Virus-Positive Merkel Cell Carcinoma Specific T Cells Are Robustly Generated from Healthy Donor Blood

Abstract

Merkel cell carcinoma (MCC) is a rare and aggressive neuroendocrine skin tumor with a five-year relative survival rate of 20%. Clonal integration of Merkel cell polyomavirus (MCPyV) drives approximately 80% of MCC cases in the United States of America. Functional T cell immunity is central for clearance of MCC, however immune checkpoint inhibitors benefit only half of advanced virus positive (VP-MCC) patients. Non-responding patients may lack endogenous functional tumor-specific T cells, therefore supplementation by adoptive T cell transfer may improve patient outcomes. Patients with dysfunctional endogenous T cells or who are HLA-mismatched to available transgenic T cell receptor therapies are ineligible to receive current VP-MCC specific adoptive T cell transplants. To serve these patients, we developed a methodology to generate MCPyV-specific T cells from healthy donors and evaluated their suitability for adoptive T cell transfer. We expanded MCPyV TAg specific T cells from donor blood using monocyte derived dendritic cells (moDCs) pulsed with synthetic overlapping peptide libraries spanning MCPyV TAg. Pulsed moDCs were co-cultured with autologous T cells in the presence of cytokines for 14 days. In concordance with previous literature, expanded healthy donor MCPyV-specific T cells were exclusively CD4+. Expansion of enriched CD4+ T cells in the presence of a novel pro-inflammatory cytokine cocktail dramatically improved their yield, producing T-bet+ Th1 cells. TAg-reactive T cells recognized epitopes across all splice variants of TAg and were validated by specific recognition of moDCs natively expressing full-length TAg. VP-MCC cell lines upregulate MHC class II mRNA when stimulated with the Th1 effector cytokine, IFNγ. However, only one of three cell lines produce surface MHC class II. These data demonstrate that MCC-specific CD4+ T cells may interface either directly with some tumors or tumor-resident antigen presenting cells. Expanded MCPyV TAg specific T cells possessed characteristics favorable for clinical use including polyfunctionality, memory phenotype, and minimal exhaustion marker expression. In conclusion, we have identified a novel production method to readily generate MCPyV TAg specific T cells from healthy donors for potential use as an allogeneic adoptive cell therapy in patients with VP-MCC.