A living probiotic factory for sustained intestinal delivery of PD1/P53 outer membrane vesicles potentiates colorectal cancer immunotherapy

Engineered bacterial outer membrane vesicles (OMVs) have emerged as promising platforms for cancer immunotherapy; however, their clinical translation is hindered by low production yields, poor tumor homing, rapid clearance, and an inability to sustain local payload delivery. Here, we present a living probiotic-based system (Ba-PD1-P53, engineered E. coli Nissle 1917 co-expressing PD-1 and P53) that colonizes the murine intestine and continuously secretes OMVs co-displaying surface PD1 and encapsulating P53 (OMV-PD1-P53). Orally administered Ba-PD1-P53 persisted in the gastrointestinal tract without systemic toxicity, as confirmed by unaltered body weight, intestinal barrier integrity, hematological parameters, and organ histology. Mechanistically, surface-displayed PD1 on secreted OMVs directly blocked tumor cell PDL1, enhancing OMV uptake while concurrently reducing PDL1 expression; meanwhile, encapsulated P53 transcriptionally suppressed PDL1 and induced tumor cell apoptosis. These dual actions were complemented by the intrinsic immunostimulatory properties of OMVs, which activated dendritic cells and T cells, as evidenced by elevated intratumoral TNF-α, IFN-γ, and IL-6. In orthotopic CT26 colorectal cancer models, Ba-PD1-P53 significantly reduced tumor burden—by > 90% relative to PBS controls—and outperformed both monotherapies (Ba-PD1, Ba-P53) and systemic anti-PDL1 antibody. Notably, the treatment also enriched beneficial gut commensals (Muribaculaceae and Bacteroidaceae) and significantly reduced tumor multiplicity in the AOM-DSS carcinogenesis model. Collectively, this work establishes a safe, orally administered probiotic platform for sustained, in situ delivery of multifunctional OMVs, achieving synergistic checkpoint blockade and tumor suppressor restoration with high efficacy and a favorable safety profile.