Les stratégies thérapeutiques mises en place contre le cancer ont de nos jours besoin de nouveaux médicaments, à la fois plus actifs que ceux déjà existants et induisant moins d’effets secondaires. Ces nouvelles stratégies visent à cibler spécifiquement les cellules cancéreuses. Parmi ces stratégies, ces travaux de thèse concernent la vectorisation active, à l’aide d’un vecteur protéique dérivé de la toxine de Shiga, STxB. STxB reconnait spécifiquement son récepteur biologique Gb3, surexprimé à la surface des cellules cancéreuses humaines. Ce projet de recherche porte sur la conception et la synthèse de conjugués, combinant STxB et un agent cytotoxique. Le linker chimique, qui relie ces deux espèces, a été soigneusement conçu pour respecter les deux critères suivants : être suffisamment stable et néanmoins pouvoir être clivé pour libérer l’agent cytotoxique une fois les cellules cancéreuses atteintes. Un premier linker a été construit autour du motif mercaptoethanol, lié au vecteur STxB par une liaison disulfure. La libération de l’agent cytotoxique peut donc être initiée par un réducteur biologique comme le glutathion, puis par une étape d’auto-immolation. Ce linker a été appliqué à deux composés cytotoxiques très puissants, dérivés de l’auristatine, et a conduit à des résultats prometteurs in vitro. La labilité de la liaison ester à pH acide a également été mise à profit dans l’élaboration de deux linkers, conçus autour de motifs glutamate et thréoninate. L’utilisation d’un agent cytotoxique modérément puissant a été l’occasion de développer une stratégie de multivalence, consistant à augmenter la charge d’agents cytotoxiques sur STxB. Une autre option a été de considérer les nano-batônnets d’or comme une plate-forme nanométrique multimodale, capable de lier plusieurs milliers d’agents cytotoxiques et STxB. Enfin l’incorporation d’une séquence peptidique, connue pour être substrat d’une protéase, a donné lieu à une troisième étude, reposant sur un linker clivable plus sélectivement. Plusieurs linkers ont été étudiées, selon qu’ils libèrent l’agent cytotoxique sous sa forme native ou non. / We need new therapeutic strategies to treat cancerous patients by the discovery of new drugs that would be more active than those existing and especially assigning fewer side effects. These new therapies aim to specifically target cancer cells. Among the strategies for cancer targeting, we investigated drug-targeted strategies using a proteic carrier, STxB, derived from Shiga toxin. This protein recognizes specifically its biological receptor Gb3, which is over-expressed on human cancer cells. This work consisted in the design and synthesis of conjugates combining STxB and a cytotoxic drug. The chemical linker binding these two moieties was carefully designed in order to fit requirements of both stability and ability to trigger a drug-delivery. A first linker was designed around a mercaptoethanol core, able to be conjugated to STxB by a disulfide bond. This constitutes a drug-delivery trigger, activated by a biological reducing agent such as glutathion, and followed by a self-immolative step. Two highly potent conjugates of auristatin derivatives were obtained and showed promising results in vitro. The ester bonds lability in acidic pH was exploited for the design of two amino acid based linker. With the aim of increasing the ratio of drug on STxB, we investigated several multivalent linkers. Another option was to consider gold nanorods as a nanometric platform, able to carry thousands of drugs and STxB. The incorporation of a protease substrate to produce an enzyme-cleavable linker was investigated. Several spacers, which induced release of the drug under native form or under prodrug form, were designed and tested.

Colorectal cancer is the second most common cause of cancer-related deaths in the United States, with the relative 5-year survival rate for distant stage cancer being only 14%. The most common treatment for colorectal cancer is with chemotherapeutic drugs; however, the discovery of these drugs is costly, time-consuming, and often requires the use of animal models that do not yield results that translate to clinical trials. Due to these shortcomings, researchers seek to develop physiologically relevant in vitro tumor models that more accurately mimic the tumor microenvironment for cheaper and faster high-throughput drug screening. The aim of this research was to develop a colorectal cancer tumor model co-cultured with endothelial and stromal cells, followed by validation with clinically relevant chemotherapeutic agents within microfluidic devices. The first experiment consisted of a lipofection of fibroblasts to yield fluorescently tagged cells that could be later imaged using a fluorescence microscope. The next experiment consisted of a co-culture of tumor, endothelial, and fibroblast cells at varying densities in a twodimensional (2D) culture to determine the optimal plating densities that would yield quantifiable tumor and endothelial network formation. The following experiment used these optimal densities to test the effects of the chemotherapeutic agents oxaliplatin and SN38 on the tumor and endothelial cells in 2D. After the various densities and drug concentrations were tested in 2D, the model was introduced into microfluidic devices. The first experiment in the devices was similar to the first experiment plated in 2D, as it involved the establishment of optimal plating densities of all three cell types within the devices. Similarly, the goal of this experiment was to yield quantifiable tumor and endothelial network formation within the devices. The final experiment performed in this research was the introduction of oxaliplatin and SN38 to the optimized densities v of cells determined from the previous experiment, with the aim of evaluating the effects of these chemotherapeutic agents on the tumor and endothelial cells within microfluidic devices. The two experiments plated in 2D established plating densities to be tested in the devices. These experiments also showed that increasing drug concentrations resulted in reduced tumor count and size and revealed no disruption in the endothelial networks when exposed to oxaliplatin concentrations as high as 50 µM. The final two experiments in microfluidic devices revealed that endothelial network formation is not yet possible within the devices with the current protocols, but that tumor cells still showed dose-dependent responses to drug exposure as they did in 2D. Due to the lack of network formation in this device model, future work is required to allow for endothelial cell organization into networks, to further increase the physiological relevancy of this model to in vivo tumor conditions.

