK cells were isolated from frozen or fresh human peripheral blood mononuclear cells (PBMC) using Human NK-cell negative selection kit (Stemcell Technologies Inc.) and EasySep magnet (Stemcell Technologies Inc.). About 1 million enriched NK cells were left untreated or cultured with 1 ng/mL of TGFβ (BPS Biosciences), TGFβ plus cetuximab or BCA101 (56 nmol/L) for 72 hours in a 12-well plate in NK MACS (Miltenyi Biotec) media containing 1% pen strep (GIBCO, Thermo Fisher Scientific), 1% heat-inactivated human serum (Sigma-Aldrich), and 1% NK supplement (Miltenyi Biotec). These pretreated NK cells were used as effectors in ADCC assay. BCA101 (56 nmol/L) was added to target tumor cells and preincubated for 1–2 hours, after which, pretreated NK cells were added at a ratio of 1:5 (5,000 target cells: 25,000 NK cells) in a 96-well plate. The assay plates were incubated for around 24 hours. Cytotoxicity was measured by evaluating protease release using CytoToxGlo (Promega). Luminescence readout was taken using a plate reader (Cytation5 BioTek plate reader) and data were analyzed using GraphPad Prism software (version9). Data are plotted as percentage change in cytotoxicity over human IgG control group.