TriSC–mediated colocalization of targeting and therapeutic nanobodies.
Orthogonal Tri-Modular Coiled-Coil Assembly for Programmable Multi-Cargo Display on <i>Escherichia coli</i> Nissle 1917
Paper
2026
Small |
DOI
Background
This study presents Tri-specific Scaffold Cells (TriSCs), an engineered live biotherapeutic platform based on <i>Escherichia coli</i> Nissle 1917, featuring three orthogonal α-helical motifs on the bacterial surface. These motifs enable spontaneous, reversible, and multiplex assembly of functional payloads through coiled-coil interactions without cross-interference. The 'mix-and-go' approach allows simultaneous display of distinct modules such as fluorescent reporters and therapeutic nanobodies. Significantly, co-display of a DR5 agonistic nanobody and an EGFR-targeting nanobody amplifies anticancer activity beyond soluble mixtures, demonstrating the therapeutic potential of spatially organized targeting. TriSCs thus offer a programmable, dynamic scaffold for precision theranostics.
Key Findings
TriSCs robustly displayed three orthogonal coiled-coil motifs (A-A', B-B', C-C') on the EcN surface, enabling dose-dependent and specific payload binding. Multiplex loading with sfGFP, mCherry, and EBFP payloads confirmed high orthogonality with 45–80% retention of singleplex signals. Payloads were dynamically and reversibly assembled by sequential displacement with competitor peptides. In cancer cell assays, αEGFR nanobody surface display drove strong colorectal cancer cell binding, while co-display of DR5 agonistic nanobody and αEGFR nanobody elicited synergistic cytotoxicity, significantly enhancing apoptosis over solo display or soluble combinations. This synergy depended on spatial colocalization on the scaffold, demonstrating that TriSCs convert receptor engagement into amplified signaling. The modular platform also integrated fluorescent reporters for imaging, establishing TriSCs as a versatile, programmable living scaffold for multi-cargo therapeutic applications.
Contribution
Creative Biogene supplied the pQE80L plasmid (catalog #VET1018), which served as the backbone for constructing the pQE80 coil expression vectors used in this study.
This study presents Tri-specific Scaffold Cells (TriSCs), an engineered live biotherapeutic platform based on <i>Escherichia coli</i> Nissle 1917, featuring three orthogonal α-helical motifs on the bacterial surface. These motifs enable spontaneous, reversible, and multiplex assembly of functional payloads through coiled-coil interactions without cross-interference. The 'mix-and-go' approach allows simultaneous display of distinct modules such as fluorescent reporters and therapeutic nanobodies. Significantly, co-display of a DR5 agonistic nanobody and an EGFR-targeting nanobody amplifies anticancer activity beyond soluble mixtures, demonstrating the therapeutic potential of spatially organized targeting. TriSCs thus offer a programmable, dynamic scaffold for precision theranostics.
Key Findings
TriSCs robustly displayed three orthogonal coiled-coil motifs (A-A', B-B', C-C') on the EcN surface, enabling dose-dependent and specific payload binding. Multiplex loading with sfGFP, mCherry, and EBFP payloads confirmed high orthogonality with 45–80% retention of singleplex signals. Payloads were dynamically and reversibly assembled by sequential displacement with competitor peptides. In cancer cell assays, αEGFR nanobody surface display drove strong colorectal cancer cell binding, while co-display of DR5 agonistic nanobody and αEGFR nanobody elicited synergistic cytotoxicity, significantly enhancing apoptosis over solo display or soluble combinations. This synergy depended on spatial colocalization on the scaffold, demonstrating that TriSCs convert receptor engagement into amplified signaling. The modular platform also integrated fluorescent reporters for imaging, establishing TriSCs as a versatile, programmable living scaffold for multi-cargo therapeutic applications.
Contribution
Creative Biogene supplied the pQE80L plasmid (catalog #VET1018), which served as the backbone for constructing the pQE80 coil expression vectors used in this study.