Two-step enhanced cancer immunotherapy with engineered Salmonella typhimurium secreting heterologous flagellin

Abstract

We report a method of cancer immunotherapy using an attenuated strain engineered to secrete flagellin B (FlaB) in tumor tissues. Engineered FlaB-secreting bacteria effectively suppressed tumor growth and metastasis in mouse models and prolonged survival. By using Toll-like receptor 5 (TLR5)-negative colon cancer cell lines, we provided evidence that the FlaB-mediated tumor suppression upon bacterial colonization is 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, indicating that TLR4 signaling is a requisite for tumor suppression mediated by FlaB-secreting bacteria, whereas TLR5 signaling augmented tumor-suppressive host reactions. Tumor microenvironment colonization by engineered appeared to induce the infiltration of abundant immune cells such as monocytes/macrophages and neutrophils via TLR4 signaling. Subsequent secretion of FlaB from colonizing resulted in phenotypic and functional activation of intratumoral macrophages with M1 phenotypes and a reciprocal reduction in M2-like suppressive activities. Together, these findings provide evidence that nonvirulent tumor-targeting bacteria releasing multiple TLR ligands can be used as cancer immunotherapeutics.

Rapid genome recoding by iterative recombineering of synthetic DNA

“…This strain contains the largest number of codon substitutions in a single recoded organism published to date. A recoded designer S. typhimurium could have diagnostic and therapeutic applications in the human body [11][12][13] . To demonstrate the power of our recoding approach, we replaced leucine codons due to their high frequency and redundancy in the Salmonella genome 14 .…”

Advances in bacterial cancer therapies using synthetic biology

“…Bacterial secretion is another promising means of therapeutic delivery to tumor microenvironment [40,43,54]. Secretion can be achieved by use of a leader signal sequence, a short peptide fused to the N-terminus of the protein of interest [40,55,56].…”

“…Secretion can be achieved by use of a leader signal sequence, a short peptide fused to the N-terminus of the protein of interest [40,55,56]. These leader signal sequences are analogous to zip codes that traffic the translated protein to the bacterial periplasm followed by secretion outside of the cell [57].…”

“…Many reviews have detailed therapeutics used; they broadly fall under two types: proteins and nucleic acids [64,84,85]. Proteins include cytotoxic agents, immune-stimulatory molecules, growth pathway regulators, and prodrug enzymes [40,55,86]. One example of a cytotoxic agent is HlyE, a pore-forming toxin, which has been used by several groups [23,29].…”

“…Immunostimulatory molecules can include cytokines, antibodies and tumor-specific antigens. Recently, delivery in vivo of S. typhimurium expressing flagellin B, a structural component of the flagellum from Vibrio Vulnificus was shown to lead to recruitment of macrophages and tumor regression, possibly via activation of the toll like receptor pathways [40]. DNA encoding for cytokines and bacteria antigens can be delivered for gene transfer via a co-opted invasion system from Listeria , or shRNA can be delivered for RNAi-mediated gene knockdown [61,64].…”