ATHERANOS project aims at developing a theranostic approach for atherosclerosis which is the main cause of death in the Western world and tends to increase rapidly in developing countries. Atherosclerosis can be regarded as an inflammatory disease that results from an initial activation of the endothelial cells with further enhancement of oxidative stress, lipid and leucocyte recruitment. The lesions evolve to vulnerable plaques presenting large lipid cores covered by a thin fibrous cap at high risk of rupture and thrombi formation, thus precipitating the clinical conditions of stroke and myocardial infarction. Nowadays, there is an increasing interest in molecular imaging in order to assess the cellular components that underlie the risk of rupture. Molecular imaging requires highly sensitive and specific probes made of a signal detection compound and an affinity ligand for targeting. Our project joins in the international competition but what makes it distinct is that it proposes human antibodies (Abs) to functionalize multi-modal nanoparticles loaded with iron oxide and drugs for Magnetic Resonance Imaging (MRI)-guided therapy of atherosclerosis. Human Abs targeting relevant biomarkers of the pathology are mandatory for repeated use in humans in order to avoid immunogenic reactions. The targeted nanoparticles developed until now with murine Abs cannot be directly translated into the clinic. Our project is carried by this will of transfer. MRI is a non-invasive imaging modality which offers the possibility to use safe contrast agents like iron oxide particles. Nucleolipid-based nanoparticles loaded with drug and iron oxide and further functionalized with human antibodies present all the necessary requirements for innocuity. Each partner in ATHERANOS project has the required skills to reach the aim the consortium has set. This multidisciplinary project is supported by the expertise of physicists, chemists, biochemists and molecular biologists. Partner 1 has the know-how for in vivo phage-display selection of human antibodies in animal models of atherosclerosis. This technology is powerful to select antibodies not only for their specificity but also for their targeting capacity in the context of the pathology. Partner 2 has developed and patented a proprietary library comprising several billion of different fully human antibodies and innovative technologies that allow the generation and improvement of high-affinity, human antibody candidates. As an alternative route, in vitro selection will be conducted on pertinent targets. Partner 3 is deeply involved in original approaches for the isolation and production of new molecules for therapeutic application in humans. Partner 4 possesses a large experience in nucleolipid-based nanoparticle construction and efficient drug encapsulation. The expertise developed by each partner in his field offers the necessary guaranties to drive ATHERANOS project to successful pre-clinical development.

Antithrombin, a double agent, anticoagulant and cytoprotective : The THERAT project the coagulation inhibitor, antithrombin (AT). In addition to inactivate thrombin (factor II activated or factor IIa of coagulation) that converts fibrinogen into fibrin, AT physiological role is much broader: this plasma protein, which belongs to the family of serpins (Serine Protease Inhibitor) inhibits also several serine proteases of coagulation, including activated FX (FXa). The EA4531, University Paris Sud (partner 2) is particularly involved in the study of structure-function relationship of AT to strengthen and extend the properties of the therapeutic product, which is the natural protein extracted from human plasma and marketed by LFB (partner 1). Indeed, the reaction of inhibition of coagulation mediated by AT is significantly increased in the presence of heparin derivatives, which include heparan sulfate cellular that bind to AT by their pentasaccharide motive. This antigoagulante protein has also cytoprotective properties (anti-inflammatory activity and vasorelaxant) mediated through binding to heparan sulfates on the surface of cells. Thus there is a rational for the development of recombinant molecules derived from natural antithrombin but by decoupling the anticoagulant and cytoprotective activities of AT. Indeed, anti-inflammatory properties of natural AT are observed in vitro only at high doses (250 IU / kg), that used in current practice would be likely to cause a hemorrhagic risk associated with the administration of supraphysiological doses of an anticoagulant protein. THERAT project focuses therefore on recombinant AT-derived molecules ("variants") to be developed for the management of severe sepsis, but also to be used as an antidote for heparins, particularly in the context of extra-body circulation. In this context, recombinant variants of AT devoid of anticoagulant activity and have an increased affinity for heparin, have recently been described {Bianchini, 2011} and which constitute the proof of concept of project THERAT. Its objective is to generalize the approach and develop two parallel strategies of random mutagenesis and directed variants of AT with high therapeutic potential, and then test them in vitro and in vivo (EA 3859, University of Angers, partner 4), while implementing innovative methodologies of characterization (UMR CNRS 8612, partner 3) and purification (LFB BIOTECHNOLOGIES, partner 1). Eventually, two clinical indications will be targeted by these new generation product : 1 - The neutralization of heparin derivatives used in the proceedings of extra-body circulation (CEC) 2 - The treatment of severe sepsis. For enhanced therapeutic benefit, selected AT variants will have to display a reduced anticoagulant activity and an increased binding to heparin compared to plasma AT, while having a satisfactory and PK being devoid of any immunogenic potential.

