In situ-activated engineered bacteria release combined dual-therapeutic proteins for tumor immunotherapy

The combination of antitumor agents is pivotal for effective tumor immunotherapy. However, achieving precise, in situ co-delivery of therapeutic payloads remains a considerable challenge. Here, we developed a dual-payload-loaded Salmonella typhimurium strain capable of releasing granulocyte-macrophage colony-stimulating factor (GM-CSF) and CD47 nanobody (CD47nb) in response to tumor hypoxia and bacterial quorum sensing (QS). GM-CSF promotes dendritic cell (DC) maturation and enhances M1-like macrophage infiltration, facilitating antigen presentation and CD8 + T cells activation. CD47nb blocks the CD47-SIRPα signaling axis, thereby enhancing macrophage-mediated tumor cells phagocytosis. The combined delivery increased mature DC infiltration from 13.5% to 34.9%, promoted M1-like macrophage activation and CD8 + T cell accumulation in the tumor microenvironment (TME), suppressed tumor growth by 83.7%, and extended the median survival from 14 to 28 days compared with PBS-treated mice. In summary, our findings support precise, in situ delivery of complementary immunomodulators as a promising strategy for tumor immunotherapy.