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 ϳ56 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 (1, 2). 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 (3) 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 release of potent inflammatory mediators (IL1␤ and CD40L) at the injured site. The endothelial cells then show an enhanced expression of adhesion molecules including VCAM-1, I-CAM, and P-selectin as well as the release of MCP-1 and other chemokines. Their altered chemotactic and adhesive properties facilitate lymphocyte and monocyte transmigration into the intima of blood vessels. The "signature" provided by an atherosclerotic lesion surface is difficult to study in vitro (4), even if progress in cell co-culture procedures allow mimicking of the disease (5). An alternative for overcoming this limitation is to probe the lesions in a high cholesterol fed rabbit model using a human phage-displayed single chain antibody library. Peptide phage display libraries have been ...

MRI of inducible P‐selectin expression in human activated platelets involved in the early stages of atherosclerosis

“…In an ultimate aim to translate such agents into human clinical use, a 'humanized' version of murine VH10 is envisaged in order to attenuate potential immunogenicity We plan to use phage display technology and directed mutagenesis approaches to develop a human antibody presenting the same specificity and affinity as its murine counterpart on human platelets, but also able to recognize with high affinity murine P-selectin (77,78). Toxicologic and pharmacokinetic in vivo data on ApoE mice will be further evaluated in order to lay the basis for preclinical development.…”

Selection of phage-displayed peptides on live adherent cells in microfluidic channels

“…The selection of phage-displayed peptides that target a cellsurface marker is typically performed through a biopanning process, either in cell suspension (19,20,22) or on a solid support (23,24). This biopanning process involves four major steps: preparation of phage-displayed peptide libraries, capture of specific phage that bind to the target, washing of low-affinity or nonspecific phage from the cell surface, and recovery of enriched target binders for the next round of selection (25).…”

The dimeric platelet collagen receptor GPVI-Fc reduces platelet adhesion to activated endothelium and preserves myocardial function after transient ischemia in mice

“…However, the authors do not provide direct evidence for exposure of collagen under I/R. Recently, Vn was identified as a potential marker for endothelial activation during early atherosclerosis (43). Thus Vn seems a plausible candidate as a binding partner on activated endothelium.…”