Cy5.5-Single-chain Cys-tagged vascular endothelial growth factor-121


Leung K.

Publication Details



In vitro Rodents



Optical fluorescence imaging is increasingly used to observe biological functions of specific targets (1, 2) in small animals. However, the intrinsic fluorescence of biomolecules poses a problem when fluorophores that absorb visible light (350–700 nm) are used. Near-infrared (NIR) fluorescence (700–1,000 nm) detection avoids the background fluorescence interference of natural biomolecules, providing a high contrast between target and background tissues. NIR fluorophores have wider dynamic range and minimal background as a result of reduced scattering compared with visible fluorescence detection. They also have high sensitivity, resulting from low infrared background, and high extinction coefficients, which provide high quantum yields. The NIR region is also compatible with solid-state optical components, such as diode lasers and silicon detectors. NIR fluorescence imaging is becoming a non-invasive alternative to radionuclide imaging in small animals.

Vascular endothelial growth factor (VEGF) consists of at least six isoforms with various numbers of amino acids (121, 145, 165, 183, 189, and 206 amino acids) produced through alternative splicing (3). VEGF121, VEGF165, and VEGF189 are the forms secreted by most cell types and are active as homodimers linked by disulfide bonds. VEGF121 does not bind to heparin like the other VEGF species (4). VEGF is a potent angiogenic factor that induces proliferation, sprouting, migration, and tube formation of endothelial cells. There are three high-affinity tyrosine kinase VEGF receptors on endothelial cells (VEGFR-1, Flt-1; VEGFR-2, KDR/Flt-1; and VEGFR-3, Flt-4). Several types of non-endothelial cells such as hematopoietic stem cells, melanoma cells, monocytes, osteoblasts, and pancreatic β cells also express VEGF receptors (3).

VEGF receptors were found to be overexpressed in various tumor cells and tumor-associated endothelial cells (5). Inhibition of VEGF receptor function has been shown to inhibit pathological angiogenesis as well as tumor growth and metastasis (6, 7). Radiolabeled VEGF has been developed as a tracer for imaging solid tumors and angiogenesis in humans (8-10). Cys-tag, a fusion tag comprising 15 amino acids, was developed for site-specific conjugation via the free sulfhydryl group of Cys. Backer et al. (11) prepared a Cys-tagged vector of VEGF121 by cloning two single-chain fragments (amino acid sequence 3–112) of VEGF121 joining head-to-tail to express as scVEGF, which can be labeled as 64Cu-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA)-scVEGF (64Cu-DOTA-scVEGF), 99mTc-hydrazinonicotinic acid (HYNIC)-scVEGF (99mTc-HYNIC-scVEGF), and Cy5.5-scVEGF for imaging VEGFR expression to study tumor angiogenesis (12). Cy5.5 is a NIR fluorescent dye with an absorbance maximum at 675 nm and an emission maximum at 694 nm with a high extinction coefficient of 250,000 M-1cm-1 according to in vitro measurement. Cy5.5-scVEGF is being developed for NIR fluorescence imaging of VEGFR-2 in tumor vasculature.



Backer et al. (11) prepared a Cys-tagged vector of VEGF121 by cloning two single-chain fragments (amino acid sequence 3–112) of VEGF121 joining head-to-tail to express as scVEGF in Escherichia coli. Dithiothreitol-treated scVEGF was incubated with a five-fold molar excess of Cy5.5-maleimide for 90 min (12). Cy5.5-scVEGF was purified with column chromatography. Cy5.5 was identified in the Cys-tag thiol group only and not in the other 16 Cys groups of scVEGF. Hence, there was ~1 Cy5.5/scVEGF. Cy5.5-inVEGF, an inactive control, was prepared by conjugation of 7–8 biotins to Cy5.5-scVEGF.

In Vitro Studies: Testing in Cells and Tissues


In vitro competition binding studies were performed in 293/KDR cells expressing cloned human VEGFR-2 in competition with the chimeric toxin SLT-VEGF for binding to VEGFR-2 (12). 64Cu-DOTA-PEG-scVEGF, DOTA-PEG-scVEGF, HYNIC-scVEGF, and Cy5.5-scVEGF inhibited the binding of SLT-VEGF to VEGFR-2. DOTA-PEG-scVEGF, HYNIC-scVEGF, and Cy5.5-scVEGF stimulated tyrosine phosphorylation of VEGFR-2 in 293/KDR cells in a manner similar to VEGF. Cy5.5-inVEGF exhibited no effects in these two assays. The extensive biotinylation of scVEGF probably destroys the binding capacity of scVEGF. Confocal laser scanning microscopy confirmed intracellular accumulation of Cy5.5-scVEGF in porcine aortic endothelial cells overexpressing VEGFR-2 and not in control cells lacking the receptor.

