Interrogation de la plaque d'athérome par phage- display in vivo (une approche pour un ciblage moléculaire à l'aide d'anticorps humains armés pour l'imagerie et la thérapie)

L athérosclérose est une maladie inflammatoire complexe qui résulte dans la formation de plaques d athérome à risque de rupture. Le concept récent que ce risque soit lié au contenu de la plaque et non à sa taille se traduit par un nouvel impératif dans le domaine de l imagerie moléculaire et de la thérapie ciblée. Aujourd hui l avènement des approches protéomiques et de criblage à haut débit de banques combinatoires permet l identification à la fois de nouvelles cibles et d agents bioactifs. Notre objectif consiste à identifier in situ chez des modèles animaux malades, des fragments d anticorps capables de cibler spécifiquement les plaques vulnérables d athérome par phage display in vivo. Ces anticorps seront à la base de nouveaux formats moléculaires pour des études de diagnostic et thérapeutique.Atheroclerosis is a chronic and progressive inflammatory artery disease. These arteries have morphologically raised lesions: the atherosclerotic plaque. The early atherogenesis is characterized by formation of a lipid-rich core constituted by lipid-laden macrophages. These changes can thin the fibrous cap and render the plaque susceptible to rupture and thrombosis, and eventually if the thrombus occludes the vessels to an acute myocardial infarction. So, there still remains a great need to develop novel diagnosis and therapeutic tools to assess these molecular changes. We have applied a novel in vivo selection scheme to select Antibody-ligands that selectively home into atherosclerotic lesions induced in animal models. Such tissue-specific homing ligands may lead to the characterization of new up-regulated lesion-associated markers and open the way to diagnostic and therapeutic strategies based on non-invasive molecular imaging and selective drug delivery for early lesion detection.BORDEAUX2-Bib. électronique (335229905) / SudocSudocFranceF

Recherche de biomarqueurs pour l'imagerie moléculaire de l'athérosclérose

Ce travail comprend deux volets principaux, et concerne le développement d'anticorps monoclonaux humains ayant un intérêt en cardiologie. La première partie concerne la production, la purification et l'analyse de fragments d'anticorps humains reconnaissant l'intégrine a llbb3. La seconde partie décrit la sélection de scFvs humains spécifiques de la plaque d'athérome par phage display. Nous avons procédé à une sélection in vivo en injectant directement les phage-anticorps à un modèle de l'athérosclérose. Les scFvs ont été testés après le premier tour de sélection sur des extraits protéiques d'artères saines et athéromateusses. Cette approche soustractive au niveau protéique, nous a permis d'identifier deux anticorps spécifiques de protéines athéroscléreuses. Leur développement aurait une utilité : (1) en diagnostic en les conjuguant à des produits de contraste, de façon à marquer par IRM les lésions précoces (2) en thérapie génique pour un ciblage moléculaire amélioré.This thesis is divided in two parts. Its aim is the development of human monoclonal antibodies of cardiovascular interest. The first part involves the production, the purification and the biochemical analysis of human antibody fragments directed against the aIIbb3 integrin. The second part described the in vivo selection by phage display of human scFvs targeting atherosclerotic lesions. We have screened in vivo the atherosclerotic plaques of this model with two different combinatorial libraries of human antibodies. Our strategy is based on a substractive screening with normal and atherosclerotic protein-coated filters, allowing the detection of scFvs after one round of in vivo biopanning. This substractive aproach has permitted us to isolate two scFvs specific to atherosclerotic proteins. The development of such antibodies could be useful : (1) in diagnosis for the early detection of atherosclerotic lesions by MRI, (2) in gene therapy, to improve the molecular targeting.BORDEAUX2-BU Santé (330632101) / SudocSudocFranceF

Les anticorps monoclonaux dans le domaine de la santé

BORDEAUX2-BU Santé (330632101) / SudocSudocFranceF

Nanoparticles functionalised with an anti-platelet human antibody for in vivo detection of atherosclerotic plaque by magnetic resonance imaging.

Atherosclerosis is an inflammatory disease associated with the formation of atheroma plaques likely to rupture in which platelets are involved both in atherogenesis and atherothrombosis. The rupture is linked to the molecular composition of vulnerable plaques, causing acute cardiovascular events. In this study we propose an original targeted contrast agent for molecular imaging of atherosclerosis. Versatile USPIO (VUSPIO) nanoparticles, enhancing contrast in MR imaging, were functionalised with a recombinant human IgG4 antibody, rIgG4 TEG4, targeting human activated platelets. The maintenance of immunoreactivity of the targeted VUSPIO against platelets was confirmed in vitro by flow cytometry, transmission electronic and optical microscopy. In the atherosclerotic ApoE(-/-) mouse model, high-resolution ex vivo MRI demonstrated the selective binding of TEG4-VUSPIO on atheroma plaques. It is noteworthy that the rationale for targeting platelets within atherosclerotic lesions is highlighted by our targeted contrast agent using a human anti-αIIbβ3 antibody as a targeting moiety. Current clinical assessment of atherosclerotic plagues is suboptimal. The authors in the article designed functionalized superparamagnetic iron oxide nanoparticles with TEG4, a recombinant human antibody, to target activated platelets. By using MRI, these nanoparticles can be utilized to study the process of atheroma pathogenesis

Nanoparticle phagocytosis and cellular stress: involvement in cellular imaging and in gene therapy against glioma.

