Site-Specific Labeling of scVEGF with Fluorine-18 for Positron Emission Tomography Imaging

Vascular endothelial growth factor (VEGF) is one of the most important mediators of angiogenesis. Single-chain (sc)-VEGF protein containing an N-terminal Cys-tag has been designed for site-specific modification with a variety of imaging and therapeutic moieties. Site-specific labeling of scVEGF with thiol-reactive prosthetic group, N-[2-(4-(18)F-fluorobenzamido) ethyl] maleimide ([(18)F]FBEM) for positron emission tomography (PET) imaging of VEFGR may provide a new tracer which has great potential for clinical translation. Methods: [(18)F]FBEM-scVEGF was synthesized by site-specific conjugation of (18)F-FBEM to a thiol group in Cys-tag of scVEGF at room temperature. The functional activity after labeling was tested by immunofluorescence staining, cellular uptake and efflux. The tumor targeting and in vivo properties were evaluated by biodistribution and microPET studies in tumor-bearing mice. Results: The radiolabeling yield and specific activity of [(18)F]FBEM-scVEGF were 20.6 ± 15.1% (based on starting [(18)F]FBEM, uncorrected, n = 5) and 58.8 ± 12.4 GBq/µmol, respectively. Noninvasive microPET and direct tissue sampling experiments demonstrated that [(18)F]FBEM-scVEGF had VEGFR specific tumor uptake in MDA-MB-435, U87MG and 4T1 xenograft models. The optimal tumor uptake was achieved at 2 h p.i., which can be partially, but significantly blocked by co-injection of non-labeled scVEGF protein. Overall, [(18)F]FBEM-scVEGF showed VEGFR specific tumor uptake. Conclusion: The scVEGF was site-specifically labeled with (18)F via [(18)F]FBEM prosthetic group and the tracer [(18)F]FBEM-scVEGF exhibited high receptor binding affinity and tumor targeting efficacy. Further study of [(18)F] FBEM-scVEGF to evaluate angiogenesis in cancer and other disease types is warranted.