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Simultaneous targeting of two ligand-binding sites on VEGFR2 using biparatopic Affibody molecules results in dramatically improved affinity.

ABSTRACT: Angiogenesis plays an important role in cancer and ophthalmic disorders such as age-related macular degeneration and diabetic retinopathy. The vascular endothelial growth factor (VEGF) family and corresponding receptors are regulators of angiogenesis and have been much investigated as therapeutic targets. The aim of this work was to generate antagonistic VEGFR2-specific affinity proteins having adjustable pharmacokinetic properties allowing for either therapy or molecular imaging. Two antagonistic Affibody molecules that were cross-reactive for human and murine VEGFR2 were selected by phage and bacterial display. Surprisingly, although both binders independently blocked VEGF-A binding, competition assays revealed interaction with non-overlapping epitopes on the receptor. Biparatopic molecules, comprising the two Affibody domains, were hence engineered to potentially increase affinity even further through avidity. Moreover, an albumin-binding domain was included for half-life extension in future in vivo experiments. The best-performing of the biparatopic constructs demonstrated up to 180-fold slower dissociation than the monomers. The new Affibody constructs were also able to specifically target VEGFR2 on human cells, while simultaneously binding to albumin, as well as inhibit VEGF-induced signaling. In summary, we have generated small antagonistic biparatopic Affibody molecules with high affinity for VEGFR2, which have potential for both future therapeutic and diagnostic purposes in angiogenesis-related diseases.

SUBMITTER: Fleetwood F

PROVIDER: S-EPMC4268634 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Simultaneous targeting of two ligand-binding sites on VEGFR2 using biparatopic Affibody molecules results in dramatically improved affinity.

Fleetwood Filippa F Klint Susanne S Hanze Martin M Gunneriusson Elin E Frejd Fredrik Y FY Ståhl Stefan S Löfblom John J

Scientific reports 20141217

Angiogenesis plays an important role in cancer and ophthalmic disorders such as age-related macular degeneration and diabetic retinopathy. The vascular endothelial growth factor (VEGF) family and corresponding receptors are regulators of angiogenesis and have been much investigated as therapeutic targets. The aim of this work was to generate antagonistic VEGFR2-specific affinity proteins having adjustable pharmacokinetic properties allowing for either therapy or molecular imaging. Two antagonist ...[more]

PMID: 25515662

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Project description: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.

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Simultaneous targeting of two ligand-binding sites on VEGFR2 using biparatopic Affibody molecules results in dramatically improved affinity.

Publications

Simultaneous targeting of two ligand-binding sites on VEGFR2 using biparatopic Affibody molecules results in dramatically improved affinity.

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