Engineering Precise Production of Lipopolysaccharides for Intratumoral Immune Activation By Salmonella Typhimurium

Immunotherapies are promising treatments for cancer; however, many tumors are non-responsive due to immunosuppression. Bacterial therapies demonstrate promise for cancer treatment due to their tumor localization and immunostimulatory properties. Many therapeutic strains of Salmonella typhimurium have been attenuated with deletion of the gene msbB which catalyzes acylation of surface lipopolysaccharides (LPS). This attenuation allows for safe intravenous injection of the bacteria; but limits their immunotherapeutic efficacy. In this work, we engineered attenuated Salmonella to produce msbB upon induction (Sal-msbB) for an inflammatory response once localized to the tumor microenvironment. Utilizing an array of ribosomal binding sites (RBS) and ssrA degradation tags we tuned our system to produce optimal levels of surface LPS to balance safety with therapeutic efficacy. Our system produces 3.75-times as much TNF-α once induced, with uninduced bacteria remaining unchanged from attenuated controls. Induced Sal-msbB increased presentation of the activation markers CD80 and CD86 by both monocytes and dendritic cells in vitro, which is key for triggering the adaptive immune system. In mice, uninduced Sal-msbB was injected intravenously at therapeutically relevant doses and did not increase systemic cytokine production or induce sepsis after induction, demonstrating a safe delivery profile. Induction of Sal-msbB reduced tumor growth by two-thirds and increased mouse survival versus attenuated controls. Within the tumor, Sal-msbB increases activation of monocytes and production of the pro-inflammatory cytokine IL-6. Overall, the adjuvant activity of controlled production of bacterial LPS increases efficacy of bacterial anti-cancer therapies and provides a natural niche for Salmonella therapies in tumor immunotherapy.