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Purpose: Although Mycobacterium bovis Bacillus Calmette-Guérin (BCG) is the most widely used bladder cancer immunotherapy, innate immune responses involving antimicrobial peptides (AMPs) cause BCG failure. Here, we developed genetically modified recombinant BCG (rBCG) strains which escape AMPs and evaluate the efficacy and effects of rBCG. Materials and Methods: We constructed rBCG strains expressing Streptococcal inhibitor of complement (Sic), which confers resistance to human α-defensin-1 and cathelicidin, and d-alanyl carrier protein ligase (dltA), which confers resistance to cationic AMPs. Sic and dltA were separately cloned into the pMV306 plasmid and introduced into BCG via electroporation. The efficacy of the Sic and dltA gene electroporation into BCG was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). The internalization rates and anticancer effects of the rBCG strains containing Sic (rBCG-Sic) and dltA (rBCG-dltA) was evaluated by the orthotopic bladder cancer mouse model. Results: The cycle quantification (Cq) values of rBCG-Sic (y=-4.8823x+13.645, R=0.9996) and rBCG-dltA (y=-5.438x+11.641, R=0.9995) were inverse correlations to the amount of Sic and dltA genes dose dependently. The mean introduction proportions of Sic and dltA genes into BCG by electroporation were 22.2%, 27.5% and showed constant efficacy. In the orthotopic bladder cancer mouse model, the relative internalization number of rBCG-Sic, and rBCG-dltA into bladder cell in mouse bladder were higher than that of BCG and the tumor volume at rBCG-Sic were lower than at BCG and rBCG-dltA at 11, 14 and 18 days. Conclusions: Our results showed that constructed rBCG-Sic and rBCG-dltA by electroporation and the rBCG-Sic and rBCG-dltA can effectively escape BCG-stimulated AMPs, and significantly improved immunotherapeutic tools to treat bladder cancer in orthotopic bladder cancer mouse model. (Korean J Urol Oncol 2021;19:40-47)

The Effects of Recombinant Bacillus Calmette-Guérin Resistant to Antimicrobial Peptides on Orthotopic Bladder Cancer Mouse Model