RBC binding is maintained in Fc-mutated and glycoengineered anti-RhD mAbs. (A) Detection of fucosylation levels (top blot) and Ab heavy/light chains (bottom blot) in purified AFUC mAbs. (B) Binding of mAb variants to RBCs was analyzed in flow cytometry. Serial dilutions of mAbs were incubated with RhD^pos or RhD^neg RBCs, and the binding was detected with Fab goat anti-human IgG (H&L) Texas Red. (C) EC50 binding of mAb variants. P values show the significance of RhD^pos RBC binding of mAb variants vs WT format in each group. Statistical analyses were conducted using 1-way analysis of variance (ANOVA), followed by Dunnett multiple comparison test (GraphPad Prism v9). Data are representative of 3 replicates. ns, not significant.