Biohybrid Salmonella typhimurium for synergistic chemoimmunotherapy via localized chemotherapy and in situ PD-L1 blockade

Abstract

Chemotherapy can substantially enhance the efficacy of cancer immunotherapy; however, the clinical translation of chemoimmunotherapy is frequently hampered by inefficient drug delivery and dose-limiting systemic toxicity. To address these challenges, we engineered a tumor-targeting Salmonella typhimurium–based platform (VNP@PD-L1nb/DOX) that simultaneously enables localized chemotherapy and in situ PD-L1 blockade. The attenuated Salmonella typhimurium strain VNP20009 was genetically engineered to secrete PD-L1-targeting nanobodies (PD-L1nb), and surface-functionalized with a phenylboronic acid (PBA)-conjugated doxorubicin prodrug (DOX-PBA) via reversible boronate ester bonds. This design allows hydrogen peroxide (H2O2)-responsive release of active doxorubicin within the tumor microenvironment while maintaining negligible toxicity toward normal tissues and the bacterial carrier itself. The engineered bacteria exhibited pronounced tumor-selective accumulation and continuously secrete PD-L1nb into the extracellular space for PD-L1 selective blockade. In a murine triple-negative breast cancer (TNBC) model, VNP@PD-L1nb/DOX significantly suppressed tumor growth, induced immunogenic cell death (ICD), and promoted robust systemic immune activation. The ICD-driven immune priming, together with localized PD-L1 blockade, synergistically enhanced intratumoral infiltration and activation of CD8+ T cells while concurrently depleting regulatory T (Treg) cells. Collectively, our findings underscore the promise of engineered bacterial vectors as a versatile integrated platform for targeted chemoimmunotherapy in advanced solid tumors.