Selective imaging of VEGFR-1 and VEGFR-2 receptors using 89Zr-labeled single-chain VEGF mutants

Vascular endothelial growth factor A (VEGF) acts via two vascular endothelial growth factor receptors VEGFR-1 and VEGFR-2 that play important and distinct roles in tumor biology. We reasoned that selective imaging of these receptors could provide unique information for diagnostics and for monitoring and optimizing responses to anti-cancer therapy, including antiangiogenic therapy. Herein, we report the development of two first-in-class 89Zr-labeled PET tracers that enable the selective imaging of VEGFR-1 and VEGFR-2. Methods. Functionally active mutants of scVEGF (an engineered single-chain version of pan-receptor VEGF A with an N-terminal cysteine-containing tag for site-specific conjugation), named scVR1 and scVR2 with enhanced affinity to, respectively, VEGFR-1 and VEGFR-2 were constructed. Parental scVEGF and its receptor-specific mutants were site-specifically derivatized with the 89Zr-chelator desferroxamine B (DFO) via a 3.4 kDa PEG linker. 89Zr-labeling of the DFO-conjugates, furnished scV/Zr, scVR1/Zr, and scVR2/Zr tracers with high radiochemical yield (>87%), high specific activity (≥9.8 MBq/nmol), and purity (>99%). Tracers were tested in an orthotopic breast cancer model using 4T1luc-bearing syngeneic Balb/c mice. For testing tracer specificity, tracers were co-injected with an excess of “cold” proteins of the same or opposite receptor specificity, or pan-receptor scVEGF. Positron emission tomography (PET) imaging, biodistribution and dosimetry studies in mice, as well as immunohistochemical analysis of harvested tumors were performed. Results. All tracers rapidly accumulated in orthotopic 4T1luc tumors, allowing for the successful PET imaging of the tumors as early as 2 h post-injection (p.i.). Blocking experiments with excess of pan-receptor or receptor-specific “cold” proteins indicated that more than 80% of tracer tumor uptake is VEGFR-mediated, while uptake in all major organs is not affected by blocking within the margin of error. Critically, blocking experiments indicated that VEGFRmediated tumor uptake of scVR1/Zr and scVR2/Zr was mediated exclusively by the corresponding receptor, VEGFR-1 or VEGFR-2, respectively. In contrast, uptake of panby on October 14, 2017. For personal use only. jnm.snmjournals.org Downloaded from