Multiplexed Evaluation
of Serum and CSF Pharmacokinetics
of Brain-Targeting Single-Domain Antibodies Using a NanoLC–SRM-ILIS
Method
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Abstract
FC5 and FC44 are single-domain antibodies (V<sub>H</sub>Hs), selected
by functional panning of phage-display llama V<sub>H</sub>H 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
V<sub>H</sub>Hs in multiplexed assays was developed and applied to
comparatively evaluate brain delivery of FC5 and FC44, and two control
V<sub>H</sub>Hs, EG2 and A20.1. FC5 and FC44 compared to control V<sub>H</sub>Hs demonstrated significantly (<i>p</i> < 0.01)
enhanced transport (50–100-fold) across rat in vitro BBB model
as well as in vivo brain targeting assessed by optical imaging. The
multiplexed SRM-ILIS analyses of plasma and CSF levels of codosed
V<sub>H</sub>Hs demonstrated that while all 4 V<sub>H</sub>Hs have
similar blood pharmacokinetics, only FC5 and FC44 show elevated CSF
levels, suggesting that they are potential novel carriers for delivery
of drugs and macromolecules across the BBB
