Nanobody-targeted photodynamic therapy: exploring cancer targets and the combination with endothelial cell targeting

Abstract

Photodynamic therapy (PDT) is a minimally invasive approach for cancer treatment. In this approach three essential elements are needed to induce local cytotoxicity: a light-activatable photosensitizer (PS), light of a specific wavelength, and oxygen molecules. The activated PS can transfer energy to oxygen and subsequently form cytotoxic reactive oxygen species (ROS), among other reactive molecular species which can destroy tumor cells, damage tumor vasculature, and also induce immune responses. In Chapter 2, the potential of G protein-coupled receptors (GPCRs) as target for nanobody-targeted PDT was presented, by using the HCMV-encoded chemokine receptor US28. US28, one of the four HCMV-encoded viral GPCRs, has been detected in multiple tumors of HCMV-positive patients, including gliomas. Nanobody-targeted PDT specifically induced cell death in the US28 expressing glioblastoma cells in 2D and 3D cell models, upon illumination with near-infrared light. In parallel, we also described the characterization of a nanobody-PS conjugate specific for the hepatocyte growth factor receptor (HGFR), also known as c-Met or Met, as an alternative target (Chapter 3). Overexpression of Met has been reported in large number of carcinomas, sarcomas, haematopoietic malignancies and other neoplasms. We showed that the nanobody-PS conjugate, induced cell death specifically and effectively in Met overexpressing cancer cells. In Chapter 4, further insights were obtained into nanobody-targeted PDT mechanism of tumor destruction, focusing on vascular effects in more detail. We investigated the biodistribution of two different formats of nanobodies targeting EGFR (7D12 as monovalent and 7D12-9G8 as biparatopic), in vivo using intravital microscopy. Subsequently, cellular responses and vascular effects of EGFR-targeted PDT were investigated. Both nanobody-PS conjugates showed more fluorescence in tumor than in distant normal tissue at 1 or 2 h after administration. 7D12-9G8-PS showed an intense, membrane localized, fluorescence pattern in tumor cells, while 7D12-PS fluorescence was more diffuse. After illumination at 1 h post injection, distinct changes to the morphology of tumor cells were observed in the GFP fluorescence images recorded in time. Importantly, besides the tumor response after PDT, vascular responses were also observed. The proportional area of tumor that showed either lack of flow or reduced flow 2 h post PDT was similar for both conjugates when light was applied 1 h after administration. The results showed that the overall acute response to EGFR-targeted NB-PS mediated PDT is a complex mixture of tumor cell response and vascular effects. As efficacy of targeted PDT can be hampered by heterogeneity of target expression and/or moderate/low target expression levels, we explored the possibility of combined targeting of endothelial and cancer cells in vitro. For this, in Chapter 6, we developed nanobodies binding to the mouse VEGFR2, which is overexpressed on tumor vasculature, and combined these with nanobodies specific for the cancer cell target EGFR. The cytotoxicity of these conjugates in monocultures and in co-cultures with cancer cells showed that the anti-VEGFR2 conjugates are specific and potent PDT agents. Nanobody-targeted PDT in co-culture of endothelial and cancer cells showed improved efficacy, when VEGFR2 and EGFR targeting nanobodies were applied simultaneously, compared to the separate treatments.