Clustering-Induced, Clathrin-Mediated Endocytosis (CIC-ME) for Cancer Therapy

Abstract

Cancer is characterized by uncontrolled growth or proliferation of cells. Besides conventional cancer therapy, antibodies can be used to target tumor-related molecules like the epidermal growth factor receptor (EGFR) and the hepatocyte growth factor (HGF) receptor (Met). Unfortunately, this attenuates tumor growth instead of eliminating tumors completely. Improvement can be made by using so-called antibody-drug conjugates (ADC) that combine the tumor-specificity of antibodies with the cytotoxicity of therapeutic agents. The variable domain of the heavy chain of heavy chain only antibodies (VHH or nanobody) are small antibody fragments that display high binding affinity in combination with good tumor penetration. This thesis provides a detailed overview of the use of nanobodies in cancer-therapy. Nanobodies for the delivery of therapeutic molecules are referred to as Nanobullets. Because Nanobullets often act on intracellular processes, they should facilitate both tumor targeting and intracellular delivery. Therefore, a proper understanding on cellular uptake by tumor cells is important. Ligand-induced signaling of both EGFR and Met is silenced by a negative-feedback mechanism consisting of rapid internalization and subsequent degradation of the receptor-ligand complex. This process is mediated by ligand-induced post-translational modifications like phosphorylation, ubiquitination and acetylation. Recently, ligand-induced clustering of EGFR on the plasma membrane was also found to be involved in receptor internalization. In this thesis, the mechanism behind clustering-induced endocytosis of EGFR was studied in more detail. EGFR clustering induces kinase-independent, clathrin-mediated endocytosis (CIC-ME), which is followed by an unconventional, ubiquitin-independent trafficking towards lysosomal degradation. CIC-ME of EGFR requires a previously unrecognized role for the transmembrane dimerization motifs in receptor internalization. Subsequently, this knowledge was put to practice by developing different examples of Nanobullets for intracellular drug delivery. Nanobody-decorated, albumin-based nanoparticles (NANAPs) facilitated the intracellular release of the platinum-linked multikinase inhibitor 17864-Lx in the lysosomes of EGFR-expressing tumor cells. Similarly, anti-Met NANAPs constitute a novel type of biomaterial for lysosomal drug delivery in Met-expressing tumor cells. Finally, nanobody-photosensitizer (PS) conjugates against EGFR exhibited specific and strong cytotoxicity, which could be improved by intracellular delivery of the PS via CIC-ME (100% specific cell death, IC50 of ~1nM). The small size, high specificity and high potency of these Nanobullets make them promising candidates for further in vivo testing.