High-affinity TfR^A bispecific facilitates TfR trafficking to lysosomes. (A and B) Surface levels of TfR in bEND.3 cells were monitored by TIRFM using the QD (QD605)-conjugated anti–murine TfR Fab fragment (TfRFab:QD) of an antibody with a different epitope for TfR from the anti-TfR bispecifics. High- and low-affinity anti-TfR bispecifics were incubated at their respective IC₅₀ concentrations to normalize for affinity differences. TfRFab:QD on basal membranes was tracked, imaged at 0 and 20 min (A), and quantified over 22 min with a 3-s laser illumination time interval (B). Images shown are pseudocolored. Quantification of the last 10 time points in B showed that TfR^A/control (Ctr) had 0.54 ± 0.01 surface TfR remaining relative to control IgG, whereas TfR^D/control had 0.80 ± 0.01 surface TfR relative to control IgG (mean ± SEM). n = 8 cells analyzed for each condition. (C and D) Movement of TfRFab:QD after 1-h incubation with high- and low-affinity anti-TfR bispecific in the presence of LysoTracker. (C) TfRFab:QD was imaged relative to LysoTracker. The total internal reflection angle was adjusted to illuminate the inside and surface of the same cells in C. (D) Quantification of the remaining TfR fraction on the cell surface after 1-h incubation with anti-TfR bispecifics, TfR^A bispecific (0.32 ± 0.02, n = 74 cells analyzed) and TfR^D bispecific (0.57 ± 0.03, n = 40 cells analyzed). Mean ± SEM; ***, P < 0.0001 by one-tailed Student’s t test for TfR^A bispecific versus TfR^D bispecific. (E) TfRFab:QD and TfRFab did not decrease TfR levels under TIRFM imaging conditions (1 h, 100 nM) as confirmed by Western blot (gel image is representative of three samples). (F) pHrodo-labeled anti-TfR bispecifics and control IgG were incubated for 1 h (at IC₅₀ concentrations) in the presence of LysoTracker, and intracellular pools were imaged for extent of colocalization. More pHrodo–anti-TfR^A/control overlapped with LysoTracker-positive, perinuclear compartments, likely representing lysosomes, than pHrodo–anti-TfR^D/control or pHrodo–control IgG. Boxed perinuclear regions are shown to the right as rendered images (G) to compare the relative locations between pHrodo–anti-TfR^A/control and pHrodo–anti-TfR^D/control, with respect to LysoTracker. White arrows highlight tubular-shaped pools of endosomes containing significantly more pHrodo–anti-TfR^D/control or pHrodo–control IgG than pHrodo–anti-TfR^A/control. Representative images were chosen from n > 20 cells imaged per condition. (H) pHrodo-conjugated TfR bispecifics and control IgG were assayed for their relative effects on TfR levels after a 24-h incubation at 1 µM (gel image is representative of three samples). A reduction in TfR was observed for pHrodo–anti-TfR^A bispecific but not pHrodo–anti-TfR^D bispecific or control IgG. Bars, 10 µm.