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

Abstract

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 (ZVEGFR2-Bp2) for in vivo visualization of VEGFR2 expression in GBM.Methods: ZVEGFR2-Bp2 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: [111In]In-NODAGA-ZVEGFR2-Bp2 bound specifically to VEGFR2 (KD=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 [111In]In-NODAGA-ZVEGFR2-Bp2 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 [111In]In-NODAGA-ZVEGFR2-Bp2 specifically targeted VEGFR2 in vivo and visualized its expression in a murine GBM orthotopic model. Tumor-to-blood ratios for [111In]In-NODAGA-ZVEGFR2-Bp2 were higher compared to other VEGFR2 imaging probes. [111In]In-NODAGA-ZVEGFR2-Bp2 appears to be a promising probe for in vivo noninvasive visualization of tumor angiogenesis in glioblastoma.

Affibody Molecules as Targeting Vectors for PET Imaging

“…Another feature of affibody molecules is the very efficient reabsorption in the proximal tubules of kidneys. This feature has been observed for all binders, independent of the molecular target [23][24][25][26][27]29,37]. Therefore, it is reasonable to suppose that the reabsorption is determined by the scaffold rather than by a binding site.…”

“…Affinity maturation permits a further increase of the affinity from a few nM to a few pM. High-affinity affibody binders have been selected for potential cancer-associated molecular targets such as human epidermal growth factor receptor type 2 (HER2) [22], epidermal growth factor receptor (EGFR or HER1) [23], human epidermal growth factor receptor type 3 (HER3) [24], insulinlike growth factor-1 receptor (IGF-1R) [25], platelet-derived growth factor receptor β (PDGFRβ) [26], vascular endothelial growth factor receptor 2 (VEGFR2) [27], programmed death-ligand 1 (PD-L1) [28], and carbonic anhydrase IX (CAIX) [29]. Several features of affibody molecules make them potentially attractive as targeting vectors for imaging probes:…”

Generating a Novel Bispecific Nanobody to Enhance Antitumor Activity

“…Expression and purification of 3VGR19-3 (3,4). Expression and purification of bispecific nanobody (5,6). L is (G4S)3 (GGCGGCGGTGGTAGCGGCGGCGGCGGTTCCGGCGGTGGTGGTTCT) linker, M is size standards.…”

“…Vascular endothelial growth factor receptor 2 (VEGFR2) is involved in the regulation of angiogenesis, therefore, inhibiting VEGFR2 activity, and the downstream signaling pathway has become an important approach in cancer treatment. [3][4][5] Overexpression of HER2 can upregulate VEGF through the PI3K signaling pathway, so blocking HER2 significantly decreases VEGF expression and helps killing the tumor cells. 3,6 In addition, based on the synergetic effect and cross-talk between the signaling pathways, targeting both receptors simultaneously may more effectively inhibit tumor cell proliferation and promote apoptosis.…”

PET Imaging of VEGFR with a Novel 64Cu-Labeled Peptide

“…Besides, several research efforts have been put toward affibody and peptide-based radiotracers. 35,36 These molecules showed satisfactory PK and image contrast, but their imaging mode is based on SPECT, which significantly limits signal sensitivity and image quality. Compared to SPECT, PET exhibits several advantages, including improved image quality, greater interpretive certainty, higher diagnostic accuracy, and lower patient dosimetry.…”