Background

Oxidation-specific epitopes (OSEs) are proinflammatory, and elevated levels in plasma predict cardiovascular events.

Objectives

The purpose of this study was to develop novel positron emission tomography (PET) probes to noninvasively image OSE-rich lesions.

Methods

An antigen-binding fragment (Fab) antibody library was constructed from human fetal cord blood. After multiple rounds of screening against malondialdehyde-acetaldehyde (MAA) epitopes, the Fab LA25 containing minimal nontemplated insertions in the CDR3 region was identified and characterized. In mice, pharmacokinetics, biodistribution, and plaque specificity studies were performed with Zirconium-89 (⁸⁹Zr)-labeled LA25. In rabbits, ⁸⁹Zr-LA25 was used in combination with an integrated clinical PET/magnetic resonance (MR) system. ¹⁸F-fluorodeoxyglucose PET and dynamic contrast-enhanced MR imaging were used to evaluate vessel wall inflammation and plaque neovascularization, respectively. Extensive ex vivo validation was carried out through a combination of gamma counting, near infrared fluorescence, autoradiography, immunohistochemistry, and immunofluorescence.

Results

LA25 bound specifically to MAA epitopes in advanced and ruptured human atherosclerotic plaques with accompanying thrombi and in debris from distal protection devices. PET/MR imaging 24 h after injection of ⁸⁹Zr-LA25 showed increased uptake in the abdominal aorta of atherosclerotic rabbits compared with nonatherosclerotic control rabbits, confirmed by ex vivo gamma counting and autoradiography. ¹⁸F-fluorodeoxyglucose PET, dynamic contrast-enhanced MR imaging, and near-infrared fluorescence signals were also significantly higher in atherosclerotic rabbit aortas compared with control aortas. Enhanced liver uptake was also noted in atherosclerotic animals, confirmed by the presence of MAA epitopes by immunostaining.

Conclusions

⁸⁹Zr-LA25 is a novel PET radiotracer that may allow noninvasive phenotyping of high-risk OSE-rich lesions.

Objectives

This study sought to develop an integrative positron emission tomography (PET) with magnetic resonance imaging (MRI) procedure for accurate atherosclerotic plaque phenotyping, facilitated by clinically approved and nanobody radiotracers.

Background

Noninvasive characterization of atherosclerosis remains a challenge in clinical practice. The limitations of current diagnostic methods demonstrate that, in addition to atherosclerotic plaque morphology and composition, disease activity needs to be evaluated.

Methods

We screened 3 nanobody radiotracers targeted to different biomarkers of atherosclerosis progression, namely vascular cell adhesion molecule (VCAM)-1, lectin-like oxidized low-density lipoprotein receptor (LOX)-1, and macrophage mannose receptor (MMR). The nanobodies, initially radiolabeled with copper-64 (⁶⁴Cu), were extensively evaluated in Apoe^-/- mice and atherosclerotic rabbits using a combination of in vivo PET/MRI readouts and ex vivo radioactivity counting, autoradiography, and histological analyses.

Results

The 3 nanobody radiotracers accumulated in atherosclerotic plaques and displayed short circulation times due to fast renal clearance. The MMR nanobody was selected for labeling with gallium-68 (⁶⁸Ga), a short-lived radioisotope with high clinical relevance, and used in an ensuing atherosclerosis progression PET/MRI study. Macrophage burden was longitudinally studied by ⁶⁸Ga-MMR-PET, plaque burden by T2-weighted MRI, and neovascularization by dynamic contrast-enhanced (DCE) MRI. Additionally, inflammation and microcalcifications were evaluated by fluorine-18 (¹⁸F)-labeled fluorodeoxyglucose (¹⁸F-FDG) and ¹⁸F-sodium fluoride (¹⁸F-NaF) PET, respectively. We observed an increase in all the aforementioned measures as disease progressed, and the imaging signatures correlated with histopathological features.

Conclusions

We have evaluated nanobody-based radiotracers in rabbits and developed an integrative PET/MRI protocol that allows noninvasive assessment of different processes relevant to atherosclerosis progression. This approach allows the multiparametric study of atherosclerosis and can aid in early stage anti-atherosclerosis drug trials.