Synthetic bacteria with programmed cell targeting and protein injection suppress tumor growthin vivo

Bacterial living therapeutics (BLTs) hold promise for treating cancer and other human diseases because they can be engineered and transported into the microbiota (e.g., of tumors, gastrointestinal tract) to deliver therapeutic payloads. Current approaches rely on the natural tropism of the bacterial chassis used and trigger the local release of protein cargoes, typically through active extracellular secretion or bacterial lysis. BLTs capable of targeting specific cellular subsets and delivering payloads intracellularly might provide new therapeutic opportunities and improve efficacy while reducing off-target effects. We used synthetic biology to develop BLTs that can deliver defined cargo proteins into the cytoplasm of target cells. We designed a modular synthetic bacterium with programmed adhesion to cells by targeting defined cell surface antigen and armed with an inducible type III secretion system (T3SS) for injection of a protein cargo of interest. As a proof of principle, we programmed synthetic bacteria to recognize the epidermal growth factor receptor (EGFR) and inject the catalytic fragments of the potent adenosine diphosphate-ribosyltransferase toxins ExoA and TccC3. These BLTs demonstrated the ability to trigger robust tumor cell deathin vitro. Intratumoral administration of these synthetic bacteria suppressed tumor growthin vivoand prolonged the survival of treated animals when the tumor cells were recognized by the engineered bacteria. These results demonstrate the potential of programming cell targeting and controlled protein injection for the development of effective and specific BLTs.