Internalization of bevacizumab by retinal endothelial cells and its intracellular fate: Evidence for an involvement of the neonatal Fc receptor

Bevacizumab is one of the VEGF-binding proteins that are established in clinical practice to treat various ocular diseases. In view of therapeutic long-term application, potential accumulation of the antibody in retinal cells gave reason for safety concerns. Internalization of considerable amounts of bevacizumab by retinal endothelial (REC) and pigment epithelial cells has been observed which may affect their important functions. Therefore we investigated the transport and intracellular localization of bevacizumab in immortalized bovine REC (iBREC) in detail, considering possible roles of vesicles and receptors mediating uptake and intracellular transport. By performing transcytosis assays with iBREC monolayers cultivated on porous membrane inserts, we demonstrated that bevacizumab was transported efficiently through a tight monolayer from the lower to the upper chamber or vice versa. When added to the lower chamber in excess, the internalized antibody was transported through the cells, but it was also recycled to be set free at the same side of the cell into a bevacizumab-free environment. The rates of both processes strongly depended on the concentration of fetal bovine serum (FBS) in the environment. This observation is important because in vivo REC might be exposed to varying amounts of serum, e.g. in patients with macular edema. FBS also affected the intracellular localization of bevacizumab as shown by analyses of subcellular fractions and direct immunofluorescence staining. When iBREC were cultivated in low-serum medium, most of the antibody was found in the fraction of cytoskeleton proteins and spots of high intensity of bevacizumab-specific staining close to the nuclei were observed. Cultivation in medium with FBS resulted in internalized bevacizumab predominately found in the membrane/organelle fraction in addition to its weaker association with proteins from the cytoskeleton and uniform staining of the cell. Bevacizumab-specific staining close to the cytoskeleton proteins alpha-tubulin or vimentin was also observed. Accumulation and association of the antibody with the cytoskeleton induced by serum reduction could be reversed by subsequent FBS addition. In uptake and transport of bevacizumab vesicles and binding to a receptor seems to be involved: Internalization was strongly temperature-dependent which ruled out paracellular passage and a fraction of the internalized bevacizumab was associated with early endosomes. Protein A inhibited transcytosis and affected intracellular localization suggesting a key role of the neonatal Fc receptor (FcRn). Interestingly, FcRn expression was decreased when iBREC were cultivated without FBS. Our results suggest this pathway of bevacizumab uptake and transition through iBREC: Independent of serum, bevacizumab is taken up through a nonspecific mechanism. The subsequent sorting into transport vesicles depends on the presence of serum as regulator of FcRn expression. Without sufficient amounts of the receptor being expressed, a likely obstructed exocytosis results in intracellular accumulation and an increased association with cytoskeleton proteins. Interaction of substantial amounts of bevacizumab with the cytoskeleton may be the reason for under these conditions suppressed migration of iBREC. If long-term therapies by intravitreal injection lead to accumulation of bevacizumab in REC in vivo and potentially harmful consequences, will have to be revealed by future investigations. (C) 2015 The Authors. Published by Elsevier Ltd.