title: In vivo phage display to identify new human antibody fragments homing to atherosclerotic endothelial and subendothelial tissues [corrected].
abstract: In vivo phage display selection is a powerful strategy for directly identifying agents that target the vasculature of normal or diseased tissues in living animals. We describe here a new in vivo biopanning strategy in which a human phage single-chain antibody (scFv) library was injected into high-fat diet-fed ApoE(-/-) mice. Extracellular and internalized phage scFvs were selectively recovered from atherosclerotic vascular endothelium and subjacent tissues. After three successive biopanning rounds, a panel of six clones with distinct gene sequences was isolated. Four scFvs produced and purified in soluble form were shown to interact in vitro with a rabbit atheromatous protein extract by time-resolved fluorescence resonance energy transfer and to target the endothelial cell surface and inflamed intima-related regions of rabbit and human tissue sections ex vivo. These new scFvs selected in a mouse model recognized both rabbit and human tissue, underlying the interspecies similarities of the recognized epitopes. By combining immunoprecipitation and mass spectrometry, one of the selected scFvs was shown to recognize carbonic anhydrase II, an up-regulated enzyme involved in resorption of ectopic calcification. These results show that in vivo biopanning selection in hypercholesterolemic animals makes it possible to identify both scFvs homing to atherosclerotic endothelial and subendothelial tissues, and lesion-associated biomarkers. Such scFvs offer promising opportunities in the field of molecular targeting for the treatment of atherosclerosis.
authors.affiliation: Magnetic Resonance Center of Biological Systems, UMR 5536, National Center for Scientific Research, Bordeaux Segalen University, Bordeaux, France.
authors.forename: Kamel
authors.initials: K
authors.lastname: Deramchia
authors.forename: Marie-Josée
authors.initials: MJ
authors.lastname: Jacobin-Valat
authors.forename: Amélie
authors.initials: A
authors.lastname: Vallet
authors.forename: Hervé
authors.initials: H
authors.lastname: Bazin
authors.forename: Xavier
authors.initials: X
authors.lastname: Santarelli
authors.forename: Stéphane
authors.initials: S
authors.lastname: Sanchez
authors.forename: Pierre
authors.initials: P
authors.lastname: Dos Santos
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Franconi
authors.forename: Stéphane
authors.initials: S
authors.lastname: Claverol
authors.forename: Stéphane
authors.initials: S
authors.lastname: Bonetto
authors.forename: Gisèle
authors.initials: G
authors.lastname: Clofent-Sanchez
mesh_terms: D000818:Animals; D001013:Aorta, Thoracic / Q000378:metabolism; D001057:Apolipoproteins E / Q000172:deficiency; D050197:Atherosclerosis / Q000378:metabolism*; D015415:Biomarkers / Q000378:metabolism; D024402:Carbonic Anhydrase II / Q000378:metabolism; D003324:Coronary Artery Disease / Q000378:metabolism; D004041:Dietary Fats / Q000008:administration & dosage; D004730:Endothelium, Vascular / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006801:Humans; D006937:Hypercholesterolemia / Q000378:metabolism; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D019151:Peptide Library; D058226:Plaque, Atherosclerotic / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D057127:Single-Chain Antibodies / Q000302:isolation & purification / Q000493:pharmacokinetics*
chemical_list: D001057:Apolipoproteins E; D015415:Biomarkers; D004041:Dietary Fats; D019151:Peptide Library; D057127:Single-Chain Antibodies; D024402:Carbonic Anhydrase II