Ultrasound Molecular Imaging of Atherosclerosis With Nanobodies: Translatable Microbubble Targeting Murine and Human VCAM (Vascular Cell Adhesion Molecule) 1.

Mukesh Punjabi,Amanda Ochoa-Espinosa,Nick Devoogdt,Ahmed Murtaja,Alexandra Kosareva,Beat A Kaufmann,Lifen Xu,Thomas Wolff,Alexis Broisat

doi:10.1161/atvbaha.119.313088

Abstract

Contrast-enhanced ultrasound molecular imaging (CEUMI) of endothelial expression of VCAM (vascular cell adhesion molecule)-1 could improve risk stratification for atherosclerosis. The microbubble contrast agents developed for preclinical studies are not suitable for clinical translation. Our aim was to characterize and validate a microbubble contrast agent using a clinically translatable single-variable domain immunoglobulin (nanobody) ligand. Approach and Results: Microbubble with a nanobody targeting VCAM-1 (MBcAbVcam1-5) and microbubble with a control nanobody (MBVHH2E7) were prepared and characterized in vitro. Attachment efficiency to VCAM-1 under continuous and pulsatile flow was investigated using activated murine endothelial cells. In vivo CEUMI of the aorta was performed in atherosclerotic double knockout and wild-type mice after injection of MBcAbVcam1-5 and MBVHH2E7. Ex vivo CEUMI of human endarterectomy specimens was performed in a closed-loop circulation model. The surface density of the nanobody ligand was 3.5×105 per microbubble. Compared with MBVHH2E7, MBcAbVcam1-5 showed increased attachment under continuous flow with increasing shear stress of 1-8 dynes/cm2 while under pulsatile flow attachment occurred at higher shear stress. CEUMI in double knockout mice showed signal enhancement for MBcAbVcam1-5 in early (P=0.0003 versus MBVHH2E7) and late atherosclerosis (P=0.007 versus MBVHH2E7); in wild-type mice, there were no differences between MBcAbVcam1-5 and MBVHH2E7. CEUMI in human endarterectomy specimens showed a 100% increase in signal for MBcAbVcam1-5versus MBVHH2E7 (20.6±27.7 versus 9.6±14.7, P=0.0156). CEUMI of the expression of VCAM-1 is feasible in murine models of atherosclerosis and on human tissue using a clinically translatable microbubble bearing a VCAM-1 targeted nanobody.

Highlights

In vivo Contrast-enhanced ultrasound molecular imaging (CEUMI) of the aorta was performed in atherosclerotic double knockout and wild-type mice after injection of MBcAbVcam[1,2,3,4,5] and MBVHH2E7
CEUMI in double knockout mice showed signal enhancement for MBcAbVcam[1,2,3,4,5] in early (P=0.0003 versus MBVHH2E7) and late atherosclerosis (P=0.007 versus MBVHH2E7); in wild-type mice, there were no differences between MBcAbVcam[1,2,3,4,5] and MBVHH2E7
CEUMI of the expression of VCAM-1 is feasible in murine models of atherosclerosis and on human tissue using a clinically translatable microbubble bearing a VCAM-1 targeted nanobody

Summary

OBJECTIVE

Contrast-enhanced ultrasound molecular imaging (CEUMI) of endothelial expression of VCAM (vascular cell adhesion molecule)-1 could improve risk stratification for atherosclerosis. The feasibility of contrast-enhanced ultrasound molecular imaging (CEUMI) for detecting the expression of VCAM-1 using full-size antibodies has been shown in animal models.[6,7,8,9] no clinical studies using this technique have been performed far. VCAM-1 targeting with nanobodies attached to microbubbles using biotin-streptavidin bridging has been recently accomplished in a mouse tumor model.[18] In the current study, we, studied the use of a microbubble with maleimide-thiol conjugation of an anti– VCAM-1 nanobody to detect VCAM-1 expression in a mouse model of atherosclerosis and ex vivo in human endarterectomy specimens using noninvasive ultrasound imaging

Study Design

RESULTS

DISCUSSION

Disclosures