High-affinity anti-TfR^A bispecific causes dose-dependent lysosomal degradation of TfR in vitro. (A and B) Mouse brain endothelial cells (line bEND.3) were incubated with increasing levels of anti-TfR^A/control (Ctr), anti-TfR^D/control, or control IgG and collected at 1 (A) or 24 h (B) for Western blot analysis. Gel images and quantification are representative of three independent experiments. (C and D) Time course of TfR reductions after incubation with anti-TfR^A/control. Gel image and quantification of one experiment with triplicate wells are shown. All antibodies were used at 1 µM. (E) bEND.3 cells were incubated with the anti-TfR/control bispecific variants at 1 µM for 24 h in the absence or presence of 100 nM bafilomycin A1 (Baf), a lysosome protease inhibitor, and assessed by Western blot for TfR levels. Gel image and quantification are representative of three independent experiments, each performed in triplicate. All data are shown as mean ± SEM. P-values were assessed by Student’s t test: *, P < 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.