CONTEXT

Atherothrombotic events remain the leading cause of death and disability throughout most industrialized countries. Atherosclerosis is a systemic disorder affecting arterial beds throughout the body, potentially resulting in manifest coronary artery disease (CAD), cerebrovascular disease (CVD), peripheral arterial disease (PAD) or a combination of these conditions (polyvascular or diffuse vascular disease). Despite identification of multiple factors and widespread use of drug therapies, it still remains a global health concern with costs estimated at € 196 billion in European union in 2012 and at $ 503 billion in USA in 2013. It is well known that the risks of atherosclerosis are more correlated with atheroma plaque rupture rather than stenosis severity. Indeed, two major types of plaques have been identified, the stable ones that become obstructive and might cause symptoms because of impaired maximal blood flow (stable angina….) and unstable ones also called vulnerable plaques that become thrombosis-prone because they contain large lipid cores, thin fibrous cap and inflammatory cell infiltrates, exposing thrombogenic material.
Lumenography (angiography) has a central role for diagnosing stable plaques by defining the site and severity of vascular stenosis. However, this invasive imaging modality is unable to provide information regarding underlying processes occurring in vulnerable plaques. Moreover, vulnerable plaque composition varies depending on the anatomical site with striking heterogeneity even within the same individual sites. Therefore reliable non-invasive imaging tools able to detect early atherosclerotic disease in various regions and to characterize the composition of the plaques are clinically desirable.
The recent concept that risk of plaque rupture is more related to plaque content than plaque size has led to a new imperative for molecular imaging. The present invention relates to antibodies (Abs) for a non-invasive molecular imaging of vulnerable plaques in atherosclerosis. The isolated human Abs of the present invention are able to target molecules involved in the development of high-risk vulnerable plaques. These Abs bind specifically to targets on atherosclerotic lesions which are both abundant and accessible under in vivo conditions. 

TECHNICAL DESCRIPTION

A human scFv library was used to develop a novel in vivo biopanning approach (in vivo phage display technology) to investigate scFvs able to target the vascular endothelial cell surface proteins and the subendothelial molecular repertoire expressed within atherosclerotic tissues of animal models fed a high cholesterol diet. The present invention relates to seven human scFvs Abs (H2.1, K3.1, I8.1, C3.3, A5.31.F1, B2.31.F1, C4.31.F2) specifically targeting atherosclerotic lesions. One of the selected scFvs, K3.1, was found to bind to carbonic anhydrase II (CA-II) protein, a cytosolic enzyme regulating carbon dioxide levels. CA-II protein is a relevant target for atherosclerosis imaging because it is involved in dissolution of arterial calcium deposits and closely associated to macrophage-rich areas.
The selected scFvs constitute the basis for developing new clinical tools dedicated to molecular imaging and therapy of human atherosclerosis. 

BENEFITS

A first benefit of the present invention is that the Abs are recombinant human Abs specifically binding to atherosclerosis lesions. Abs have great potential in targeting, due to their ability to recognize their target with both high specificity and affinity. However, murine Abs have been withdrawn from the clinical practice due to adverse immunological responses. The use of fully human Abs is thus mandatory for further clinical applications.
A second benefit relies on antibody engineering which allows to create different formats of antibody fragments (recombinant human IgG, recombinant human Fab’2, recombinant human scFv-Fab’2, recombinant human scFv-Fc, recombinant human scFv) in order to select the most suitable one in terms of biodistribution and pharmacokinetic.
A third benefit is to have at disposal a panel of Abs recognizing a panel of targets, potentially over-expressed in vulnerable plaques. One of them, the CA-II protein, has been recently reported as strongly expressed in vulnerable plaque.
Finally, the Abs of the present invention could be easily labeled with a tracer thanks to tags added to engineered Abs for in vivo Nuclear or Magnetic Resonance Imaging (MRI). 

INDUSTRIAL APPLICATIONS

The Abs as described herein are preferably for use in medical diagnostic for in vivo imaging of atherosclerotic lesions or vulnerable plaques. The Abs are preferably labeled for in vivo medical imaging with 18F, 123/131I or 99mTc for Nuclear Imaging or with on the Versatile UltraSmall SuperParamagnetic Iron Oxide (VUSPIO) platform (Versatile USPIO, WO2004107368) for MRI. Once adequately labelled, the Abs of the present invention may be used in nuclear or MRI in vivo medical imaging method.
Another application is for use in treatment of atherosclerosis, either as therapeutic antibody or in targeting therapeutic agents to atherosclerotic lesions (drug delivery).
The present invention could be used for in vivo imaging of atherosclerotic lesions and treatment of patients comprising (i) visualization by detection, for example, of labeled K3.1, binding specifically to human CA II protein and (ii) reduction of the lesions size by delivering a drug in situ.

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