A novel radiofluorinated agouti-related protein for tumor angiogenesis imaging
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A novel protein scaffold based on the cystine knot domain of the agouti-related protein (AgRP) has been used to engineer mutants that can bind to the αvβ3 integrin receptor with high affinity and specificity. In the current study, an 18F-labeled AgRP mutant (7C) was prepared and evaluated as a positron emission tomography (PET) probe for imaging tumor angiogenesis. AgRP-7C was synthesized by solid phase peptide synthesis and site-specifically conjugated with 4-nitrophenyl 2-18/19F-fluoropropionate (18/19F-NFP) to produce the fluorinated peptide, 18/19F-FP-AgRP-7C. Competition binding assays were used to measure the relative affinities of AgRP-7C and 19F-FP-AgRP-7C to human glioblastoma U87MG cells that overexpress αvβ3 integrin. In addition, biodistribution, metabolic stability, and small animal PET imaging studies were conducted with 18F-FP-AgRP-7C using U87MG tumor-bearing mice. Both AgRP-7C and 19F-FP-AgRP-7C specifically competed with 125I-echistatin for binding to U87MG cells with half maximal inhibitory concentration (IC50) values of 9.40 and 8.37 nM, respectively. Non-invasive small animal PET imaging revealed that 18F-FP-AgRP-7C exhibited rapid and good tumor uptake (3.24 percentage injected dose per gram [% ID/g] at 0.5 h post injection [p.i.]). The probe was rapidly cleared from the blood and from most organs, resulting in excellent tumor-to-normal tissue contrasts. Tumor uptake and rapid clearance were further confirmed with biodistribution studies. Furthermore, co-injection of 18F-FP-AgRP-7C with a large molar excess of blocking peptide c(RGDyK) significantly inhibited tumor uptake in U87MG xenograft models, demonstrating the integrin-targeting specificity of the probe. Metabolite assays showed that the probe had high stability, making it suitable for in vivo applications. 18F-FP-AgRP-7C exhibits promising in vivo properties such as rapid tumor targeting, good tumor uptake, and excellent tumor-to-normal tissue ratios, and warrants further investigation as a novel PET probe for imaging tumor angiogenesis.
KeywordsKnottin αvβ3 integrin PET 18F
This work was supported, in part, by National Cancer Institute (NCI) 5R01 CA119053, NCI In Vivo Cellular Molecular Imaging Center (ICMIC) grant P50 CA114747, NCI 5K01 CA104706, and a Stanford Molecular Imaging Scholars postdoctoral fellowship R25 CA118681. SJM has been supported by an NSF Graduate Research Fellowship, the Medtronic Stanford Graduate Fellowship, and a Siebel Scholars Fellowship. This work is also partly sponsored by Grants from the Zhejiang Provincial Natural Science Foundation of China (Z2110230), Health Bureau of Zhejiang Province (2010ZA075, 2011ZDA013), National Science Foundation of China (NSFC) (no. 81101023, 81170306, 81173468), and Ministry of Science and Technology of China (2012BAI13B06). The authors acknowledge Tamara Locke for help on editing the manuscript.
Conflict of interest
The authors declare that they have no conflict of interest.
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