64Cu-1,4,7,10-Tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid-polyethylene glycol-single-chain Cys-tagged vascular endothelial growth factor-121


Leung K.

Publication Details



In vitro Rodents



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 (1). 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 (2). 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 (1).

VEGF receptors were found to be overexpressed in various tumor cells and tumor-associated endothelial cells (3). Inhibition of VEGF receptor function has been shown to inhibit pathological angiogenesis as well as tumor growth and metastasis (4, 5). Radiolabeled VEGF has been developed as a tracer for imaging solid tumors and angiogenesis in humans (6-8). 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. (9) prepared a Cys-tagged vector of VEGF121 by cloning two single-chain fragments (3–112 amino acids long) of VEGF121 joining head-to-tail to express as scVEGF, which can be labeled as (64Cu-DOTA-scVEGF), 99mTc-hydrazinonicotinic acid (HYNIC)-scVEGF (99mTc-HYNIC-scVEGF), and Cy5.5-scVEGF for imaging VEGFR expression to study tumor angiogenesis (10). 64Cu-DOTA-PEG-scVEGF is being developed for positron emission tomography (PET) imaging of VEGFR-2 in tumor vasculature.



Backer et al. (9) 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 for mammalian cell production. 64Cu labeling of scVEGF was performed through DOTA chelation. DOTA was activated by a five-fold molar excess of N-hydroxysuccinimide-polyethylene glycol (PEG)-maleimide (pH 8) for 60 min (10). A mixture of scVEGF and the activated DOTA in a molar ratio of 1:10 was incubated for 60 min. The DOTA-PEG-scVEGF conjugate was purified with high-performance liquid chromatography. The number of DOTA molecules per protein was ~1. For radiolabeling, 20 μg of scVEGF was added to 18.5 MBq (0.5 mCi) of 64Cu diluted in 0.1 M sodium acetate buffer (pH 5.5). The reaction mixture was incubated at 55ºC for 1 h. 64Cu-DOTA-PEG-scVEGF was purified with column chromatography with a radiolabeling yield of 40%. 64Cu-DOTA-PEG-inVEGF, used as an inactive control, was prepared by conjugation of 7–8 biotins to 64Cu-DOTA-PEG-scVEGF. The extensive biotinylation of 64Cu-DOTA-PEG-scVEGF probably destroys the binding capacity of 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 (10). 64Cu-DOTA-PEG-scVEGF, DOTA-PEG-scVEGF, HYNIC-scVEGF, and Cy5.5-scVEGF inhibited the binding of SLT-VEGF to VEGFR-2. Saturation binding was performed with 64Cu-DOTA-PEG-scVEGF to provide a dissociation constant of 2.8 nM and a maximum binding of 1.3 × 106 binding sites per cell. 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. 64Cu-DOTA-PEG-scVEGF was >95% intact after incubation in mouse plasma or radiolabeling buffer at 37ºC for 5 h.

Animal Studies



Backer et al. (10) performed biodistribution studies of 64Cu-DOTA-PEG-scVEGF in mice bearing 4T1 murine breast tumors expressing VEGFR-2. The tumor accumulated ~3% injected dose per gram (% ID/g), whereas the muscle accumulated ~0.5% ID/g. The organs with the highest accumulation of 64Cu-DOTA-PEG-scVEGF were the kidneys (~60% ID/g), liver (~8% ID/g), and lung (~5% ID/g) at 2 h after injection. PET imaging studies were performed at 1, 3, and 9 h after injection of 64Cu-DOTA-PEG-scVEGF or 64Cu-DOTA-PEG-inVEGF. 64Cu-DOTA-PEG-scVEGF revealed higher and more heterogeneous focal accumulation than 64Cu-DOTA-PEG-inVEGF. Pre-incubation of 64Cu-DOTA-PEG-scVEGF with KDR-Fc (VEGFR-2) before injection greatly reduced the tumor accumulation.

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


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