However, the 128.1 antibody localized to a CD63-positive late endosomal/lysosomal compartment one hour after internalization, where it presumably is degraded. In contrast to 128.1, the mouse monoclonal antibody MEM-189 is processed like the ligand. It is endocytosed at a slightly slower rate than transferrin and the 128.1 antibody in hCMEC/D3 cells, which is likely attributable to the lower-affinity binding profile of the antibody. However, once internalized, the antibody is processed through the endocytic pathway with transcytosis and recycling kinetics comparable to that of transferrin. The 128.1 and MEM-189 antibodies target overlapping epitopes, as shown by a competition ELISA, which lowers the probability that the MEM-189 local epitope on TfR is solely responsible for its transcytosis potential, although it cannot be excluded. Even antibodies with closely overlapping epitopes can demonstrate fundamental functional differences, as illustrated for the CD20 antibodies GA101 and rituximab. Intracellular degradation of TfR antibodies after endocytosis has been described by others, and the 128.1 antibody follows those examples in terms of lysosomal localization and degradation. Furthermore, incubation of murine lymphoma cells with full IgGs against the TfR has been shown to even downregulate surface expression of the TfR. In contrast, Yu et al. have demonstrated that in vivo, antibodies against the transferrin receptor are transported into the brain to an extent inversely correlated to their binding affinity. We compared the bivalent affinities of the different TfR antibodies by direct ELISA and observed a similar correlation with regard to transcytosis potential. A low-affinity antibody, LT-71 was capable of shuttling through hCMEC/ D3 monolayer, albeit after low uptake, while the highaffinity antibodies 128.1 and 13E4 remain inside the cells. However, two antibodies with intermediate binding affinities demonstrated significantly different sorting behavior: while MEM-189 was efficiently transcytosed and recycled, MA712 was incapable of leaving the endothelial cells. It is unlikely that the small difference in binding affinity should be responsible for the striking difference in transcytosis potential. The transferrin:transferrin receptor complex undergoes dramatic conformational changes upon endosomal acidification, which are partly driven by iron release from the ligand, but which are accompanied by significant changes in the TfR conformation. These conformational changes might be responsible for the observed pH-dependence of several TfR antibodies. In addition, histidine residues in antibody CDR could contribute to pH-dependent target binding. The mechanism of transferrin transcytosis, especially which sorting events determine routing for basolateral transcytosis or apical recycling, have not yet been investigated. In polarized cells, TfR is known to be recycled via recruitment of the adaptor protein AP1B in the recycling endosome.