Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease. Tumors are poorly immunogenic and immunosuppressive, preventing T cell activation in the tumor microenvironment. Here, we present a microbial-based immunotherapeutic treatment for selective delivery of an immunogenic tetanus toxoid protein (TT _856-1313 ) into PDAC tumor cells by attenuated Listeria monocytogenes . This treatment reactivated preexisting TT-specific memory T cells to kill infected tumor cells in mice. Treatment of KrasG12D,p53R172H, Pdx1-Cre (KPC) mice with Listeria -TT resulted in TT accumulation inside tumor cells, attraction of TT-specific memory CD4 T cells to the tumor microenvironment, and production of perforin and granzyme B in tumors. Low doses of gemcitabine (GEM) increased immune effects of Listeria -TT, turning immunologically cold into hot tumors in mice. In vivo depletion of T cells from Listeria -TT + GEM–treated mice demonstrated a CD4 T cell–mediated reduction in tumor burden. CD4 T cells from TT-vaccinated mice were able to kill TT-expressing Panc-02 tumor cells in vitro. In addition, peritumoral lymph node–like structures were observed in close contact with pancreatic tumors in KPC mice treated with Listeria -TT or Listeria -TT + GEM. These structures displayed CD4 and CD8 T cells producing perforin and granzyme B. Whereas CD4 T cells efficiently infiltrated the KPC tumors, CD8 T cells did not. Listeria -TT + GEM treatment of KPC mice with advanced PDAC reduced tumor burden by 80% and metastases by 87% after treatment and increased survival by 40% compared to nontreated mice. These results suggest that Listeria -delivered recall antigens could be an alternative to neoantigen-mediated cancer immunotherapy.

Background: Treatments for pancreatic ductal adenocarcinoma are poorly effective, at least partly due to the tumor’s immune-suppressive stromal compartment. New evidence of positive effects on immune responses in the tumor microenvironment (TME), compelled us to test the combination of gemcitabine (GEM), a standard chemotherapeutic for pancreatic cancer, with nicotinamide (NAM), the amide form of niacin (vitamin B₃), in mice with pancreatic cancer. Methods: Various mouse tumor models of pancreatic cancer, that is, orthotopic Panc-02 and KPC (Kras^G12D, p53^R172H, Pdx1-Cre) grafts, were treated alternately with NAM and GEM for 2 weeks, and the effects on efficacy, survival, stromal architecture and tumor-infiltrating immune cells was examined by immunohistochemistry (IHC), flow cytometry, Enzyme-linked immunospot (ELISPOT), T cell depletions in vivo, Nanostring analysis and RNAscope. Results: A significant reduction in tumor weight and number of metastases was found, as well as a significant improved survival of the NAM+GEM group compared with all control groups. IHC and flow cytometry showed a significant decrease in tumor-associated macrophages and myeloid-derived suppressor cells in the tumors of NAM+GEM-treated mice. This correlated with a significant increase in the number of CD4 and CD8 T cells of NAM+GEM-treated tumors, and CD4 and CD8 T cell responses to tumor-associated antigen survivin, most likely through epitope spreading. In vivo depletions of T cells demonstrated the involvement of CD4 T cells in the eradication of the tumor by NAM+GEM treatment. In addition, remodeling of the tumor stroma was observed with decreased collagen I and lower expression of hyaluronic acid binding protein, reorganization of the immune cells into lymph node like structures and CD31 positive vessels. Expression profiling for a panel of immuno-oncology genes revealed significant changes in genes involved in migration and activation of T cells, attraction of dendritic cells and epitope spreading. Conclusion: This study highlights the potential of NAM+GEM as immunotherapy for advanced pancreatic cancer.

Ovarian cancer is known for its poor neoantigen expression and strong immunosuppression. Here, we utilized an attenuated non-pathogenic bacterium Listeria monocytogenes to deliver a highly immunogenic Tetanus Toxoid protein (Listeria-TT), as a neoantigen surrogate, into tumor cells through infection in a metastatic mouse ovarian cancer model (Id8p53-/-Luc). Gemcitabine (GEM) was added to reduce immune suppression. Listeria-TT+GEM treatments resulted in tumors expressing TT and reactivation of pre-existing CD4 and CD8 memory T cells to TT (generated early in life). These T cells were then attracted to the TT-expressing tumors now producing perforin and granzyme B. This correlated with a strong reduction in tumor burden, and significant improvement of the survival time compared to all control groups. Checkpoint inhibitors have little effect on ovarian cancer partly because of low neoantigen expression. Here we demonstrated that Listeria-TT+GEM+anti-PD1 was significantly more effective (efficacy and survival) than anti-PD1 or Listeria-TT+GEM alone. Of clinical interest, high doses of anti-PD1 (PD1H) (when added to Listeria-TT+GEM) were less effective than the low doses (PD1L). IHC and ELISPOT demonstrated that high doses of anti-PD1 inhibited T cell function in the TME. Using RNAseq, Differentially Expressed Genes (DEG) analysis and Genes Set Enrichment Analysis (GSEA) showed that gene expression levels and biological pathways were predominantly upregulated in the PD1H compared to the PD1L group, in correlation with low immune infiltration in tumors, more immune suppression, and more aggressive ovarian cancer. In summary, this study suggests that our approach may benefit ovarian cancer patients. Abstract Figure: Graphical Human concept: Childhood vaccinations with the highly immunogenic tetanus toxoid (TT) generate TT-specific memory T cells, which circulate in the blood for life. After appearance of ovarian cancer (late in life), the patients will receive one high dose with Listeria-TT to deliver TT into tumor cells, followed by multiple low doses of Listeria-TT over a period of 2 weeks to restimulate the pre-existing memory T cells to TT. MDSC are involved in the delivery of Listeria-TT to the TME. Reactivated memory T cells will in turn destroy the tumor cells expressing TT. Multiple low doses of GEM will be added after TT has been delivered at the tumor site, which reduce immune suppression by eliminating MDSC and TAM (not shown here). Since individuals have seen TT earlier in life (during childhood vaccinations) and since TT is highly immunogenic (attracting T cells to the TME) but not expressed in normal cells, TT functions here as a vaccine recall antigen and as a neoantigen surrogate, respectively.

PDF file - 146K, Figure S9: Activation of CD8 T cells by c-di-GMP-treated MDSC compared to non-treated MDSC.

PDF file - 164K, Figure S5: Developing of a therapeutic protocol with c-di-GMP and Listeria for clinical application.