Pancreatic ductal adenocarcinoma is poorly immunogenic, and immune suppression prevents T cell activation. We developed a microbial-based immunotherapeutic concept for selective delivery of immunogenic tetanus toxoid (TT856-1313) antigen into tumor cells by attenuated Listeria monocytogenes, and reactivation of TT-specific memory T cells to kill infected tumor cells. Treatment of KPC mice with Listeria-TT resulted in TT accumulation inside tumor cells, and attraction of TT-specific CD4 and CD8 T cells.Lymph node-like structures were frequently observed in contact with pancreatic tumors of treated mice, and exhibited CD4 and CD8 T cells producing IFNg. Gemcitabine combined with Listeria-TT significantly improved migration of CD4 T cells into tumors, and enhanced perforin and granzyme B production. The combination treatment also significantly reduced pancreatic tumors and metastases in Panc-02 and KPC mouse models, with minimal side effects, turning "cold" tumors into "hot" tumors. This study provides insight into mechanisms for improving immunotherapy for pancreatic cancer.selectively attracts MDSC through the production of cytokines and factors 11,20 , where MDSC deliver Listeria to the TME as a Trojan horse 10,19 . Once at the tumor site, Listeria spreads from MDSC into tumor cells through a cell-to-cell mechanism unique to Listeria 21 . Listeria can also infect tumor cells directly 22 . Listeria would be rapidly killed in healthy tissues, but is protected from immune clearance in the TME through strong immune suppression. Because of this combination of selective attraction of MDSC by bacteria and cancer with the strong immune suppression occurring in the TME but not in normal tissues, Listeria can selectively enter, multiply, and survive in the TME but not in normal tissues 10,19,23,24 . Based on these results we now use Listeria as a platform for the selective delivery of anticancer agents to the TME 19,23,25 .In the current study, we tested the combination of Listeria-TT+GEM (Fig 1b) in two mouse models of pancreatic cancer, a syngeneic Panc-02 model 26 and a transgenic KPC model 27,28 . Listeria-TT was administered intraperitoneally, and the in vivo uptake by tumor cells was verified by intravital imaging. We demonstrate that the combination of Listeria-TT+GEM robustly reduced pancreatic cancer at early and advanced stages in both Panc-02 and KPC mouse models. Our results unveil new mechanisms of Listeria to improve immunotherapy for PDAC. RESULTS Development and characterization of Listeria-TTThe Listeria construct used to develop Listeria-TT includes pGG34 29 , a truncated non-cytolytic Listeriolysin O (LLO) fused to a non-toxic TT856-1313 fragment, and a myc sequence for detection as outlined in Fig 2a. Secretion of LLO-TT856-1313 protein into the culture medium by the bacteria was detected by western blotting (Fig 2b). Infection of Panc-02 tumor cells with Listeria-TT856-1313 resulted in the expression of TT protein in the tumor cells (Fig 2c).

Cancer vaccination or immunotherapy may be our best and most benign option for preventing or curing metastatic cancer. However, a real breakthrough is hampered by immune suppression in the tumor microenvironment (TME). Myeloid- derived suppressor cells (MDSC) are one of the most important players in immune suppression in the TME. In the study presented here we found that c-di-GMP (ligand for STING), an ubiquitous bacterial signaling molecule and promising vaccine adjuvant, improved the therapeutic efficacy of an attenuated L. monocytogenes-based vaccine expressing tumor-associated antigen (TAA) Mage-b, in a mouse model of metastatic breast cancer (4T1), by targeting MDSC. An extreme low dose of c-di-GMP (0.01 nmol) combined with a low dose of Listeria-Mage-b (104 CFU), dramatically reduced the number of metastases, in correlation with a significant reduction of MDSC and a significant increase in CD8 T cell responses to Mage-b. Moreover, low dose of c-di-GMP increased the production of IL-12, while high dose killed tumor cells directly. Administration of one high dose c-di-GMP to kill the tumor cells, followed by repeated administrations of low dose c-di-GMP to improve CD8 T cell responses, provided similar efficacy against the metastatic breast cancer as Listeria-Mage-b and c-di- GMP in the 4T1 model. These results demonstrate that c-di-GMP is an attractive adjuvant for cancer therapy.

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Patients with pancreatic ductal adenocarcinoma (PDAC) have a five-year survival of only 6%. Cancer immunotherapy has shown promising results for metastatic cancer but only in 20% of the patients. Our lab has developed a new concept of immunotherapy using an attenuated non-pathogenic bacterium Listeria monocytogenes for the selective delivery of a highly immunogenic recall antigen tetanus toxoid (TT) into tumor cells of pancreatic tumors and metastases. Most individuals have been exposed to TT during childhood vaccinations and memory T-cells to TT circulate in blood for live. These memory T-cells to TT can be reactivated by Listeria-TT at any time in life and will now destroy the infected tumor cells. Low doses of gemcitabine (GEM) were added to further improve T-cell responses. Panc-02 and KPC mice were immunized with the “human TT vaccine” to generate memory T-cells before development of the pancreatic cancer. After cancer development, mice were treated with Listeria-TT and GEM, and tumors (weight) and metastases (number) were analyzed by eye in the Panc-02 mice, and by SUVmax (measurement of tumor growth) in the KPC mice. T-cell responses in the spleen were analyzed by flow cytometry and ELISPOT, and KPC tumors were analyzed by RNAseq. Listeria-TT and GEM nearly completely eliminated both early and advanced pancreatic cancer in Panc-02 and KPC mice, in correlation with abundant CD4 and CD8 T-cell responses producing perforin, granzyme B, or IFNγ, in vivo or in vitro, respectively. RNAseq analysis of “treated” KPC tumors showed a strong influx of CD4 and CD8 T-cells, upregulation of multiple granzymes, perforin, co-stimulatory molecules, and cell death pathways. These results suggest the multiple synergistic effects of Listeria-TT and GEM on pancreatic cancer. This novel treatment modality of Listeria-TT and GEM warrants further investigation in a clinical setting. Citation Format: Benson C. Selvanesan, Dinesh Chandra, Wilber Quispe, Ankur Patel, Kiran Meena, Steven K. Libutti, Ziqiang Yuan, Jennifer Chuy, Claudia Gravekamp. A new concept of cancer immunotherapy by targeting pancreatic ductal adenocarcinoma through a childhood recall antigen [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B132.