Antibodies (Abs) exert their function by binding to distinct Fc receptors and complement. The affinity of these interactions is traditionally ascribed to the Ab isotype. At least for the IgGs this view is evolving. N-linked glycans are increasingly proposed to influence binding to the classical Fc-gamma receptors (Fc?R) and allow for binding to non-classical type II receptors. This proposal will study the functional significance of Fc-glycosylation in potentiating or regulating the chronic inflammatory response in Multiple Sclerosis (MS) and its animal models. MS is a chronic disabling disease during which inflammatory lesions in the central nervous system cause local tissue damage, including demyelination and neuronal (axonal) loss. The genetic susceptibility points to a causal role for the immune response, but the triggers that provoke T and B cells to attack myelin, the factors that cause disease relapses, and the anatomical localisation and composition of the inflammatory lesions in relation to the heterogeneous clinical expression of MS remain active fields of investigation. Antibodies contribute to MS. Their presence in the cerebrospinal fluid is a diagnostic hallmark. Active lesions contain antibodies and reveal complement activation. Autoantibodies recognising potassium channel KIR4.1, Neurofascin, as well as glycolipids have been identified. Within MS lesions antibodies specific for Myelin oligodendrocyte glycoprotein (MOG) can be detected. The pathogenicity of MOG Abs is raising attention as their titers are increased in juvenile MS, but also optic neuritis, and in a subset of patients with Neuromyelitis Optica (NMO). The crystallisable fragment (Fc) of Abs binds to distinct Fc receptors (FcRs) and complement. For human IgGs this tightly regulated process relies on the structural heterogeneity of the Ig-Fc domain that arises from differences among the four subclasses (IgG1, IgG2, IgG3 and IgG4), and the complex bi-antennary N-linked glycan attached to the conserved Asn297. The glycosylation of the Fc-domain is proposed to create two structural conformations, an “open” conformation that favors binding to type I FcR that include the activating receptors Fc?RIA (CD64), Fc?RIIA (CD32a), Fc?RIIC, Fc?RIIIA (CD16a) and Fc?RIIIB, as well as the inhibitory receptor Fc?RIIB (CD32b). Accordingly, the absence of sialic acids and low levels of galactosylation confer important pro-inflammatory properties to IgG by facilitating their binding to activating Fc?Rs. In addition, the absence of core-fucose improves the affinity to the Fc?IIIA, thereby enhancing antibody-dependent cellular cytotoxicity. In contrast, a “closed” conformation favours binding to type II FcRs that comprise the C-type lectin receptor CD209 (DC-SIGN) and CD23. These sialylated Abs are endowed with a potential anti-inflammatory effect. The overarching aim of this research project is to assess whether this diversity of Fc-effector functions impacts on the severity of chronic inflammatory diseases of the central nervous system. Our strategy is to study this impact functionally using a translational approach. In MS patients we will analyse the N-glycome and the Fc-glycome of the antibody response. In animal models of MS we will study the pathogenicity of Fc-glycovariants.