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64Cu-Labeled 1,4,7,10-Tetraazacyclododedane-N,N’,N’’,N’’’-tetraacetic acid–conjugated vascular endothelial growth factor A isoform 121-gelonin fusion protein

, PhD
National Center for Biotechnology Information, NLM, NIH
Corresponding author.

Created: ; Last Update: August 10, 2011.

Chemical name:64Cu-Labeled 1,4,7,10-Tetraazacyclododedane-N,N’,N’’,N’’’-tetraacetic acid–conjugated vascular endothelial growth factor A isoform 121-gelonin fusion protein
Abbreviated name:64Cu-DOTA-VEGF121/rGel
Agent Category:Proteins
Target:Vascular endothelial growth factor receptor (VEGFR)
Target Category:Receptors
Method of detection:Positron emission tomography (PET)
Source of signal / contrast:Copper-64 (64Cu)
  • Checkbox In vitro
  • Checkbox Rodents
No structure is available.



64Cu-Labeled 1,4,7,10-tetraazacyclododedane-N,N’,N’’,N’’’-tetraacetic acid (DOTA)–conjugated vascular endothelial growth factor A isoform 121 (VEGF121)-gelonin fusion protein (VEGF121/rGel), abbreviated as 64Cu-DOTA-VEGF121/rGel, is an imaging agent developed by Hsu et al. for monitoring the targeting efficiency and treatment efficacy of the VEGF121/rGel immunotoxin (1).

VEGFs are a group of five potent inducers of cell migration, invasion, vascular permeability, and neovascular formation (2). They act via three receptor tyrosine kinases: VEGFR-1, VEGFR-2, and VEGFR-3 (3). These receptors are overexpressed on the endothelial cells of tumor neovasculature and are almost undetectable in the endothelium of adjacent normal tissues. The critical role of the VEGF/VEGFR signal pathway in tumor angiogenesis has prompted great efforts in the development of antiangiogenic therapies, and agents have been tested by acting on different steps of the pathway, such as by binding to the VEGF ligand, inhibiting VEGFR tyrosine kinase, inhibiting downstream effectors (mammalian target of rapamycin inhibitors), and modulating VEGF production (4-6). These agents are highly effective against tumor growth in animal models when they are used alone; however, they seldom lead to tumor regression and exhibit insufficient efficacy in the clinical setting, although combination with chemotherapy has been shown to improve patient survival in certain tumor types. The most likely explanation for this phenominon is that tumor endothelial cells can adapt to antiangiogenic treatment and form functional vasculature that loses sensitivity to the inhibitors of VEGF/VEGFR (4, 6). It is hypothesized that VEGF/VEGFR-targeted therapy should be administrated before the development of a well-established vascular network.

Molecular imaging provides a means to reveal the mechanism underlying this phenomenon and to monitor the antiangionic therapy (1, 7). VEGF121/rGel has been generated with VEGF121, which is linked with recombinant plant toxin gelonin through a G4S tether (4, 8-10). Gelonin is a member of the ribosome-inactivating protein family, which depurinates rRNA and other polynucleotide substrates and subsequently inhibits protein synthesis (11). A series of preclinical studies showed that VEGF121/rGel could specifically inhibit the growth of tumor endothelial cells (8-10, 12). Like other immunotoxins, VEGF121/rGel is also expected to be effective against tumors resistant to VEGF/VEGFR-targeting inhibitors if the tumor cells express sufficient levels of VEGFR (11). To monitor the VEGFR-targeting efficiency of VEGF121/rGel with imaging techniques, Hsu et al. and Cho et al. labeled the VEGF121/rGel with 64Cu (64Cu-DOTA-VEGF121/rGel) and with MnFe2O4 nanoparticles (VEGF121/rGel-MNPs), respectively (1, 7). Both imaging studies have concluded that noninvasive imaging with VEGF121/rGel will be useful to monitor the treatment efficacy and to identify patients who may benefit from the VEGF121/rGel therapy. This chapter summarizes data obtained with 64Cu-DOTA-VEGF121/rGel (1).



