title: The role of Fc-receptors in the uptake and transport of therapeutic antibodies in the retinal pigment epithelium.
abstract: In the ophthalmological clinic, intravitreally applied antibodies or Fc-containing fusion proteins are frequently used, but the biology and pharmacokinetics of these therapeutics in the retina are not well understood. We have previously shown intracellular uptake of Fc-containing molecules in RPE cells. In this study, we investigated the involvement of Fc-receptors, both Fcγ-receptors and the neonatal Fc-receptor (FcRn) in the uptake and intracellular trafficking of the VEGF-antagonists bevacizumab, aflibercept and the anti-CD20 antibody rituximab in three different model systems, primary porcine RPE cells, ARPE-19 cells and porcine RPE/choroid explants. The expression of Fcγ-receptors was tested in primary porcine RPE cells, and the expression of Fcγ-receptors I and II could be shown in RT-PCR and qRT-PCR, while the expression of FcRn was additionally confirmed in Western blot and immunocytochemistry. All three compounds, bevacizumab, rituximab and aflibercept, were taken up into the cells and displayed a characteristic time-dependent pattern, as shown in Western blot and immunohistochemistry. The uptake was not altered by the inhibition of Fcγ-receptors using different inhibitors (TruStain FcX, genistein, R406). However, the inhibition of FcRn with an antagonistic antibody reduced intracellular IgG in porcine RPE cells (rituximab) and ARPE-19 cells (bevacizumab, rituximab). Colocalisations between the tested compounds and myosin7a could be found. In addition, limited colocalization with FcRn and the tested compounds, as well as triple localization between compound, FcRn and myosin7a could be detected, indicating a role of myosin7a in FcRn mediated transport. However, the colocalizations are restricted to small fractions of the Fc-containing compounds. Furthermore, the FcRn is mainly found in the membrane section, where only minute amounts of the Fc-containing compounds are seen, suggesting a limited interaction. An apical to choroidal transport of IgG through the RPE/choroid can be found in RPE/choroid explants. Inhibition of FcRn increases the amount of bevacizumab found on the choroidal side, suggesting a role of FcRn in the recycling of bevacizumab. In conclusion, our data indicate a role for FcRn, but not Fcγ-receptors, in the uptake and transport of Fc-containing molecules in the RPE and indicate a recycling function of FcRn in the retina.
authors.affiliation: University of Kiel, University Medical Center, Department of Ophthalmology, Kiel, Germany.
authors.forename: Michaela
authors.initials: M
authors.lastname: Dithmer
authors.affiliation: University of Kiel, Department of Anatomy, Kiel, Germany.
authors.forename: Kirsten
authors.initials: K
authors.lastname: Hattermann
authors.affiliation: University of Kiel, University Medical Center, Department of Ophthalmology, Kiel, Germany.
authors.forename: Prasti
authors.initials: P
authors.lastname: Pomarius
authors.affiliation: University of Kiel, University Medical Center, Department of Ophthalmology, Kiel, Germany; University of Cairo, Kasr Al-Aini Faculty of Medicine, Cairo, Egypt.
authors.forename: Shereen Hassan
authors.initials: SH
authors.lastname: Aboul Naga
authors.affiliation: Department of Gastroenterology, Charite University Clinic, Berlin, Germany.
authors.forename: Tim
authors.initials: T
authors.lastname: Meyer
authors.affiliation: University of Kiel, Department of Anatomy, Kiel, Germany.
authors.forename: Rolf
authors.initials: R
authors.lastname: Mentlein
authors.affiliation: University of Kiel, University Medical Center, Department of Ophthalmology, Kiel, Germany.
authors.forename: Johann
authors.initials: J
authors.lastname: Roider
authors.affiliation: University of Kiel, University Medical Center, Department of Ophthalmology, Kiel, Germany. Electronic address: aklettner@auge.uni-kiel.de.
authors.forename: Alexa
authors.initials: A
authors.lastname: Klettner
mesh_terms: D020533:Angiogenesis Inhibitors / Q000493:pharmacokinetics*; D000818:Animals; D000068258:Bevacizumab / Q000493:pharmacokinetics*; D015153:Blotting, Western; D002478:Cells, Cultured; D002829:Choroid / Q000378:metabolism; D007074:Immunoglobulin G / Q000378:metabolism; D007150:Immunohistochemistry; D023421:Models, Animal; D000081412:Myosin VIIa; D009218:Myosins / Q000378:metabolism; D011961:Receptors, Fc / Q000502:physiology*; D040262:Receptors, Vascular Endothelial Growth Factor; D011993:Recombinant Fusion Proteins / Q000493:pharmacokinetics*; D055213:Retinal Pigment Epithelium / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000069283:Rituximab / Q000493:pharmacokinetics*; D013552:Swine
chemical_list: D020533:Angiogenesis Inhibitors; D007074:Immunoglobulin G; D000081412:Myosin VIIa; D011961:Receptors, Fc; D011993:Recombinant Fusion Proteins; C533178:aflibercept; D000068258:Bevacizumab; D000069283:Rituximab; D040262:Receptors, Vascular Endothelial Growth Factor; D009218:Myosins
keywords: Aflibercept; Bevacizumab; Neonatal Fc-receptor (FcRn); Retinal pigment epithelium (RPE); Rituximab