(553e) Extended Brain Delivery of Off-the-Shelf IgGs Using CD98hc Bispecific Antibodies

The modest ability of antibodies and other biologics to penetrate the blood-brain barrier (BBB) severely limits their use in diagnostic, imaging, and therapeutic applications. One promising strategy for delivering IgGs into the brain parenchyma is to use a bispecific BBB shuttle, which involves fusing an IgG to a second affinity ligand that engages a cerebrovascular endothelial target and facilitates transport across the BBB. Nearly all prior efforts have focused on the transferrin receptor (TfR-1) as the prototypical endothelial target despite inherent delivery and safety challenges. Here we report bispecific antibody shuttles that engage CD98hc, the heavy chain of the large neutral amino acid transporter (LAT1), and efficiently transport IgGs into the mouse brain parenchyma. Our work builds on a 2016 pioneering study by Genentech that demonstrated the potential of CD98hc-mediated brain shuttling of antibody (Fab) fragments in mice (Zuchero et al., Neuron, 2016). Our studies aimed to address several unique questions left unanswered by this outstanding study. First, we have sought to deliver intact IgGs, instead of Fab fragments, to the mouse brain parenchyma, using a 2×1 CD98hc shuttle that maintains the native structure of the brain-targeted IgG. This is critical, as reformatting bivalent IgGs into monovalent Fabs for incorporation into a 1×1 shuttle is likely to compromise biological function in the brain parenchyma. Second, we have extensively characterized the pharmacokinetics of untargeted IgGs shuttled into the brain via CD98hc and TfR-1, revealing unique advantages of each transport pathway. For our 2x1 shuttles, TfR-1 shuttles achieve earlier and higher peak brain concentrations, whereas CD98hc shuttles demonstrate a far slower decline in their brain concentrations, resulting in >100-fold higher blood:brain ratios at 7 days than those for their TfR-1 counterparts. Third, we have characterized the parenchymal engagement and cellular uptake of untargeted 2x1 CD98hc and TfR-1 shuttles. While TfR-1 targeting mediates internalization into various cell types in the brain parenchyma for non-targeted IgGs, the untargeted IgG/CD98hc shuttles remain associated with the cerebrovasculature and do not demonstrate cellular uptake in the absence of a parenchymal target. Fourth, we have incorporated IgGs that bind cell-type specific surface proteins on neurons, astrocytes, or oligodendrocytes into the 2x1 CD98hc shuttle and determined the extent to which they reach their target cells following intravenous injection. In each case, we observe a profound temporal redistribution of the CD98hc shuttles, which shifts from initial localization at the cerebrovasculature to final localization at each cell type in the brain parenchyma. Moreover, some IgGs mediate internalization into their target cells, while others do not. Finally, we have demonstrated parenchymal delivery and receptor activation of a CD98hc-shuttled TrkB agonist IgG for extended periods of time (several days) after a single intravenous dose and are currently evaluating the neuroprotective activity of this agonist IgG in various acute and chronic neurological disorders.