Despite constant advances in cancer research, the treatment of pancreatic adenocarcinoma remains extremely challenging. The intratumoral immunotherapy approach that was developed by our research group and was based on a combination of mannan-BAM, TLR ligands, and anti-CD40 antibody (MBTA) showed promising therapeutic effects in various murine tumor models, including a pancreatic adenocarcinoma model (Panc02). However, the efficacy of MBTA therapy in the Panc02 model was negatively correlated with tumor size at the time of therapy initiation. Here, we aimed to further improve the outcome of MBTA therapy in the Panc02 model using the glutamine antagonist 6-diazo-5-oxo-L-norleucine (DON). The combination of intratumoral MBTA therapy and intraperitoneal administration of DON resulted in the complete elimination of advanced Panc02 subcutaneous tumors (140.8 ± 46.8 mm3) in 50% of treated animals and was followed by development of long-term immune memory. In the bilateral Panc02 subcutaneous tumor model, we observed a significant reduction in tumor growth in both tumors as well as prolonged survival of treated animals. The appropriate timing and method of administration of DON were also addressed to maximize its therapeutic effects and minimize its side effects. In summary, our findings demonstrate that the intraperitoneal application of DON significantly improves the efficacy of intratumoral MBTA therapy in both advanced and bilateral Panc02 subcutaneous tumor murine models.

Center for Cancer and Immunology Research Children's Research Institute Children's National Hospital Washington DC 20010 USA

Department of Medical Biology Faculty of Science University of South Bohemia Ceske Budejovice 37005 Czech Republic

Department of Medical Biology Faculty of Science University of South Bohemia Ceske Budejovice 37005 Czech Republic; Section on Medical Neuroendocrinology Eunice Kennedy Shriver National Institute of Child Health and Human Development National Institutes of Health Bethesda MD 20814 USA

Immunoaction LLC Charlotte VT 05445 USA

Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic v v i 166 10 Prague Czech Republic

Neuro Oncology Branch National Cancer Institute National Institutes of Health Bethesda MD 20814 USA

Section on Medical Neuroendocrinology Eunice Kennedy Shriver National Institute of Child Health and Human Development National Institutes of Health Bethesda MD 20814 USA

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