Selective imaging of VEGFR-1 and VEGFR-2 receptors in orthotopic breast tumor using 89Zr-labeled receptor-specific VEGF mutants

Objectives Vascular endothelial growth factor A (VEGF) acts via two vascular endothelial growth factor receptors VEGFR-1 and VEGFR-2 and is the main regulator of angiogenesis in cancer and other pathologies.¹ Targeting VEGF/VEGFR signaling is a well-established therapeutic modality, known as anti-angiogenic therapy.² Unfortunately, to date, only a small fraction of cancer patients benefit from such a therapy, while serious side effects are possible.² Hence, it is critically important to identify and validate reliable imaging biomarkers for the optimization of anti-angiogenic therapy for individual patients, and VEGF receptors could play this role. Currently, VEGF receptors are imaged with VEGF-based tracers that do not discriminate between VEGFR-1 and VEGFR-2. We reasoned that since VEGFR-1 and VEGFR-2 play different roles in tumor biology, selective imaging of these receptors could provide unique information on tumor development and response to therapy.

Methods Herein, we report the development of two novel ⁸⁹Zr-labeled PET tracers, which are based on receptor-specific mutants of scVEGF (an engineered 28 kDa single-chain VEGF A with an N-terminal Cys-tag for site-specific conjugation³) that enable the selective imaging of VEGFR-1 or VEGFR-2 in tumor vasculature. Single-chain (sc) VEGF and its receptor-specific mutants were site-specifically derivatized with the ⁸⁹Zr-chelator deferoxamine (DFO) via 3.4 kDa PEG linker, yielding, respectively, scV, scVR1, and scVR2 targeting conjugates. Subsequent ⁸⁹Zr-labeling of the DFO-conjugates furnished the novel tracers scV/Zr, scVR1/Zr, and scVR2/Zr in high radiochemical yields (>87%), high specific activities (>12 mCi/mg) and purities (>99%).

Results All tracers rapidly accumulated in orthotopic 4T1luc mouse breast tumors in balb/c mice, allowing for the successful PET imaging of the tumors as early as 2 h post-injection (p.i.). with calculated tumor uptake values determined by ex vivo biodistribution studies of 2.94 ± 0.27 %ID/g for scVR1 and 3.46 ± 0.69 %ID/g for scVR2, changing over time to 1.92 ± 0.26 %ID/g for scVR1 and 6.39 ± 1.24 %ID/g for scVR2 at p.i. 48 h, respectively. The short blood half-life of the tracers (<2 h) gave high tumor-to-blood ratios at 12 h p.i. of 6.57 ± 2.78 for scVR1/Zr and 10.58 ± 4.12 for scVR2/Zr, respectively, resulting in good tumor delineation at early imaging time points. Blocking experiments with excess of pan-receptor or receptor-specific “cold” proteins indicated that more than 80% of tracer tumor uptake is VEGF receptor mediated, while uptake in all major organs is receptor-independent. Critically, blocking experiments indicated that tumor uptake of scVR1/Zr and scVR2/Zr was mediated exclusively by VEGFR-1 or VEGFR-2, respectively. In contrast, uptake of pan-receptor scV/Zr was mediated by both VEGFR-1 and VEGFR-2 at approximately 2:1 ratio. As expected for relatively small protein-based tracers, the major non-specific uptake was observed in kidney (58.4-64.9 %ID/g for scVR1/Zr and 53.8-68.3 %ID/g for scVR2/Zr). Despite this relatively high kidney uptake, dosimetry calculations based on ex vivo biodistribution data gave total effective doses of 1.29 rem/mCi and 1.43 rem/mCi for scVR1/Zr and scVR2/Zr, respectively, demonstrating the clinical potential of these PET tracers.

Conclusions In sum, a new approach for the selective PET imaging of VEGFR-1 and VEGFR-2 has been developed. The newly constructed receptor-selective tracers show promising tumor uptake and retention, which could be suitable for observing the separate effects of anti-angiogenic therapy on VEGFR-1 and VEGFR-2 in tumor vasculature in real time. $$graphic_7EF8986A-1F76-4FD2-B0E7-3D03916D621A$$