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Purpose: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) binds to death receptor (DR) 4 and DR5 and induces tumor-selective apoptosis. The fusion proteins NCTR25–TRAIL and NCTR25–TGF3L–TRAIL self-assembled into polymers and triggered super-active cancer cell killing. The role of TGF3L in self-assembly and super-activity was unclear. These multivalent TRAILs elicited apoptosis with great potency, but their specificity towards receptors and subsequent efficacy in signal activation were unclear. Methods: NCTR25–TRAIL fusion was constructed and prokaryotically expressed. The size of fusion protein polymers was estimated. Their cytotoxicity was assessed in eight cancer cell lines and two noncancerous cell lines. Receptor binding and activation specificity were determined by antibody blockade. Apoptosis was evaluated, and the associated pathway was verified by quantifying caspase activity. The NF-κB signaling pathway was assessed by dual-luciferase assay. The in vivo antitumor activity was also evaluated in nude mice. Results: NCTR25 fusion to TRAIL promoted its self-assembly into polymers and showed similar super-cytotoxicity to NCTR25–TGF3L–TRAIL in vitro. The multivalent TRAILs exclusively activated both DR4 and DR5 and showed a bias towards DR4 in mediating cytotoxicity in NCI-H460 cells. They activated caspase pathway and induced apoptosis with higher potency but in similar efficacy than TRAIL. A higher potency and a greater efficacy were observed in activating NF-κB pathway by NCTR25–TRAIL comparing to TRAIL. Both the polymers showed better in vivo antitumor activity than TRAIL. Conclusions: NCTR25 fusion alone facilitates the formation of TRAIL polymers. Multivalent TRAIL polymers bind and activate DR4 and DR5 specifically and exclusively, triggering the signaling pathways with higher potency, and greater efficacy than TRAIL. [ABSTRACT FROM AUTHOR]