Epidermal growth factor receptor: Target for delivery and silencing

Abstract

Epidermal growth factor receptor in cancer therapy Recently, cancer research has been able to identify molecular targets that are specific for (or highly expressed by) cancer cells. These molecular targets serve as models for the development of rationally designed anticancer drugs that target important mediators of tumour development. Also, some of these molecular targets have been explored for targeted drug delivery, to ensure that anticancer drugs are specifically delivered to the tumours. The epidermal growth factor receptor (EGFR) is an extensively investigated growth factor and a recognized target for cancer therapy. Many human epithelial cancers overexpress EGFR and the dysregulated EGFR signalling is associated with uncontrolled cell proliferation, inhibition of apoptosis, stimulation of angiogenesis, tissue invasion and metastasis. Two main strategies have been employed to inhibit EGFR: tyrosine kinase inhibitors (TKIs) and monoclonal antibodies (mAbs), some of them have already been approved for clinical use. EGFR: target for silencing A different strategy to inhibit EGFR was employed in this thesis. Here, the expression of EGFR protein was interrupted by use of small interfering RNA (siRNA). For efficient gene silencing, siRNA must be delivered intact into the target cell cytoplasm, where RNA interference (RNAi) takes place. Carriers, such as Lipofectamine, are generally used to promote cellular uptake. However, when internalization occurs via endocytosis, the endosomal escape becomes one of the limiting steps for efficient knockdown of the target protein. In this thesis, two different methods were successfully employed to facilitate the endosomal escape of siRNA: an influenza-derived fusogenic peptide and photochemical internalization (PCI). PCI combines a photosensitizer with light to induce photochemical reactions that destabilize endosomal membranes and allow the release of entrapped molecules. PCI was also successfully applied in vivo for enhancing the endosomal escape of anti-EGFR siRNA-Lipofectamine, delivered by intratumoral injection. This study demonstrates that also in vivo endosomal escape is a limiting step that can be facilitated by PCI, improving silencing efficiencies. Local administration of siRNA is not always possible and, therefore, novel targeted drug delivery systems are developed for cancer-specific molecular targets, to direct siRNA and other therapeutic molecules to cancer cells. EGFR: target for delivery Liposomes are attractive delivery systems that have been employed for delivery of a wide range of therapeutic molecules. Liposomes surface has been modified with shielding polymers and targeting ligands that increase their circulation time in the bloodstream and mediate specific target-cell interactions, respectively. In this thesis, a novel targeted drug delivery system was described: anti-EGFR nanobody-liposomes. Nanobodies are the smallest functional antigen binding immunoglobulin fragments that have been isolated from heavy chain-only antibodies, first discovered in camelids. Nanobodies are 10 times smaller than conventional antibodies, though perfectly capable of binding to their antigens with high specificity and efficiency. This new drug delivery system can induce a remarkable downregulation of EGFR, which combined with encapsulated therapeutic molecules offers attractive properties for use in combined anticancer strategies. In conclusion, this thesis has addressed the inhibition of EGFR as therapeutic strategy. EGFR was inhibited by silencing of protein expression with siRNA and by downregulation induced by nanobody-liposomes.