Animal Studies



Backer et al. (12) performed in vivo whole-body imaging of mice bearing mouse 4T1 or human MDA-MB-231 mammary adenocarcinoma tumors after i.v. injection of 10 ug of Cy5.5-scVEGF. Persistent fluorescence signal was evident in the tumors for several hours and could be detected up to 7 d after injection. Tumor accumulation of Cy5.5-scVEGF was highly heterogeneous. Co-administration of Cy5.5-scVEGF with 100 ug scVEGF markedly reduced the fluorescence signal. Ex vivo imaging and autoradiography of frozen tumor sections showed that the Cy5.5-scVEGF NIR fluorescence signal intensity in the tumor colocalized with immunoreactivity for VEGFR-1, VEGFR-2, and CD31 (an endothelial cell marker) in endothelial capillary cells. No tumor accumulation of Cy5.5-inVEGF was observed after injection.

Other Non-Primate Mammals


No publication is currently available.

Non-Human Primates


No publication is currently available.

Human Studies


No publication is currently available.

NIH Support

R21 EB001946, R43 CA113080, P50 CA114747, CA064436


Ntziachristos V. , Bremer C. , Weissleder R. Fluorescence imaging with near-infrared light: new technological advances that enable in vivo molecular imaging. Eur Radiol. 2003;13(1):195–208. [PubMed: 12541130]
Achilefu S. Lighting up tumors with receptor-specific optical molecular probes. Technol Cancer Res Treat. 2004;3(4):393–409. [PubMed: 15270591]
Ferrara N. Vascular endothelial growth factor: basic science and clinical progress. Endocr Rev. 2004;25(4):581–611. [PubMed: 15294883]
Cohen T. , Gitay-Goren H. , Sharon R. , Shibuya M. , Halaban R. , Levi B.Z. , Neufeld G. VEGF121, a vascular endothelial growth factor (VEGF) isoform lacking heparin binding ability, requires cell-surface heparan sulfates for efficient binding to the VEGF receptors of human melanoma cells. J Biol Chem. 1995;270(19):11322–6. [PubMed: 7744769]
Soria J.C. , Fayette J. , Armand J.P. Molecular targeting: targeting angiogenesis in solid tumors. Suppl 4Ann Oncol. 2004;15:iv223–7. [PubMed: 15477311]
Ferrara N. Vascular endothelial growth factor as a target for anticancer therapy. Suppl 1Oncologist. 2004;9:2–10. [PubMed: 15178810]
Hicklin D.J. , Ellis L.M. Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis. J Clin Oncol. 2005;23(5):1011–27. [PubMed: 15585754]
Li S. , Peck-Radosavljevic M. , Koller E. , Koller F. , Kaserer K. , Kreil A. , Kapiotis S. , Hamwi A. , Weich H.A. , Valent P. , Angelberger P. , Dudczak R. , Virgolini I. Characterization of (123)I-vascular endothelial growth factor-binding sites expressed on human tumour cells: possible implication for tumour scintigraphy. Int J Cancer. 2001;91(6):789–96. [PubMed: 11275981]
Li S. , Peck-Radosavljevic M. , Kienast O. , Preitfellner J. , Hamilton G. , Kurtaran A. , Pirich C. , Angelberger P. , Dudczak R. Imaging gastrointestinal tumours using vascular endothelial growth factor-165 (VEGF165) receptor scintigraphy. Ann Oncol. 2003;14(8):1274–7. [PubMed: 12881392]
Li S. , Peck-Radosavljevic M. , Kienast O. , Preitfellner J. , Havlik E. , Schima W. , Traub-Weidinger T. , Graf S. , Beheshti M. , Schmid M. , Angelberger P. , Dudczak R. Iodine-123-vascular endothelial growth factor-165 (123I-VEGF165). Biodistribution, safety and radiation dosimetry in patients with pancreatic carcinoma. Q J Nucl Med Mol Imaging. 2004;48(3):198–206. [PubMed: 15499293]
Backer M.V. , Patel V. , Jehning B.T. , Claffey K.P. , Backer J.M. Surface immobilization of active vascular endothelial growth factor via a cysteine-containing tag. Biomaterials. 2006;27(31):5452–8. [PubMed: 16843524]
Backer M.V. , Levashova Z. , Patel V. , Jehning B.T. , Claffey K. , Blankenberg F.G. , Backer J.M. Molecular imaging of VEGF receptors in angiogenic vasculature with single-chain VEGF-based probes. Nat Med. 2007;13(4):504–9. [PubMed: 17351626]