Raimond Heukers

Cancer is characterized by uncontrolled growth or proliferation of cells. Cancer cells have acquired self-sufficiency in growth signalling and/or unlimited replication. Besides surgery or chemo- or radiation therapy, cancer therapies can focus on targeting specific tumor-related molecules. Examples of targeted therapies include small molecule inhibitors, small interfering RNAs (siRNA) and immunotherapy, the latter of which involves the use of antibodies that either activate the immune system or block tumor-related signalling cascades. Unfortunately, antibody treatments are often not sufficient to remove a tumor completely and more aggressive measures are welcome. A way to improve the specific anti-tumor activity is using so-called antibody-drug conjugates (ADC). Combining the tumor-specificity of an antibody with the cytotoxicity of a therapeutic agent enhances the potency of the antibody and reduces the off-target toxicity of chemotherapy.

For optimal tumor targeting and penetration, specificity, binding affinity and size of the targeting ligand matter. In that respect, conventional antibodies are relatively large and therefore display poor tumor penetration/distribution. This is why several types of smaller antibody fragments or scaffold proteins have been developed in the last decades. An example of such a small antibody fragment is the variable domain of the heavy chain of heavy chain only antibodies (VHH or nanobody). An overview of the use of nanobodies for cancer-therapy is given in Chapter 2. Nanobodies that are being used for the delivery of therapeutic molecules can be referred to as so-called “Nanobullets”. Because many therapeutic, and in this case cytotoxic compounds require an intracellular localization for their activity, ideal Nanobullets should not only target to tumor cells but should also facilitate intracellular delivery. In order to achieve an efficient intracellular delivery of Nanobullets, a basic understanding of the biology behind cellular uptake is essential.

The epidermal growth factor receptor (EGFR) and the hepatocyte growth factor (HGF) receptor (Met) are receptor tyrosine kinases that are overexpressed in many cancers, making them attractive targets for cancer therapy. EGFR serves as a model receptor and its activation and internalization are well studied. Ligand-induced signalling of both receptors is silenced by a negative-feedback mechanism consisting of rapid internalization and subsequent degradation of the receptor-ligand complex. Internalization of EGFR is regulated by many ligand-induced post-translational modifications like phosphorylation, ubiquitination and acetylation. However, these modifications do not explain the observed internalization completely. Because ligand-binding also results in the formation of higher order clusters of EGFR on the plasma membrane, clustering was recently suggested to be involved in receptor internalization also. However, the mechanism behind clustering-induced internalization is still unclear.

In this thesis, the mechanism behind clustering-induced endocytosis was studied using multi-epitopic antibody constructs directed against the extracellular part of EGFR (Chapter 3). EGFR clustering via biparatopic (binding two non-overlapping epitopes) VHH constructs results in the clustering-induced, clathrin mediated endocytosis (CIC-ME) of the receptor-antibody complex. CIC-ME is kinase-independent and therefore does not activate signalling cascades towards growth and proliferation. CIC-ME also revealed a previously unrecognized role for the transmembrane dimerization motifs of EGFR in internalization. Subsequently, EGFR clustering via tri-epitopic antibody-fibronectin constructs induces unconventional, ubiquitin-independent trafficking of EGFR towards lysosomal degradation (Chapter 4). The knowledge of achieving kinase-independent internalization into tumor cells was put to practice by developing different internalizing Nanobullets for intracellular drug delivery. Nanobody-decorated, albumin-based nanoparticles (NANAPs) were generated for the intracellular release of the platinum-linked multikinase inhibitor 17864-Lx in lysosomes of EGFR-expressing tumor cells (Chapter 5). Anti-Met NANAPs displayed similar internalization, lysosomal trafficking and degradation and could therefore serve as a novel biomaterial for drug delivery into Met-expressing cells (Chapter 6). Finally, nanobodies were turned into Nanobullets for photodynamic therapy (PDT) by conjugating them to the relatively hydrophilic photosensitizer IRDye700DX (Chapter 7). Illumination of such photosensitizers (PS) with near-infra red light locally generates the very toxic 1O2. The nanobody-PS conjugates display a specific and strong anti-tumor activity, which can be improved even further by employing internalizing, biparatopic Nanobullets (100% specific cell death, IC50 of ~1nM).

Taken together, this thesis (I) describes the use of nanobodies for cancer therapy, (II) provides a fundamental background behind clustering-mediated uptake and trafficking in tumor cells and (III) gives three examples of nanobullets for cancer therapy. Because of their small size, high specificity and high potency, internalizing nanobody-PS conjugates are considered to be the most promising examples of Nanobullets, which clearly deserve further in vivo testing.