MicroPET Imaging of Integrin αvβ3 Expressing Tumors Using 89Zr-RGD Peptides

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Abstract
Purpose The dimeric transmembrane integrin, αvβ3, is a well-investigated target by different imaging modalities through suitably labeled arginine–glycine–aspartic acid (RGD) containing peptides. In this study, we labeled four cyclic RGD peptides with or without PEG functional groups: c(RGDfK) (denoted as FK), PEG3-c(RGDfK) (denoted as FK-PEG3), E[c(RGDfK)]2 (denoted as [FK]2), and PEG4-E[PEG4-c(RGDfK)]2 (denoted as [FK]2-3PEG4), with 89Zr (t 1/2 = 78.4 h), using the chelator desferrioxamine-p-SCN (Df) for imaging tumor integrin αvβ3. Methods The Df conjugated RGD peptides were subjected to integrin αvβ3 binding assay in vitro using MDA-MB-435 breast cancer cells. The 89Zr-labeled RGD peptides were then subjected to small animal positron emission tomography (PET) and direct tissue sampling biodistribution studies in an orthotopic MDA-MB-435 breast cancer xenograft model. Results All four tracers, 89Zr-Df-FK, 89Zr-Df-FK-PEG3, 89Zr-Df-[FK]2, and 89Zr-Df-[FK]2-3PEG4, were labeled in high radiochemical yield (89 ± 4%) and high specific activity (4.07–6 MBq/μg). Competitive binding assay with 125I-echistatin showed that conjugation of the RGD peptides to the Df chelator did not have significant impact on their integrin αvβ3 binding affinity and the dimeric peptides were shown to be more potent than the monomers. In agreement with binding results, tumor uptake of 89Zr-Df-[FK]2 and 89Zr-Df-[FK]2-3PEG4 was significantly higher (4.32 ± 1.73%ID/g and 4.72 ± 0.66%ID/g, respectively, at 2 h post-injection) than the monomers 89Zr-Df-FK and 89Zr-Df-FK-PEG3 (1.97 ± 0.38%ID/g and 1.57 ± 0.49%ID/g, respectively, at 2 h post-injection). Out of the four labeled peptides, 89Zr-Df-[FK]2-3PEG4 gave the highest tumor-to-background ratio (18.21 ± 2.52 at 2 h post-injection and 19.69 ± 3.99 at 4 h post-injection), with the lowest uptake in metabolic organs. Analysis of late time points biodistribution data revealed that the uptake in the tumor was decreased, along with increase in the bone, which implies decomplexation of 89Zr-Df. Conclusion Efficient radiolabeling of peptides with an appropriate chelator such as Df-RGD with 89Zr was observed. The 89Zr radiolabeled peptides provided high-quality and high-resolution microPET images in xenograft models. 89Zr-Df-[FK]2-3PEG4 demonstrated the highest tumor-to-background ratio of the compounds tested. Preparation of 89Zr peptides to take advantage of the longer half-life is unwarranted due to the relatively rapid clearance from the tumor region of peptide tracers prepared for this study and the increased uptake in the bone of transchelated 89Zr with time (2.0 ± 0.36%ID/g, 24 h post-injection).