A novel radiofluorinated agouti-related protein for tumor angiogenesis imaging

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.