S2-106 Talimogene laherparepvec (T-VEC)-induced tumor oncolysate as a source of tumor antigens and maturation factors for blood-isolated myeloid dendritic cells

Dendritic cell (DC) tumor vaccines have been pursued for a long time. Recently blood-isolated “natural myeloid DC” were found to be more potent immunostimulants compared to monocyte-derived DC. Intratumoral administration of unmanipulated myeloid DC (myDC) in combination with the oncolytic virus Talimogene laherparepvec (T-VEC) is currently being investigated in patients with advanced melanoma (NCT03747744). In this project, we aim to mimic these cellular interactions in vitro and explore the effect of a T-VEC-induced tumor lysate on the phenotype and function of myDC.
CD1c (BDCA-1)+ myDC or the combination of CD1c (BDCA-1)+/CD141 (BDCA-3)+ myDC are isolated from peripheral blood mononuclear cells (from leukapheresis) using the CliniMACS Prodigy®. Melanoma cell lines are cultured in the presence of T-VEC at a different multiplicity of infection (MOI) to induce cell death. Inhibition of cell growth is visualized using IncuCyte® live cell imaging. Uptake of pHrodoTM-labelled dead tumor cells is assessed by IncuCyte® live cell imaging and flow cytometry. The phenotype of myDC is measured by flow cytometry and cytokine secretion by multiplex analysis using the MesoScale Discovery system. BDCA-1/3+ myDC that have taken up dying tumor cells will be co- cultured with autologous T cells electroporated with a T cell receptor recognizing a given tumor antigen in a specific HLA type to assess antigen processing and presentation.
Melanoma cell lines 624-mel and 938-mel are susceptible to T-VEC-induced oncolysis in a dose-dependent manner. BDCA-1+ and BDCA-1/3+ myDC are not susceptible to T-VEC infection, as no secretion of GM-CSF could be demonstrated, even when infected at a MOI of 10. Melanoma cells undergo immunogenic cell death, as evidenced by partial maturation of myDC upon co-culture with the supernatant of the dying cells. Currently we are assessing the cytokine secretion pattern of these myDCs cultured in the presence of supernatant of dying tumor cells. Next, we plan to investigate the phagocytosis of dying tumor cells by BDCA-1/3+ myDC, wherein we will evaluate if both myDC subtypes take up the dying tumor cells and which receptor(s) are implicated. Finally, we will assess whether the myDCs are capable of processing and cross-presenting phagocytosed tumor antigens to T cells.
In conclusion, T-VEC has no cytolytic effect on BDCA-1/3+ myDC and supernatant of T-VEC-lysed melanoma cells induces partial maturation of BDCA-1/3+ myDC. In ongoing experiments, we are investigating which myDC subtypes phagocytose T-VEC oncolysed tumor cells and which receptors are implicated in this process. Furthermore, we will evaluate whether myDCs are capable of processing and presenting phagocytosed tumor material to T cells.