Merkel Cell Carcinoma (MCC) is an aggressive neuroendocrine skin cancer that is highly immunogenic. Approximately 80% of MCC tumors are virus positive (VP-MCC) and express Merkel cell polyomavirus (MCPyV) T antigens that drive oncogenesis. As VP-MCC have a low mutation burden with few predicted neoantigens, viral oncogenes are thought to be the primary target for anti-cancer immunity. Immune checkpoint inhibitors improve MCC survival, yet <50% of patients have durable benefit. Patients who fail checkpoint inhibition or who have immune dysfunction may benefit from adoptive cellular therapy using virus reactive T cells from HLA-matched donors. As the risk of graft versus host disease is low with virus-specific T cells, this approach is being investigated for other viral diseases. We set out to generate MCPyV specific T cells from healthy donors for potential adoptive immunotherapy. Naïve and memory T cells from donors were magnetically isolated prior to stimulation with autologous monocyte-derived dendritic cells pulsed with 15-mer overlapping peptide libraries of MCPyV T antigens. T cells were serially re-stimulated with peptide-pulsed irradiated, autologous PHA-blasts up to five times. Cultures were maintained in either standard growth cytokines (IL-2, IL-7, and IL-15) or a pro-inflammatory cytokine cocktail. T cell phenotype and reactivity were evaluated by flow cytometry. We found that standard cytokines were unable to promote growth of virus-specific mature T cells. Altering growth conditions to include IL-1β, IL-2, IL-6, IL-7, IL-15, IL-21, IL-23, and TGFβ promoted expansion of CD4+ T cells specific for MCPyV T antigen peptides. These cells produced TNFα and upregulated the activation markers CD154 and CD137 upon cognate antigen exposure. Cells were generated without bias for particular HLAs. These results suggest that peptide-based expansions may be a suitable platform to generate allogeneic adoptive T cell immunotherapies for VP-MCC patients.

Generating Merkel cell polyomavirus reactive T cells from healthy donors for adoptive immunotherapy