High-affinity anti-TfR bispecific variants reduce cortical TfR levels. (A–C) Mice were i.v. injected with various doses of anti-TfR bispecific high- and low-affinity variants, and cortical TfR levels were assessed by Western blot. Quantification of cortex TfR levels normalized to actin and control IgG–dosed animals at 1 and 4 d after dosing with anti-TfR/BACE1 bispecific affinity variants or a control (Ctr) IgG. One sample from the 4 d anti-TfR^D/BACE1 control IgG group did not produce a significant band (A, bottom); however, these same 4 d control IgG samples also appear in the top blot. (D and E) Western blot of cortical TfR levels after injection with anti-TfR/control IgG bispecific variants at 4 d after dosing. Scatter points indicate individual mice sampled in each Western blot and data panel (n = 3–4 mice per group; except in C, n = 2 for the second control IgG group). (F) Immunohistochemistry for TfR 4 d after injection with anti-TfR/control IgG bispecific variants at 50 mg/kg and quantitative analysis of TfR-positive hippocampal vascular area. n = 4 mice were sampled per group, and three stained sections per mouse were quantified. Rabbit anti-TfR detects a different TfR epitope than the injected anti-TfR bispecific. Anti–human IgG reveals antibody distribution in the hippocampus. IV, i.v.; ROI, region of interest. Bars, 100 µm. (G and H) Total cortex (G) and plasma (H) human IgG antibody levels assessed by ELISA. (I and J) Scatter plot and correlation analysis of cortical TfR levels (from E) plotted against brain pharmacokinetics at 4 d after injection (from G) for all dosages of each bispecific (n = 12 in each scatter plot). Bar graphs show means of each group, and error bars are ± SEM; p-values were obtained by Student’s t test versus control IgG groups, except in G and H where p-values compare corresponding doses of anti-TfR^A/control IgG: *, P < 0.05; ***, P ≤ 0.001.