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Yang Liu, Terra Potocky, Jingtai Cao, Joel Martin, Nicholas Papadopoulos, Stanley Wiegand; Immune-Like Complexes of Bevacizumab Bind to Retinal Pigment Epithelial Cells and Endothelial Cells in vitro. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3286.
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© ARVO (1962-2015); The Authors (2016-present)
The VEGF inhibitors ranibizumab (RAN), aflibercept (AFL), and bevacizumab (BEV) bind VEGF with different binding stoichiometries. AFL binds VEGF exclusively as a 1:1 complex (MW: 155 kDa). In contrast, BEV:VEGF complexes are heterogeneous in size (~400kDa to >2,000 kDa) (IOVS 2012, E-Abstract 440), while one or two RAN molecules may bind to a single VEGF dimer, forming 1:1 or 2:1 complexes. We compared the binding patterns of BEV, RAN, and AFL to ARPE-19 cells and HUVEC, and investigated the mechanisms underlying differential binding of drug:VEGF complexes to cells.
ARPE-19 cells were treated with equimolar concentration of BEV, RAN, and AFL. Cell surface bound VEGF blockers were detected by immunofluorescence staining. Cells and culture media were collected for TaqMan and ELISA of VEGF165 RNA and protein levels. For acute binding assay, ARPE-19 cells were incubated with serial dilutions of VEGF blocker alone, VEGF blocker +VEGF121 or VEGF165 followed by immunostaining of cell surface bound VEGF blockers. ARPE-19 cells were incubated with serial dilutions of soluble neuropilin-1 (NRP-1) or heparin with VEGF165 and BEV. Cells were then processed as above for signal analysis. Binding studies were conducted on HUVEC using the same protocols.
BEV showed a time-dependent increase in binding to ARPE-19 cells coincident with the up-regulation of VEGF165 in cultures. In contrast, AFL showed no specific binding, while RAN exhibited very light binding to RPE cells. Acute binding of BEV to ARPE-19 cells was noted in the presence of exogenous VEGF165, but not VEGF121. At the same concentrations, no staining was observed with AFL or RAN + VEGF165 or VEGF121. Similar patterns of cell surface binding of BEV were noted in cultured HUVEC. Exogenous heparin blocked the binding of BEV:VEGF165 complexes to ARPE-19 cells, whereas, NRP-1 blocked the binding of BEV:VEGF165 complexes to HUVEC.
These results show that BEV can bind to cultured ARPE-19 cells and HUVEC. Acute binding of BEV:VEGF165 complexes occurs at molar ratios previously shown to favor the formation of high molecular weight immune like complexes of BEV:VEGF. Neither AFL nor RAN bind appreciably to RPE or endothelial cells under identical conditions. Binding of BEV:VEGF165 complexes to RPE or HUVEC appears to be mediated via interactions of bound VEGF165 with lower affinity binding partners, heparin or NRP-1.
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