Cardiovascular disease (CVD) and particularly atherosclerosis is a leading cause of morbidity in the developed world. Atherosclerosis and the rupture of vulnerable atherosclerotic plaque cause 70% of deaths from CVD. The progression of atherosclerosis has been identified as a pathological inflammatory process. Targeting atherosclerotic drug therapies to inflammatory markers has emerged as an important and growing research area. The adhesion molecule CD44 has been implicated in the onset and build-up of atherosclerotic lesions throughout the course of development. The research in this dissertation is aimed at targeting anti-inflammatory therapy to activated vascular endothelium with directed with an anti-CD44 antibody, IM7, conjugated to a non ionic surfactant vesicle (niosome) drug carrier. The IM7 conjugated immunoniosome has been shown to bind to endothelial and synovial lining cells in vitro. The preliminary research is involved with the development of the drug delivery vesicle, and the antibody linkage chemistry, along with an analysis of vesicle characteristics and stability. A novel linking chemistry using polyoxyethylene sorbitan monostearate and cyanuric chloride allows antibodies to be conjugated to vesicle surface polymer groups without prior derivatization. Subsequent research tested the resulting 'immunoniosome's' ability to bind to target antigens with selectivity and specificity. Bovine aortic endothelial cells activated with cytokines provide a model of inflammation. Analysis of binding was done through fluorescent and scanning electron microscopy. In vivo uptake of vesicles at sites of inflammation is size dependent. In order to overcome this barrier to uptake, niosome suspensions were thermally extruded to create uniform 200 nm vesicles. Further analysis of the efficacy of the system looked at live cell uptake of the immunoniosomes measured by confocal and transmission electron microscopy. Preparation for in vivo murine studies required that the antibody component was modified to counteract the immune response. Finally, the conjugation of antibody fragments to niosomes and the binding and uptake of the vesicles in a live endothelial cell model is evaluated. A viable drug delivery particle showing binding and cellular uptake capabilities in inflammatory cells was produced by this research using a novel surfactant-antibody linker.

Les anticorps thérapeutiques sont des molécules de choix pour le traitement standard de nombreuses formes de cancers. Leur application est à ce jour restreinte au compartiment extracellulaire à cause de leur taille trop importante qui les empêche de traverser la membrane cellulaire. Comme la plupart des cibles thérapeutiques du cancer semblent être situées dans le milieu intracellulaire, ce serait un plus de pouvoir exploiter les propriétés des anticorps dans les cellules pour étudier et perturber l’activité de ces cibles. Néanmoins, l’utilisation des anticorps dans le milieu intracellulaire constitue un véritable challenge, notamment à cause de la membrane cellulaire et de l’environnement réducteur du cytoplasme. L’ensemble des travaux de thèse présentés dans ce manuscrit ont permis d’établir les bases de plusieurs stratégies innovantes d’immunociblage intracellulaire et de mettre en lumière l’importance des différentes étapes de validation d’anticorps ou de fragments dérivés utilisés comme anticorps intracellulaires. La vectorisation d’anticorps complets par électroporation, le développement d’un intracorps bispécifique original anti-PCNA et la mise au point d’une méthode de mutagenèse inspirée de l’hypermutation somatique constituent les principales avancées apportées par ce travail dans le domaine de la recherche technologique en immuno biotechnologie. / Therapeutic antibodies are interesting molecules used to treat numerous pathologies such as cancer. Because of their size, their application is currently limited to the extracellular space. Indeed, antibodies cannot cross the cell membrane. Almost all therapeutic targets in cancer seem to be located inside cells, it would be beneficial to take advantage of antibodies in cells in order to neutralize the activity of these targets. The use of antibodies inside the cells is a real challenge, because of the cell membrane and the reducing environment of the cytoplasm. Several strategies of intracellular immunotargeting are presented in this thesis.