Mechanism of triterpenoid saponin mediated augmentation of saporin based immunotoxin cytotoxicity

Immunotoxins are hybrid molecules comprised of a protein toxin molecule and a monoclonal antibody that selectively delivers the toxin to antigen expressing cells. These therapeutics are under investigation as agents for the treatment of cancers, but their clinical development has been hampered by a number of off-target, dose limiting toxicities particularly vascular leak syndrome. A number of plant derived triterpenoid saponins significantly augment the cytotoxicity of saporin based immunotoxins, possibly by increasing the endolysosomal escape of the toxin into the cytosol. This would have the effect of quantitatively increasing toxin availability to target ribosomes whilst avoiding its degradation by lysosomes. This may help to widen the therapeutic window of immunotoxin therapy. The primary objective of the work presented in this thesis was to investigate the mechanism(s) by which saponin augments immunotoxin cytotoxicity against lymphoma and leukaemia cells. The work focuses primarily on the potential roles that different endocytic processes and the endolysosomal escape of the immunotoxin into the cytosol play in the augmentation process. The work in this thesis presents evidence for the possible involvement of various endocytic pathways in saponin mediated augmentation of immunotoxin cytotoxicity and suggests that an acidic endolysosomal lumen is a requirement for augmentation. Also presented is evidence that membrane repair processes are not involved in the augmentation of immunotoxin cytotoxicity. Part of this thesis describes the development of a novel flow cytometric method to measure the endolysosomal escape of a fluorescently labelled immunotoxin into the cytosol. This assay revealed that treatment of target cells with saponin increased the endolysosomal escape of the immunotoxin into the cytosol and allowed a quantitative assessment of the effects of pharmacological inhibitors of endocytic processes on this escape. The effect of these agents on the saponin mediated endolysosomal escape of the immunotoxin correlated with their effect on the augmentation of immunotoxin cytotoxicity by saponin. This offers further evidence that endolysosomal escape is at least one of the mechanisms by which augmentation occurs