Hsu et al. described the synthesis of 64Cu-DOTA-VEGF121/rGel (1). The synthesis, expression, and purification of the VEGF121/rGel immunotoxin were performed as described previously by Veenendaal et al. (8). The molecular weight of VEGF121/rGel was 84 kDa. VEGF121/rGel was conjugated to DOTA to generate DOTA-VEGF121/rGel. Labeling with 64Cu was completed in the reaction of 64CuCl2 and DOTA-VEGF121/rGel for 1 h at 40°C.

The total time for 64Cu-labeling of the DOTA-VEGF121/rGel, including the final purification, was 90 ± 10 min (n = 3). The radiolabeling yield was 85.2 ± 9.2% on the basis of 37 MBq (1 mCi) 64Cu per 25 µg DOTA-VEGF121/rGel (n = 3). The specific activity of 64Cu-DOTA-VEGF121/rGel was 1.3 ± 0.1 GBq/mg (35.14 ± 2.7 mCi/mg), and the radiochemical purity was ≥98%. The number of DOTA molecules per VEGF121/rGel molecule was 3.3 ± 0.1 (n = 4).

In Vitro Studies: Testing in Cells and Tissues


A cell-binding assay with VEGF121/rGel and the DOTA-VEGF121/rGel conjugate was performed with 125I-VEGF165 (specific activity, 74 TBq/mmol (2 kCi/mmol)) as the radioligand (1). Both VEGF121/rGel and DOTA-VEGF121/rGel without the metal inhibited the 125I-VEGF165 binding to PAE/KDR cells (porcine aortic endothelial cells transfected with cDNA of VEGFR2) in a dose-dependent manner. The 50% inhibition concentrations of VEGF121/rGel and DOTA-VEGF121/rGel were 24.5 nM and 40.6 nM, respectively, indicating that DOTA conjugation induced no significant change in the VEGF121/rGel binding affinity. Western blot analysis (functional assay) of the VEGFR2 expression on PAE/KDR cells revealed a slight decrease in the expression level of phosphorylated VEGFR2 after DOTA conjugation. Increased expression levels of the phosphorylated VEGFR2 were observed at concentrations ≥5 nM for both VEGF121/rGel and DOTA-VEGF121/rGel.

Animal Studies



Positron emission tomography with 64Cu-DOTA-VEGF121/rGel was performed in athymic nude mice bearing intracranial tumors (n = 3) (1). Tumors were generated by intracranial injection into the right frontal lobe with 105 firefly luciferase-transfected U87MG human glioblastoma cells (U87MG-fLuc). The mice were intravenously injected with 5–10 MBq (0.14–0.27 mCi) 64Cu-DOTA-VEGF121/rGel and were imaged for up to 48 h after injection.

64Cu-DOTA-VEGF121/rGel exhibited high tumor accumulation and retention, as well as high tumor/background contrast from 1 h to 48 h after injection (1). Tumor accumulation at 1 h after injection was 5.8 ± 0.5% injected dose per gram (ID/g) (n = 3) and steadily increased, peaking at ~18 h after injection (11.8 ± 2.3% ID/g). At 46 h after injection, tumor uptake decreased to 8.4 ± 1.7% ID/g. There was no clear relationship between tumor size and tracer uptake. 64Cu-DOTA-VEGF121/rGel was cleared through both the hepatic and the renal pathways (data not shown). However, no evidence about the in vivo stability of this agent was reported.

A blocking study was carried out by injecting 200 µg VEGF121 before injecting 64Cu-DOTA-VEGF121/rGel. Blocking with VEGF121 resulted in a significant reduction in the 64Cu-DOTA-VEGF121/rGel uptake (P < 0.05), suggesting VEGFR-specific tumor uptake of the 64Cu-DOTA-VEGF121/rGel (1).

Other Non-Primate Mammals


No references are currently available.

Non-Human Primates


No references are currently available.

Human Studies


No references are currently available.


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Mohamedali K.A., Kedar D., Sweeney P., Kamat A., Davis D.W., Eve B.Y., Huang S., Thorpe P.E., Dinney C.P., Rosenblum M.G. The vascular-targeting fusion toxin VEGF121/rGel inhibits the growth of orthotopic human bladder carcinoma tumors. Neoplasia. 2005;7(10):912–20. [PMC free article: PMC1550288] [PubMed: 16242074]
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