The Potential of Nanobody-Targeted Photodynamic Therapy to Trigger Immune Responses

Nanobody-targeted photodynamic therapy (NB-PDT) has been recently developed as a more tumor-selective approach rather than conventional photodynamic therapy (PDT). NB-PDT uses nanobodies that bind to tumor cells with high affinity, to selectively deliver a photosensitizer, i.e., a chemical which becomes cytotoxic when excited with light of a particular wavelength. Conventional PDT has been reported to be able to induce immunogenic cell death, characterized by the exposure/release of damage-associated molecular patterns (DAMPs) from dying cells, which can lead to antitumor immunity. We explored this aspect in the context of NB-PDT, targeting the epidermal growth factor receptor (EGFR), using high and moderate EGFR-expressing cells. Here we report that, after NB-PDT, the cytoplasmic DAMP HSP70 was detected on the cell membrane of tumor cells and the nuclear DAMP HMGB1 was found in the cell cytoplasm. Furthermore, it was shown that NB-PDT induced the release of the DAMPs HSP70 and ATP, as well as the pro- inflammatory cytokines IL- 1β and IL-6. Conditioned medium from high EGFR-expressing tumor cells treated with NB-PDT led to the maturation of human dendritic cells, as indicated by the upregulation of CD86 and MHC II on their cell surface, and the increased release of IL-12p40 and IL-1β. Subsequently, these dendritic cells induced CD4+ T cell proliferation, accompanied by IFNγ release. Altogether, the initial steps reported here point towards the potential of NB-PDT to stimulate the immune system, thus giving this selective-local therapy a systemic reach.