ABSTRACT Pancreatic ductal adenocarcinoma is highly metastatic, poorly immunogenic, and immune suppression prevents T cell activation in the tumor microenvironment. We developed a microbial-based immunotherapeutic concept for selective delivery of a highly immunogenic tetanus toxoid protein (TT 856-1313 ), into tumor cells by attenuated Listeria monocytogenes , and reactivation of pre-existing TT-specific memory T cells (generated during childhood) to kill infected tumor cells. Thus, TT here functions as an alternative for neoantigens. Treatment of KPC mice with Listeria -TT resulted in TT accumulation in tumors and inside tumor cells, and attraction of predominantly TT-specific memory CD4 T cells. Moreover, gemcitabine (GEM) combined with Listeria-TT significantly improved the migration of CD4 T cells into tumors and the production of perforin and granzyme B, turning cold tumors into immunological hot tumors. In vivo depletion of T cells in Listeria-TT+GEM-treated mice demonstrated CD4 T cell-mediated eradication of tumors and metastases (Mann-Whitney p&lt;0.05). In addition, peritumoral lymph node like structures (LNS) were observed in close contact with the pancreatic tumors displaying CD4 T cells and CD8 T cells of KPC mice treated with Listeria -TT or Listeria -TT+GEM. The production of perforin and granzyme B was observed in LNS of KPC mice that received Listeria -TT+GEM. This combination not only reduced tumor burden (80%) and metastases (87%) significantly (p&lt;0.05, Mann-Whitney), but also improved the survival time of KPC mice with advanced pancreatic cancer substantially (Mantel-Cox p&lt;0.0001). Our results unveil new mechanisms of Listeria and GEM improving immunotherapy for PDAC.

Tumor-targeted delivery of childhood vaccine recall antigens by attenuatedListeriareduces pancreatic cancer