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Johanna Meyer, Alexander Cunea, Pia Welker, Kai Licha, Jens Dernedde, Frank G Holz, Steffen Schmitz-Valckenberg; Molecular in vivo imaging of anti-VEGF antibodies in an animal model. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):254.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate fluorescent molecular probes linked to antibodies directed against VEGF for in vivo imaging of VEGF.
Bevacizumab (monoclonal antibody against VEGF165), B20-4.1.1 (polyclonal antibody against VEGF165 and VEGF164) and AF564 (polyclonal antibody against VEGF164) were conjugated with a novel indocyanine green label (6S-ICG), yielding soluble conjugates. Binding properties were assessed by BIAcore and proliferation assays. Molecular in vivo imaging was performed in rats that had undergone laser photocoagulation to induce choroidal neovascularization. Retinal uptake and fluorescence were recorded following injection of the dye conjugates. Distribution and accumulation of the probes were determined by immunohistochemistry and flow cytometry analysis.
Comparable affinities of VEGF165 and VEGF164 to labeled bevacizumab were measured by BIAcore. Antibody B20-4.1.1 was able to bind VEGF from both species. Antibody labeling with 6S-ICG showed no influence on binding properties. In vivo imaging showed a strong fluorescence immediately following an intravenous or intravitreal injection. After 24 hours, a general observation after probe application was both the occurrence of fluorescence within the laser lesions and multiple hyperfluorescent spots.Over time, a continuous decrease of fluorescence intensity was observed for all probes. Bevacizumab-ICG showed longest accumulation within the lesions and highest number as well as longest duration of detectable fluorescent spots. Lowest values were recorded for AF564-ICG. Immunohistochemistry showed double-staining of fluorescent spots with macrophages and/or microglia cells. Analysis of cell sorting by flow cytometry suggested double staining of bevacizumab-ICG with retinal microglia cells.
Ex vivo analysis demonstrated successful binding of antibody conjugates with ICG while affinity to VEGF isoforms and cell viability were both preserved. Pharmacokinetics of fluorescent-labeled anti-VEGF probes can be investigated in vivo following intravenous and intravitreal injection. Strong accumulations within the laser lesions were observed for all antibody-conjugates. Also fluorescent spots were visible that might represent macrophages and/or microglia cells. This novel molecular imaging approach of VEGF may be applicable in patients for earlier diagnosis and more refined individualized anti-VEGF therapies with the aim of optimizing functional outcomes.
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