Degradation of TfR reduces BBB transcytosis capacity for a therapeutic bispecific antibody. (A) Multidose in vivo study paradigm to assess the effect of TfR degradation on BBB antibody transcytosis and Western blot analysis of cortical lysates. Mice were dosed i.v. with antibody at 50 mg/kg on day 1. After 48 h (day 3), each received either a control IgG or anti-TfR^D/BACE1 at 50 mg/kg. Each lane represents one individual mouse brain sample; n = 4 mice per group. (B) Brains were collected on day 4, and cortical lysates were probed by Western blot and quantified for TfR and actin levels. (C) Levels of anti-TfR^D/BACE1 on day 4 were determined by a BACE1-specific ectodomain capture ELISA. (D and E) Levels of total human IgG antibody concentrations in brain (D) and plasma (E) were determined by a generic human Fc ELISA. Bars represent mean ± SEM; p-values were obtained by Student’s t test versus control IgG groups or as indicated: *, P < 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. Ctr, control.