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S. Schmitz-Valckenberg, K. Licha, P. Herrmann, P. Welker, N. Eter, M. Schirner, F. G. Holz; Fluorescence-Labelled Bevacizumab for in-vivo Molecular Imaging of Vascular Endothelial Growth Factor in the Retina. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1638.
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Preclinical and clinical studies have underscored the key role of vascular endothelial growth factor (VEGF)-inhibitors in the pathogenesis of various retinal diseases, including neovascular age-related macular degeneration (AMD), diabetic macular edema and retinal vein occlusion. The aim of this study was to develop a fluorescent molecular probe for VEGF using a humanized monoclonal VEGF-antibody to be used for in-vivo molecular imaging of free VEGF.
Bevacizumab was covalently attached to a near-infrared indocyanine dye yielding a soluble conjugate of a dye-to-protein ratio of 2. In-vivo retinal reflectance (exc. 488 nm and 830 nm) and fluorescence (exc. 488 nm and 790 nm, em 500 - 700 nm and > 800 nm) imaging was performed in C57BL/6 mice using a confocal scanning laser ophthalmoscopy (Heidelberg Retina Angiograph, Heidelberg Engineering, Germany). Argon laser photocoagulation was performed to induce multiple focal lesion in the retina in both eyes at day 1. At day 7, conventional 10% sodium fluorescein and the fluorescent bevacizumab conjugate were intravenously injected and their retinal uptake recorded in-vivo.
In-vitro studies using cellular VEGF assays showed maintained binding properties of the conjugate compared to untreated antibody. In-vivo retinal imaging before dye injection showed ill-defined retinal lesions around the optic nerve head following laser application at day 7. Directly following injection, a strong fluorescence signal in the retinal vasculature, including retinal capillaries and inside the laser lesions was observed with both markers. At day 8, no more fluorescein signal was detectable, while near-infrared fluorescence imaging revealed a persistent, but weaker signal in the retinal vasculatures. Inside the laser lesions, the signal was relatively increased at the level of the outer retina.
The findings indicate that the fluorescent bevacizumab conjugate can be visualized by in-vivo imaging in the retinal vasculature and its spatio-temporal relationship at the site of VEGF-hyperexpression. Following expanded investigations in animal models, molecular imaging of VEGF may be applicable not only for earlier diagnosis of disease with VEGF-driven pathways but also for more refined individualized anti-VEGF therapies based on a biomarker that signals disease and reactivation before morphological and potentially irreversible damage occur.
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