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Nanobody-targeted photodynamic therapy (NB-PDT) has been developed as a potent and more tumor-specific approach compared to conventional PDT [1]. Interestingly, conventional PDT is able to induce immunogenic cell death, characterized by the exposure/release of damage associated molecular patterns (DAMPs) from dying cells, which can result in an anti-tumor immune response. In this study, we aim at understanding the first steps towards immune-modulation triggered by NB-PDT. For this, the EGFR-targeted NB 7D12 was conjugated to the photosensitizer IRDye700DX and used to perform NB-PDT on EGFR-overexpressing A431 tumor cells, exposed to 30 minute incubation with the conjugates and followed by a light dose of 10 J/cm2. The cytoplasmic DAMP HSP70 was detected on the cell membrane after mild NB-PDT (1 nM conjugate), while it was detected in the medium after highly toxic NB-PDT (25 nM conjugate). The nuclear DAMP HMGB1 was found in the cell cytoplasm under both NB-PDT conditions. Furthermore, cells treated with highly toxic NB-PDT showed an increased release of the pro-inflammatory cytokines IL-1β and IL-6, and a decreased release of pro-tumoral IL-8. Lastly, medium collected from tumor cells treated with highly toxic NB-PDT was able to induce the phenotypic maturation of a dendritic cell line, as indicated by the upregulation of CD40, CD86 and MHCII. Altogether, these results are the first to indicate immune-modulation by NB-PDT, which can be exploited to increase NB-PDT efficacy even further.
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