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Michaela Dithmer, Johann Roider, Alexa Karina Klettner; Intracellular uptake of therapeutic immunoglobulins and fusion proteins: Comparison between Aflibercept, Bevacizumab and Rituximab. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):835. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Bevacizumab, a VEGF antagonist used in age-related macular degeneration (AMD)-therapy, is taken up by retinal pigment epithelium (RPE) cells and stored for at least 7 days. To get new insights in the pharmacokinetics in the eye, we investigated a possible involvement of the Fc-part in the uptake and transportation and whether the mechanism of uptake is a generic process, independent of specificity of the compound.
Primary porcine RPE- and ARPE-19-cells were treated with the clinical dose of Bevacizumab (250 µg/ml), Aflibercept (500 µg/ml and 250 µg/ml) and Rituximab (250 µg/ml) for different time intervals. The uptake of the applied molecules was analyzed via Western blot and immunocytochemistry. The Fcg-receptors were inhibited with Genistein (150 µM) or R406 (1µM). Possible interactions between FcRn and the tested molecules were observed via immunocytochemistry and subcellular extraction. To get more insight in uptake and transport, clathrin, myosin VIIa, actin filament, endocytotic vesicles and lysosomes were also stained. Transport of the used molecules through RPE-choroid organ cultures in Ussing chambers were detected with IgG-ELISA.
The tested molecules were taken up by RPE-cells and detected in a similar pattern. The uptake could been observed despite inhibition of the Fc-receptors. Colocalisations between the FcRn and IgG could been detected in immunocytochemistry, with the Fc-containing molecules found on the cytoskeleton, and FcRn located on membranes and organelles in subcellular fractioning. Triple-colocalisations between IgG, FcRn and Myosin VIIa could also been observed. In the Ussing chambers transport of Bevacizumab and Rituximab could be detected. A few colocalisations between lysosomes and Aflibercept could been observed.
All therapeutical antibodies tested are taken up and retained in the RPE, with bevacizumab showing longer retention time. The uptake does not seem to be mediated via Fc-Receptors, while FcRn seems to be involved in intracellular transport. Moreover, FcRn and IgG colocalizes with the motor protein myosin 7a. As we can find a transport of Bevacizumab and Rituximab in RPE/Choroid organ cultures, our data indicate an active transport process of IgG through the RPE via FcRn and myosin 7a.
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