Pas de résumé / Recently targeted therapies appeared as attractive alternatives to classical antitumoral treatments. The approach, developed on the concept of targeting drug to cancer cells, aims to spear normal tissues and decrease the side effects. This doctoral dissertation focuses on developing new anticancer targeted treatments in the field of chemotherapy and cancer immunotherapy by exploiting an original targeting moiety, the B subunit of Shiga toxin (STxB). Its specific properties, such as, recognition with its receptor Gb3 overexpressed in cancer cells or in antigen-presenting cells, its unconventional intracellular trafficking, guided the choice of this protein as targeting carrier. This project is based in the use of copper-free Huisgen [3+2] cycloaddition as a coupling method, which led to successful preparation of various conjugates for their respective applications. The concept was first validated by STxB-biotin conjugate. The high yield of the reaction and the compatibility between the targeting carrier and the chemical ligation promoted the design of conjugates for chemotherapy and immunotherapy. Two therapeutical optimizations of previously developed strategy in STxB drug targeting delivery were investigated: synthesis of multivalent drug-conjugates and synthesis of conjugates containing a highly potent anticancer agent. Both approaches exploited three anticancer agents: SN38, Doxorubicin and Monomethyl auristatin F. The disulfide spacer, combined with various self-immolative systems, insured drug release. Two cytotoxic conjugates STxB–doxorubicin (STxB-Doxo) and STxB-monomethyl auristatin F (STxB-MMAF) were obtained in very high yield and demonstrated strong tumor inhibition activity in the nanomolar range on Gb3-positive cells. Based on the results the STxB-MMAF conjugate was investigated on a mouse model. The project aimed also to develop STxB bioconjugates for vaccine applications. Previous studies used B subunit as a targeting carrier coupled to an antigenic protein in order to induce a more potent immune response against cancer. The conjugates were prepared using a commercial linker, requiring modifying the antigen at first place, or by oxime ligation, where slightly acidic conditions promoted the coupling. Thus, the work presented herein proposed an alternative ligation via copper-free click chemistry especially for more sensitive antigenic proteins. Various types of conjugates were synthesised and investigated for their immune stimulation properties. The STxB targeting strategy was also applied to the development of a new vaccine based on coupling the targeting carrier to alpha-GalCer, one of the most potent immune stimulating agents known. The work focused on the synthesis of functionalised alpha-Galcer with an azide handle.

Ces travaux de thèse ont porté sur l'utilisation d'un nouveau vecteur particulaire lipidique (LNCs) destiné à la voie orale. Ce système devrait permettre d'encapsuler une molécule anticancéreuse et améliorer sa biodisponibilité. Notre premier axe de recherche a été d'étudier les propriétés in vitro des LNCs encapsulant du paclitaxel en vue d'une administration par voie orale. La stabilité in vitro dans les milieux gastro-intestinaux simulés a été démontrée. De plus, les études de transport à travers l'épithélium intestinal (modèle cellulaire Caco-2) ont montré que les LNCs permettaient d'augmenter le transport du paclitaxel grâce à un mécanisme d'endocytose actif clathrine- et cavéole-dépendant situé dans le microenvironnement de la P-gp. Nous avons aussi mis en évidence les interactions entre l'endocytose des nanocapsules et l'activité de la P-gp. Notre second axe de recherche a porté sur l'application des LNCs pour encapsuler un nouvel anticancéreux : le Sn38 qui est le métabolite actif de l'irinotécan. Une formulation modifiée de nanocapsules lipidiques a été obtenue en utilisant un co-tensioactif et une huile hydrophile en plus des excipients standards. Les propriétés physico-chimiques et la stabilité, l'absorption epithéliale et la toxicité in vitro de cette formulation ont ensuite été vérifiées. Enfin, une activité antitumorale de courte durée après administration orale chez la souris a été observée. Ce travail fait donc la preuve de concept de l'absorption d'anticancéreux après administration par voie orale à l'aide de vecteurs colloïdaux. Néanmoins, certaines optimisations restent encore à réaliser pour prévoir une application clinique.