Abstract 1610: Targeted cancer immunotherapy with engineered Salmonella typhimurium secreting heterologous bacterial flagellin
Authors: Min Jung-Joon , Zheng Jin Hai , Hong Yeongjin , Choy Hyon E. , Rhee Joon Haeng 
Published: July 2017

Salmonella typhimurium is a facultative anaerobic bacteria and can selectively grow in tumors following systemic administration. Different strategies have been used to deliver payloads to tumor tissues using engineered Salmonellae. We report a novel method of cancer immunotherapy using an attenuated S. typhimurium strain engineered to secrete Vibrio vulnificus flagellin B (FlaB) in tumor tissues.Previously, we developed an attenuated strain of S. typhimurium, which is defective in ppGpp synthesis (ΔppGpp S. typhimurium), and manifested significantly increased LD50 by 100,000 to 1,000,000-fold. To generate an inducible vector system for bacterial expression of the therapeutic gene, we cloned the flaB gene into the pBAD plasmid vector in which the pelB leader sequence was cloned upstream of flaB to guide extracellular secretion; gene expression from the plasmid (pFlaB) was induced only in the presence of L-arabinose. To evaluate the antitumor activity of engineered S. typhimurium, C57BL/6 mice transplanted with MC38 tumors were injected intravenously (i.v.) with PBS, ΔppGpp S. typhimurium carrying an empty vector, or ΔppGpp S. typhimurium carrying pFlaB (+/− L-arabinose induction). To test whether the FlaB-secreting ΔppGpp S. typhimurium has inhibitory effects on human metastatic cancer, we next implanted HCT116-luc2 tumors into the intestinal wall of BALB/c athymic nu-/nu- mice using a surgical orthotopic implantation (SOI) procedure. Engineered FlaB-secreting Salmonellae significantly suppressed tumor growth and metastasis in mouse models and prolonged survival. By using TLR5-negative colon cancer cell lines (MC38 and HCT116), we have proved that the FlaB-mediated tumor suppression upon bacterial colonization should be associated with TLR5-mediated host reactions in the tumor microenvironment. These therapeutic effects were completely abrogated in TLR4 and MyD88 knockout mice, and partly in TLR5 knockout mice, strongly suggesting that TLR4 signaling is a requisite for the FlaB-secreting bacteria-mediated tumor suppression where TLR5 signaling augmented tumor suppressive host reactions. Tumor colonization by engineered Salmonellae appeared to induce the infiltration of abundant immune cells such as monocytes/macrophages and neutrophils via the TLR4 signaling. Subsequent secretion of FlaB from colonizing Salmonellae resulted in phenotypic and functional activation of intratumoral macrophages with M1 phenotypes and a reciprocal reduction in M2-like suppressive activities. Taken together, these findings provide evidence that non-virulent tumor targeting bacteria liberating multiple TLR ligands can be used as novel cancer immunotherapeutics.

Citation Format: Jung-Joon Min, Jin Hai Zheng, Yeongjin Hong, Hyon E. Choy, Joon Haeng Rhee. Targeted cancer immunotherapy with engineered Salmonella typhimurium secreting heterologous bacterial flagellin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1610. doi:10.1158/1538-7445.AM2017-1610