Programmable genetic control of tumor-colonizing Bifidobacterium longum for intratumoral therapeutic delivery and biocontainment

Engineered bacteria offer a distinct modality for cancer therapy by exploiting the ability of certain species to colonize tumors and deliver therapeutic payloads. Improving their efficacy and safety requires control over bacterial activity after tumor colonization, yet few microbial chassis permit it. Bifidobacterium longum, a probiotic with intrinsic tumor-targeting and antitumor activity, is a promising chassis but lacks such control. Here, we develop a genetic control system that regulates B. longum activity within tumors, from gene expression to bacterial abundance. A human-isolate-derived replicon supports plasmid maintenance without antibiotic selection, and promoter and ribosome-binding-site libraries provide [~]150-fold and [~]48-fold expression ranges, respectively. Signal peptides enable secretion of structurally diverse therapeutic payloads and B. longum secreting CCL21 or an anti-PD-L1 nanobody reduces tumor growth relative to PBS controls. Anhydrotetracycline delivered in drinking water induces transgene expression in tumor-resident bacteria and reduces intratumoral bacterial load through CRISPRi targeting essential genes. Together, these results establish a tumor-homing probiotic as an externally controllable therapeutic chassis.