Study of the transcytosis of an anti-transferrin receptor antibody with a Fab' cargo across the blood-brain barrier in mice

Abstract

One strategy used to transport pharmacologically active substances across the blood-brain barrier (BBB) is to link the substance to a molecule capable of crossing the BBB using a receptor-mediated transcellular transport system in brain capillary endothelial cells. The transferrin receptor (TfR) is related to a transcytosis process in these cells, and the 8D3 antibody, directed against mouse TfR, is able to induce a receptor response. In this work, the potential of 8D3 to carry molecules across the BBB was investigated. This antibody (either unlabeled or FITC-labeled) was intravenously administered to ICR-CD1 mice. Immunocomplexes (ICs) consisting of 8D3 antibody (carrier) and Fab' fragments (simulated cargo), in some cases directly fluorolabeled with FITC, were also administered to ICR-CD1 mice. At different time-points of recirculation, the IC components were studied using immunohistochemical procedures and fluorescence resonance energy transfer (FRET). The results suggested that 8D3 alone or in an IC with Fab' fragments entered the endothelial cells. FRET analysis indicated that the colocalization of their fluorescent signals inside the endothelial cells decreased with time, indicating that ICs can be processed and Fab' fragments probably separated from 8D3. However, neither 8D3 alone nor the IC components crossed the BBB.

Keywords: 8D3 antibody; AF; Alexa Fluor; BBB; BCEC; BSA; Blood–brain barrier; Bovine Serum Albumin; FITC; FRET; IC; MAb; PBS; PS; ROI; Receptor-mediated transcytosis; Tf; TfR; Transferrin receptor; blood–brain barrier; brain capillary endothelial cell; fluorescein isothiocyanate; fluorescence resonance energy transfer; i.v.; immunocomplex; intravenous; monoclonal antibody; phosphate-buffered saline; positive staining; region of interest; transferrin; transferrin receptor.