In vivo imaging of fluorescent probes linked to antibodies against human and rat vascular endothelial growth factor (VEGF)

Abstract

Purpose: To investigate fluorescent molecular probe linked to antibodies against VEGF for in vivo imaging of VEGF.

Methods: Bevacizumab (a humanized monoclonal antibody, Roche), antiRatVEGF (a polyclonal antibody against rat VEGF164, R&D Systems) and B20-4.1.1 (a polyclonal antibody against human and rat VEGF, Genentech) were covalently attached to indocyanine green (ICG), a near-infrared dye, yielding soluble conjugates. Binding properties were assessed by an in vitro proliferation assay. Using confocal scanning laser ophthalmoscopy (cSLO), in vivo reflectance and fluorescence imaging was performed in Dark Agouti rats that had undergone argon laser photocoagulation to induce choroidal neovascularisations (CNV). Retinal uptake and fluorescence were recorded following intravenous and intravitreal injection of the dye conjugates for up to 100 days.

Results: In vivo imaging before dye application showed ill-defined retinal lesions at day 7. Immediately following intravenous and intravitreal injection a strong fluorescence was visible. Twenty-four hours following injection an accumulation of the antibody-conjugate at the site of the roundish laser lesions for all antibody-conjugates were observed. Furthermore, multiple fluorescent spots were visible up to 35 days following intravenous injection of Bevacizumab-ICG and for up to 60 days following intravitreal injection of Bevacizumab-ICG, B20-4.1.1-ICG and antiRatVEGF-ICG. No fluorescent spots occurred after intravenous injection of B20-4.1.1-ICG or antiRatVEGF-ICG. Over time, a continuous decrease of the fluorescence intensity was observed for all antibody-conjugates.

Conclusions: Pharmacokinetics of fluorescent-labeled bevacizumab, antiRatVEGF and B20-4.1.1 can be investigated in vivo following intravenous and intravitreal injection. Strong accumulations in the site of the laser lesion were observed for all antibody-conjugates. 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.

Keywords: 748 vascular endothelial growth factor • 453 choroid: neovascularization • 552 imaging methods (CT, FA, ICG, MRI, OCT, RTA, SLO, ultrasound)