Spatially organized nanoassembly of single-chain TRAIL that induces optimal death receptor clustering and cancer-specific apoptosis

Tumor necrosis factor-related apoptosis-induced ligands (TRAILs) are cytokines that efficiently trigger apoptosis by specifically binding to death receptors (DRs), predominantly expressed in cancer cells. Despite the potential as a cancer-targeted therapeutic, a functionally active TRAIL homotrimer is non-covalently associated and prone to disassembly upon administration, resulting in suboptimal therapeutic outcome. Here, we report a TRAIL nanoassembly (HexTR) with defined valency of two single-chain TRAILs (3 TRs), i.e., hexavalent TRAILs, to address unmet needs in TRAIL therapy. HexTR was produced by specific coiled-coil interaction between two building blocks containing human-originated, heterodimeric α-helical domains each of which was genetically linked to 3 TR, respectively. The organized self-assembly allowed HexTR to present optimally distanced two 3 TRs, inducible for formation of a high-order TRAIL-DR network. HexTR demonstrated the capability to bind both soluble and cell-surface DRs with higher affinity than the building blocks alone. Moreover, HexTR outperformed both wild-type TRAIL and 3  TR s in eliciting in vitro apoptotic efficacy against HCT116 colon cancer cells, while sparing healthy cells, and inhibiting tumor growth in HCT116 xenografted mice. These results highlight HexTR's significant advantages over existing nanomedicines as it was self-structured, biocompatible, and delivers potent cancer-specific effects through controlled geometry and valency of TRAILs favoring effective DR clustering.