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Molecular imaging of VEGF receptors in angiogenic vasculature with single-chain VEGF-based probes

Abstract

We describe a new generation of protein-targeted contrast agents for multimodal imaging of the cell-surface receptors for vascular endothelial growth factor (VEGF). These receptors have a key role in angiogenesis and are important targets for drug development. Our probes are based on a single-chain recombinant VEGF expressed with a cysteine-containing tag that allows site-specific labeling with contrast agents for near-infrared fluorescence imaging, single-photon emission computed tomography or positron emission tomography. These probes retain VEGF activities in vitro and undergo selective and highly specific focal uptake into the vasculature of tumors and surrounding host tissue in vivo. The fluorescence contrast agent shows long-term persistence and co-localizes with endothelial cell markers, indicating that internalization is mediated by the receptors. We expect that multimodal imaging of VEGF receptors with these probes will be useful for clinical diagnosis and therapeutic monitoring, and will help to accelerate the development of new angiogenesis-directed drugs and treatments.

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Figure 1: Site-specific modifications do not affect binding and internalization of scVEGF.
Figure 2: NIRF imaging with scVEGF/Cy and localization of Cy5.5 on histological sections.
Figure 3: Blood clearance, biodistribution and SPECT imaging of scVEGF/Tc.
Figure 4: Blood clearance, biodistribution and PET imaging of scVEGF/Cu.

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Acknowledgements

We thank J. Pizzonia (KODAK Molecular Imaging Systems) for help with NIRF imaging; K. Tracht (Olympus America) for help with microscopy; P.T. Pienkos (Molecular Logix) for codon optimized human EGF; and R. Barth (The Ohio State University) for F98-EGFR(F) cells. This work was supported in part by NIH grants R43 CA113080 and R21 EB001946 to J.M.B., NIH 1 P50 CA114747 to F.G.B., and by support from NIH CA064436 and the Patrick and Catharine Weldon Donaghue Foundation to K.P.C.

Author information

Authors and Affiliations

Authors

Contributions

M.V.B. and Z.L. contributed equally to this manuscript. M.V.B., F.G.B. and J.M.B. conceived of and initiated the project, coordinated discovery research and wrote the manuscript; M.V.B. designed imaging conjugates, validated all probes in tissue culture and performed colocalization studies; M.V.B. and J.M.B. designed and conducted optical imaging experiments; Z.L. and F.G.B. prepared radiolabeled SPECT and PET probes and performed all experiments with these probes; V.P. and B.T.G. made scVEGF-based conjugates; and K.C. performed confocal microscopy experiments. All authors discussed and commented on the manuscript.

Corresponding author

Correspondence to Joseph M Backer.

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Competing interests

M.V.B. and J.M.B. own equity in privately held SibTech Inc.

Supplementary information

Supplementary Fig. 1

Scatchard analysis of scVEGF/Cu binding to VEGFR-2 on 293/KDR cells. (PDF 88 kb)

Supplementary Fig. 2

Long-term retention of Cy5.5 after imaging with scVEGF/Cy. (PDF 123 kb)

Supplementary Fig. 3

NIRF imaging with scVEGF/Cy in MDA-231luc orthotopic tumor model. (PDF 100 kb)

Supplementary Fig. 4

Functionally active Cys-EGF/Cy accumulates in tumor area, but does not co-localize with endothelial cells. (PDF 139 kb)

Supplementary Fig. 5

Stability of radionuclide-loaded scVEGF probes in vivo (PDF 130 kb)

Supplementary Fig. 6

SPECT imaging of 4T1luc tumor bearing mice. (PDF 170 kb)

Supplementary Fig. 7

scVEGF/Cu stability in murine plasma ex vivo. (PDF 110 kb)

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Backer, M., Levashova, Z., Patel, V. et al. Molecular imaging of VEGF receptors in angiogenic vasculature with single-chain VEGF-based probes. Nat Med 13, 504–509 (2007). https://doi.org/10.1038/nm1522

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