In gene therapy against glioma, targeting tumoral tissue is not an easy task. We used the tumor infiltrating property of microglia in this study. These cells are well adapted to this therapy since they can phagocyte nanoparticles and allow their visualization by MRI. Indeed, while many studies have used transfected microglia containing a suicide gene and other internalized nanoparticles to visualize microglia, none have combined both approaches during gene therapy. Microglia cells were transfected with the TK-GFP gene under the control of the HSP(70) promoter. First, the possible cellular stress induced by nanoparticle internalization was checked to avoid a non-specific activation of the suicide gene. Then, MR images were obtained on tubes containing microglia loaded with superparamagnetic nanoparticles (VUSPIO) to characterize their MR properties, as well as their potential to track cells in vivo. VUSPIO were efficiently internalized by microglia, were found non-toxic and their internalization did not induce any cellular stress. VUSPIO relaxivity r(2) was 224 mM(-1).s(-1). Such results could generate a very high contrast between loaded and unloaded cells on T(2)-weighted images. The intracellular presence of VUSPIO does not prevent suicide gene activity, since TK is expressed in vitro and functional in vivo. It allows MRI detection of gene modified macrophages during cell therapy strategies

(EN) LIPID BASED NANOCARRIER COMPOSITIONS LOADED WITH METAL NANOPARTICLES AND THERAPEUTIC AGENT (FR) COMPOSITIONS DE NANOVECTEURS À BASE DE LIPIDES CHARGÉES DE NANOPARTICULES MÉTALLIQUES ET D'UN AGENT THÉRAPEUTIQUE

(EN)The invention relates to non-polymeric lipid-based nanocarrier compositions loaded with metal nanoparticles and at least one therapeutic agent, useful.as agents for transportation, vectorization, cellular delivery cellular targeting or cellular localization of at least one therapeutic agent. (FR)L'invention se rapporte à des compositions de nanovecteurs non polymères à base de lipides, chargées de nanoparticules métalliques et d'au moins un agent thérapeutique, utiles en tant qu'agents pour le transport, la vectorisation, l'administration cellulaire, le ciblage cellulaire ou la localisation cellulaire d'au moins un agent thérapeutique

A nano-emulsion platform functionalized with a fully human scFv-Fc antibody for atheroma targeting: towards a theranostic approach to atherosclerosis

Atherosclerosis is at the onset of the cardiovascular diseases that are among the leading causes of death worldwide. Currently, high-risk plaques, also called vulnerable atheromatous plaques, remain often undiagnosed until the occurrence of severe complications, such as stroke or myocardial infarction. Molecular imaging agents that target high-risk atheromatous lesions could greatly improve the diagnosis of atherosclerosis by identifying sites of high disease activity. Moreover, a “theranostic approach” that combines molecular imaging agents (for diagnosis) and therapeutic molecules would be of great value for the local management of atheromatous plaques. The aim of this study was to develop and characterize an innovative theranostic tool for atherosclerosis. We engineered oil-in-water nano-emulsions (NEs) loaded with superparamagnetic iron oxide (SPIO) nanoparticles for magnetic resonance imaging (MRI) purposes. Dynamic MRI showed that NE-SPIO nanoparticles decorated with a polyethylene glycol (PEG) layer reduced their liver uptake and extended their half-life. Next, the NE-SPIO-PEG formulation was functionalized with a fully human scFv-Fc antibody (P3) recognizing galectin 3, an atherosclerosis biomarker. The P3-functionalized formulation targeted atheromatous plaques, as demonstrated in an immunohistochemistry analyses of mouse aorta and human artery sections and in an Apoe−/− mouse model of atherosclerosis. Moreover, the formulation was loaded with SPIO nanoparticles and/or alpha-tocopherol to be used as a theranostic tool for atherosclerosis imaging (SPIO) and for delivery of drugs that reduce oxidation (here, alpha-tocopherol) in atheromatous plaques. This study paves the way to non-invasive targeted imaging of atherosclerosis and synergistic therapeutic applications.Translational Research and Advanced Imaging LaboratoryOptimization of therapeutic monoclonal antibodies development Better antibodies, better developed AND better use

Platelet function and microparticles levels for patients with paroxysmal and persistent atrial fibrillation including during acute episodes

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