title: Nanobodies targeting mouse/human VCAM1 for the nuclear imaging of atherosclerotic lesions.
abstract: BACKGROUND
  A noninvasive tool allowing the detection of vulnerable atherosclerotic plaques is highly needed. By combining nanomolar affinities and fast blood clearance, nanobodies represent potential radiotracers for cardiovascular molecular imaging. Vascular cell adhesion molecule-1 (VCAM1) constitutes a relevant target for molecular imaging of atherosclerotic lesions.
  
  
  OBJECTIVE
  We aimed to generate, radiolabel, and evaluate anti-VCAM1 nanobodies for noninvasive detection of atherosclerotic lesions.
  
  
  RESULTS
  Ten anti-VCAM1 nanobodies were generated, radiolabeled with technetium-99m, and screened in vitro on mouse and human recombinant VCAM1 proteins and endothelial cells and in vivo in apolipoprotein E-deficient (ApoE(-/-)) mice. A nontargeting control nanobody was used in all experiments to demonstrate specificity. All nanobodies displayed nanomolar affinities for murine VCAM1. Flow cytometry analyses using human human umbilical vein endothelial cells indicated murine and human VCAM1 cross-reactivity for 6 of 10 nanobodies. The lead compound cAbVCAM1-5 was cross-reactive for human VCAM1 and exhibited high lesion-to-control (4.95±0.85), lesion-to-heart (8.30±1.11), and lesion-to-blood ratios (4.32±0.48) (P<0.05 versus control C57Bl/6J mice). Aortic arch atherosclerotic lesions of ApoE(-/-) mice were successfully identified by single-photon emission computed tomography imaging. (99m)Tc-cAbVCAM1-5 binding specificity was demonstrated by in vivo competition experiments. Autoradiography and immunohistochemistry further confirmed cAbVCAM1-5 uptake in VCAM1-positive lesions.
  
  
  CONCLUSIONS
  The (99m)Tc-labeled, anti-VCAM1 nanobody cAbVCAM1-5 allowed noninvasive detection of VCAM1 expression and displayed mouse and human cross-reactivity. Therefore, this study demonstrates the potential of nanobodies as a new class of radiotracers for cardiovascular applications. The nanobody technology might evolve into an important research tool for targeted imaging of atherosclerotic lesions and has the potential for fast clinical translation.
authors.affiliation: Laboratoire des Radiopharmaceutiques Bioclinique, INSERM 1039, Grenoble, France. alexis.broisat@inserm.fr
authors.forename: Alexis
authors.initials: A
authors.lastname: Broisat
authors.forename: Sophie
authors.initials: S
authors.lastname: Hernot
authors.forename: Jakub
authors.initials: J
authors.lastname: Toczek
authors.forename: Jens
authors.initials: J
authors.lastname: De Vos
authors.forename: Laurent M
authors.initials: LM
authors.lastname: Riou
authors.forename: Sandrine
authors.initials: S
authors.lastname: Martin
authors.forename: Mitra
authors.initials: M
authors.lastname: Ahmadi
authors.forename: Nicole
authors.initials: N
authors.lastname: Thielens
authors.forename: Ulrich
authors.initials: U
authors.lastname: Wernery
authors.forename: Vicky
authors.initials: V
authors.lastname: Caveliers
authors.forename: Serge
authors.initials: S
authors.lastname: Muyldermans
authors.forename: Tony
authors.initials: T
authors.lastname: Lahoutte
authors.forename: Daniel
authors.initials: D
authors.lastname: Fagret
authors.forename: Catherine
authors.initials: C
authors.lastname: Ghezzi
authors.forename: Nick
authors.initials: N
authors.lastname: Devoogdt
mesh_terms: D000818:Animals; D001057:Apolipoproteins E / Q000172:deficiency / Q000235:genetics; D050197:Atherosclerosis / Q000175:diagnosis* / Q000378:metabolism* / Q000473:pathology; D015415:Biomarkers / Q000378:metabolism; D002460:Cell Line; D004195:Disease Models, Animal; D004730:Endothelium, Vascular / Q000378:metabolism* / Q000473:pathology; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D018345:Mice, Knockout; D057054:Molecular Imaging / Q000379:methods*; D011849:Radioactive Tracers; D016719:Radioimmunodetection / Q000379:methods; D019275:Radiopharmaceuticals; D013667:Technetium; D019010:Vascular Cell Adhesion Molecule-1 / Q000378:metabolism*
chemical_list: D001057:Apolipoproteins E; D015415:Biomarkers; D011849:Radioactive Tracers; D019275:Radiopharmaceuticals; D019010:Vascular Cell Adhesion Molecule-1; D013667:Technetium