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

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.