Identification of Human scFvs Targeting Atherosclerotic Lesions

Abstract

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 ∼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. 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 ∼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. Atherosclerosis is an inflammatory disease in which the immune system interacts with risk factors to initiate, propagate, and activate lesions in the vasculature that are referred to as atherosclerotic plaques (1Ross R. N. Engl. J. Med. 1999; 340: 115-126Crossref PubMed Scopus (19248) Google Scholar, 2Hansson G.K. N. Engl. J. Med. 2005; 352: 1685-1695Crossref PubMed Scopus (6987) Google Scholar). The rupture of such plaques and subsequent thromboembolism lead to cardiovascular disease, the major cause of death in Western countries. The early detection of atherosclerosis and the development of “smart” contrast agents to be used in a non-invasive procedure such as magnetic resonance imaging are critical for the diagnosis and management of vulnerable patients. An early event in plaque formation is the interaction of circulating platelets with activated endothelial cells (3Goncalves I. Nesbitt W.S. Yuan Y. Jackson S.P. J. Biol. Chem. 2005; 280: 15430-15437Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) with ensuing leukocyte recruitment via soluble or cell surface signaling molecules such as proinflammatory cytokines and selectins. This cascade of events including the tight adhesion of platelets leads to local re