A novel platform for engineering blood-brain barrier-crossing bispecific biologics.

The blood-brain barrier (BBB) prevents the access of therapeutic antibodies to central nervous system (CNS) targets. The engineering of bispecific antibodies in which a therapeutic "arm" is combined with a BBB-transcytosing arm can significantly enhance their brain delivery. The BBB-permeable single-domain antibody FC5 was previously isolated by phenotypic panning of a naive llama single-domain antibody phage display library.

Multiplexed evaluation of serum and CSF pharmacokinetics of brain-targeting single-domain antibodies using a NanoLC-SRM-ILIS method.

FC5 and FC44 are single-domain antibodies (VHHs), selected by functional panning of phage-display llama VHH library for their ability to internalize human brain endothelial cells (BEC) and to transmigrate the in vitro BBB model. Quantification of brain delivery of FC5 and FC44 in vivo was challenging using classical methods because of their short plasma half-life and their loss of functionality with radioactive labeling. A highly sensitive (detection limit <2 ng/mL) and specific SRM-ILIS method to detect and quantify unlabeled VHHs in multiplexed assays was developed and applied to comparatively evaluate brain delivery of FC5 and FC44, and two control VHHs, EG2 and A20.

In vitro and in vivo methods for assessing FcRn-mediated reverse transcytosis across the blood-brain barrier.

The neonatal Fc receptor, FcRn, mediates endocytic recycling pathway that prevents degradation of IgG and is expressed in most endothelial cells. The blood-brain barrier (BBB), formed by brain endothelial cells sealed with tight junctions, restricts transport of IgG from the blood to the brain. In contrast, it has been suggested that IgG undergoes efflux from the brain parenchyma via reverse transcytosis across the BBB mediated by FcRn.