Orthogonal Tri‐Modular Coiled‐Coil Assembly for Programmable Multi‐Cargo Display on Escherichia coli Nissle 1917

ABSTRACT Live biotherapeutic products (LBPs) represent emerging living medicines capable of site‐specific intervention; however, current strategies for surface functionalization rely predominantly on static genetic fusion or covalent modification, limiting multiplexing and dynamic reconfiguration. Here, we engineer TriSCs (Tri‐specific Scaffold Cells), a tri‐modular and orthogonally programmable living scaffold based on Escherichia coli Nissle 1917. Three distinct α‐helical motifs are displayed on the bacterial outer membrane, forming a reconfigurable biointerface that enables spontaneous, highly specific, and reversible payload assembly via coiled‐coil interactions. This mix‐and‐go strategy allows simultaneous and selective recruitment of multiple functional modules without structural interference. Spatial co‐display of a DR5 agonistic nanobody and an EGFR‐targeting nanobody produces enhanced anticancer activity compared to soluble combinations, underscoring the impact of surface‐organized signaling. In parallel, modular incorporation of a fluorescent reporter enables real‐time bioimaging. By integrating orthogonality, reversibility, and multiplex capability within a single living chassis, TriSCs establish a dynamic and programmable LBP platform for precision theranostics and next‐generation bioengineered therapeutics.