Adsorptive-Mediated Endocytosis of Sulfo-Cy5-Labeled IgG Causes Aberrant IgG Processing by Brain Endothelial-Like Cells

Abstract

Brain endothelial cells (BECs) hinder macromolecules from reaching brain parenchyma, necessitating the evaluation and engineering of therapeutic immunoglobulin γ (IgG) for improved brain delivery. Emerging fluorescent-based approaches to assess IgG brain exposure can expedite and complement current methods; however, alterations in IgG pharmacokinetics following fluorophore conjugation, which remain unexplained, indicate that conjugation may confound analysis of native IgG processing. Here, changes in transcytosis and intracellular processing of IgG conjugates (with sulfonated cyanine 5) were examined using human induced pluripotent stem cell-derived BECs (iBECs). Above a critical degree of labeling, transcytosis rates increased significantly but could be attenuated by nonspecific protein competition. Concurrent increases in intracellular accumulation, which was not attributable to disrupted binding by the neonatal Fc receptor (FcRn), are indicative of indirect reduction of FcRn-mediated recycling that agrees with reported aberrations in the pharmacokinetics of certain unconjugated IgGs. Overall, these findings support the notion that certain fluorophore-IgG conjugates can engage in adsorptive interactions with cell surface moieties, reminiscent of phenomena exhibited by cationized IgG, and provide in vitro criteria to identify changes in IgG processing following fluorophore conjugation.