Brain Uptake of a Fluorescent Vector Targeting the Transferrin Receptor: A Novel Application of in Situ Brain Perfusion

Abstract

Monoclonal antibodies (mAbs) targeting blood-brain barrier (BBB) transporters are being developed for brain drug targeting. However, brain uptake quantification remains a challenge, particularly for large compounds, and often requires the use of radioactivity. In this work, we adapted an in situ brain perfusion technique for a fluorescent mAb raised against the mouse transferrin receptor (TfR) (clone Ri7). We first confirmed in vitro that the internalization of fluorolabeled Ri7 mAbs is saturable and dependent on the TfR in N2A and bEnd5 cells. We next showed that the brain uptake coefficient (Clup) of 100 μg (∼220 nM) of Ri7 mAbs fluorolabeled with Alexa Fluor 750 (AF750) was 0.27 ± 0.05 μL g(-1) s(-1) after subtraction of values obtained with a control IgG. A linear relationship was observed between the distribution volume VD (μL g(-1)) and the perfusion time (s) over 30-120 s (r(2) = 0.997), confirming the metabolic stability of the AF750-Ri7 mAbs during perfusion. Co-perfusion of increasing quantities of unlabeled Ri7 decreased the AF750-Ri7 Clup down to control IgG levels over 500 nM, consistent with a saturable mechanism. Fluorescence microscopy analysis showed a vascular distribution of perfused AF750-Ri7 in the brain and colocalization with a marker of basal lamina. To our knowledge, this is the first reported use of the in situ brain perfusion technique combined with quantification of compounds labeled with near-infrared fluorophores. Furthermore, this study confirms the accumulation of the antitransferrin receptor Ri7 mAb in the brain of mice through a saturable uptake mechanism.

Antibody transcytosis across brain endothelial-like cells occurs nonspecifically and independent of FcRn

“…Development of new approaches to increase the penetration of IgG or other macromolecules across the BBB can benefit from thorough in vitro characterization to appropriately interpret transcytosis phenomena. Our observation of saturable transcytosis of transferrin is consistent with other in vivo and in vitro reports also examining RMT by the TfR 45,46 , and the increased intracellular accumulation (relative to human IgG) supports the notion that lysosomal accumulation and degradation is a detriment to RMT 46 . Although TfR saturation in iBECs (~667 nM transferrin) is similar to that observed in mice (~500 nM of an anti-transferrin IgG 45 ), saturation kinetics can vary from species to species 47 .…”

“…Our observation of saturable transcytosis of transferrin is consistent with other in vivo and in vitro reports also examining RMT by the TfR 45,46 , and the increased intracellular accumulation (relative to human IgG) supports the notion that lysosomal accumulation and degradation is a detriment to RMT 46 . Although TfR saturation in iBECs (~667 nM transferrin) is similar to that observed in mice (~500 nM of an anti-transferrin IgG 45 ), saturation kinetics can vary from species to species 47 . Accordingly, evaluation of other RMT receptors should consider appropriate species-relevant models and potential saturation by native ligands at endogenous levels.…”

Human Apolipoprotein E ε4 Expression Impairs Cerebral Vascularization and Blood—Brain Barrier Function in Mice

“…9,14,28 However, a unique advantage of the in situ brain perfusion technique is to allow complete control on not only the perfusate content and perfusion time, but also the flow rate, by adjusting the pump. 8 Since 100% of the perfused [ 3 H]-diazepam has direct access to the BBB, in accordance with Fick's first law of diffusion, a decrease in [ 3 H]-diazepam uptake can only be explained by a reduced surface permeability of the BBB. 13,22 Finally, considerable changes in BBB integrity have been ruled out by experiments conducted with perfused [ 14 C]-sucrose, systemically injected hIgG and two tight junctions proteins (occludin and claudin-5).…”

Cerebrovascular and blood–brain barrier impairments in Huntington's disease: Potential implications for its pathophysiology

“…In situ cerebral perfusion was performed using established protocols . Each mouse was perfused for 240 seconds with [ 14 C]sucrose (0.6µCi/ml of perfusion buffer; 412mCi.mmol −1 ; Moravek Biochemicals, Brea, CA), a vascular space marker that does not cross the BBB, and [ 3 H]diazepam (0.3µCi/ml of perfusion buffer; 83.7Ci.mmol −1 ; PerkinElmer, Boston, MA), a highly lipophilic drug that readily crosses the BBB in a flow‐limited fashion.…”