Greater degradation of high-affinity TfR^A bispecific as shown by ¹¹¹In accumulation in brain. (A) Schematic illustrating three potential endocytic sorting routes that anti-TfR bispecifics may take within endothelial cells at the BBB. Upon receptor binding, (1) anti-TfR bispecifics may be recycled to the luminal side (blood), (2) transcytosed to the parenchyma (brain), or (3) sorted to the lysosome for degradation. (B–D) Greater retention of [¹¹¹In]DOTA–anti-TfR^A/control (Ctr) IgG high-affinity variant in vivo. (B) Plasma pharmacokinetics of anti-TfR^A/control, anti-TfR^D/control, and control IgG labeled with either ¹¹¹In or ¹²⁵I assessed up to 96 h after dose. (C) Brain radioactivity levels over time were higher for anti-TfR^A/control compared with the other two antibodies. (D) ¹¹¹In signal exceeded and was sustained longer than ¹²⁵I for all antibodies in brain. The total uptake of anti-TfR^A/control in the brain was higher than that of anti-TfR^D/control and control IgG. The percentage of antibody degraded at each time point (shown below graph in D) was calculated by subtracting percent injected doses of ¹²⁵I signal from ¹¹¹In. All data are shown as mean ± SEM; n = 3 mice per antibody group and time point for all panels.