The extracellular domains of human FcγRIIb, FcγRIIIaF158, FcγRIIIaV158, FcγRIIaH131 or FcγRIIaR131 with AviTag and a 6×his tag fused at the C-terminus were expressed by HEK293F cells and purified with HisTrap™ HP column (GE healthcare) and buffer-exchanged to PBS. For biotinylation, the purified FcγRs were treated with biotin-protein ligase (GeneCopoeia) and the degree of biotinylation was verified by a pull-down assay using streptavidin magnetic beads (Thermo Fisher Scientific). The Fc variant genes were obtained by site-directed mutagenesis using the wild-type Fc gene as template. Fc and antibody (rituximab, trastuzumab or NK003) variants were expressed by HEK293F or CHO-K1 cells and the defucosylated Fc antibodies were expressed by FUT8-/- CHO-K1 cells and purified using protein A beads (GE healthcare).

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