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Vascular endothelial growth factor receptor-2 (VEGFR2) is a key mediator of angiogenesis and therefore a promising therapeutic target in malignancies including glioblastoma multiforme (GBM). Molecular imaging of VEGFR2 expression may enable patient stratification for antiangiogenic therapy. The goal of the current study was to evaluate the capacity of the novel anti-VEGFR2 biparatopic affibody conjugate (Z VEGFR2 -Bp 2 ) for in vivo visualization of VEGFR2 expression in GBM. Methods: Z VEGFR2 -Bp 2 coupled to a NODAGA chelator was generated and radiolabeled with indium-111. The VEGFR2-expressing murine endothelial cell line MS1 was used to evaluate in vitro binding specificity and affinity, cellular processing and targeting specificity in mice. Further tumor targeting was studied in vivo in GL261 glioblastoma orthotopic tumors. Experimental imaging was performed. Results: [ 111 In]In-NODAGA-Z VEGFR2 -Bp 2 bound specifically to VEGFR2 (K D =33±18 pM). VEGFR2-mediated accumulation was observed in liver, spleen and lungs. The tumor-to-organ ratios 2 h post injection for mice bearing MS1 tumors were approximately 11 for blood, 15 for muscles and 78 for brain. Intracranial GL261 glioblastoma was visualized using SPECT/CT. The activity uptake in tumors was significantly higher than in normal brain tissue. The tumor-to-cerebellum ratios after injection of 4 µg [ 111 In]In-NODAGA-Z VEGFR2 -Bp 2 were significantly higher than the ratios observed for the 40 µg injected dose and for the non-VEGFR2 binding size-matched conjugate, demonstrating target specificity. Microautoradiography of cryosectioned CNS tissue was in good agreement with the SPECT/CT images. Conclusion: The anti-VEGFR2 affibody conjugate [ 111 In]In-NODAGA-Z VEGFR2 -Bp 2 specifically targeted VEGFR2 in vivo and visualized its expression in a murine GBM orthotopic model. Tumor-to-blood ratios for [ 111 In]In-NODAGA-Z VEGFR2 -Bp 2 were higher compared to other VEGFR2 imaging probes. [ 111 In]In-NODAGA-Z VEGFR2 -Bp 2 appears to be a promising probe for in vivo noninvasive visualization of tumor angiogenesis in glioblastoma.

Radionuclide imaging of VEGFR2 in glioma vasculature using biparatopic affibody conjugate: proof-of-principle in a murine model