Identification of human scFvs targeting atherosclerotic lesions: selection by single round in vivo phage display

Our aim was to investigate by in vivo biopanning the lesions developed early in atherosclerosis and identify human antibodies that home to diseased regions. We have designed a two-step approach for a rapid isolation of human Monoclonal phage-display single-chain antibodies (MoPhabs) reactive with proteins found in lesions developed in an animal model of atherosclerosis. After a single round of in vivo biopanning, the MoPhabs were eluted from diseased sections of rabbit aorta identified by histology and NMR microscopy. MoPhabs expressed in situ were selected by subtractive colony filter screening for their capacity to recognize atherosclerotic but not normal aorta. MoPhabs selected by our method predominantly bind atherosclerotic lesions. Two of them, B3.3G and B3.GER, produced as scFv fragments, recognized an epitope present on the surface in early atherosclerotic lesions and within the intimal thickness in more complex plaques. These human MoPhabs homed to atherosclerotic lesions in ApoE(-/-) mice after in vivo injection. A protein of approximately 56 kDa recognized by B3.3G was affinity-purified and identified by mass spectrometry analysis as vitronectin. This is the first time that single round in vivo biopanning has been used to select human antibodies as candidates for diagnostic imaging and for obtaining insight into targets displayed in atherosclerotic plaques.