Dual Targeting of Endothelial and Cancer Cells Potentiates In Vitro Nanobody-Targeted Photodynamic Therapy

SIMPLE SUMMARY: Photodynamic therapy (PDT) eradicates tumors by the local activation of a photosensitizer with appropriate light. Although very promising, its clinical application is limited by the sub-optimal tumor specificity of the photosensitizer. Nanobody-targeted PDT was developed to improve specificity and efficacy of the treatment, using nanobodies that target the photosensitizer specifically to proteins overexpressed on cancer cells. Notably, intratumoral heterogeneity and/or low/moderate expression of some of these proteins hamper the efficacy of targeted PDT. To circumvent these, we explored the combination of cancer and endothelial cell targeting using nanobodies. For that, we developed nanobodies targeting mouse VEGFR2, which is mainly overexpressed on tumor vasculature, and we combined these with an EGFR targeted nanobody. Dual targeting of mouse endothelial and human cancer cells in a co-culture setup, using nanobody-photosensitizer conjugates, showed improved efficacy. In vivo follow up studies will reveal the full potential of this promising approach. ABSTRACT: Photodynamic therapy (PDT) induces cell death through local light activation of a photosensitizer, although sub-optimal tumor specificity and side effects have hindered its clinical application. We introduced a new strategy named nanobody-targeted PDT in which photosensitizers are delivered to tumor cells by means of nanobodies. 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. 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 nanobodies were conjugated to the photosensitizer IRDye700DX and specificity of the newly developed nanobodies was verified using several endothelial cell lines. The cytotoxicity of these conjugates was assessed in monocultures and in co-cultures with cancer cells, after illumination with an appropriate laser. The results show that the anti-VEGFR2 conjugates are specific and potent PDT agents. Nanobody-targeted PDT on co-culture of endothelial and cancer cells showed improved efficacy, when VEGFR2 and EGFR targeting nanobodies were applied simultaneously. Altogether, dual targeting of endothelial and cancer cells is a promising novel therapeutic strategy for more effective nanobody-targeted PDT.