Deletion of the Salmonella pathogenicity island 2 gene, spiC, in attenuated Salmonella Typhimurium VNP20009 optimizes its potential for bacterial schwannoma therapy.
UNLABELLED: Recent advances in systems biology and immunotherapy have spurred the investigation of bacteria as therapeutic vehicles for cancer treatment. Currently, Bacillus Calmette-Guérin remains the only FDA-approved bacterial cancer therapy; it is a live attenuated mycobacterium that is indicated for the treatment and prophylaxis of carcinoma in situ of the urinary bladder and for the prophylaxis of primary or recurrent papillary tumors following transurethral resection. Although safety concerns have been raised, attenuated Salmonella Typhimurium strains such as VNP20009 have advanced to clinical trials targeting fast-growing human tumors. Notably, this strain induces robust immunological control of slow-growing tumors such as NF2-related schwannomatosis (NF2-SWN) in preclinical murine models. Here, we genetically characterize VNP20009 with the goal of constructing genetically defined attenuated strains that retain its promising therapeutic features while improving safety. Specifically, we investigated the contribution of the Salmonella pathogenicity island I (SPI-1) and SPI-2 type III secretion systems to antitumor efficacy and biosafety. Mutation of the SPI-1 gene sipB, a key structural component required for SPI-1 type III secretion system function, partially reduced tumor control in NF2-SWN murine schwannoma models, suggesting that bacterial invasion alone does not fully account for antitumor activity. In contrast, deletion of the SPI-2 gene spiC, a key effector required for intracellular survival, preserved robust tumor regression in NF2-SWN murine schwannoma models while improving safety and reducing systemic toxicity. To create a genetically defined and tractable platform, we generated two attenuated strains-AST101 and AST101-ΔspiC-which retain key mutations present in VNP20009 but lack ill-characterized background mutations. In the syngeneic NF2-SWN mouse schwannoma model, both strains significantly suppressed tumor growth compared to PBS. Collectively, these findings support the development of rationally engineered Salmonella Typhimurium strains with enhanced safety and preserved antitumor efficacy. IMPORTANCE: Given long-standing safety concerns surrounding the therapeutic use of live bacteria, we constructed a ΔspiC mutant of VNP20009 and demonstrated that it provides a markedly improved safety profile while retaining antitumor efficacy in NF2-related schwannomatosis mouse schwannoma models. In addition, we created two genetically defined Salmonella Typhimurium strains, AST01 and AST01-ΔspiC, which incorporate the key-targeted mutations found in VNP20009 and VNP20009-ΔspiC, respectively. These engineered strains offer a well-defined genetic background, enabling precise investigation of the bacterial traits responsible for Salmonella Typhimurium-mediated tumor control and thus further improvement of attenuated strains optimized for bacteriotherapy of neoplasms.