Prompting higher-order
death receptor (DR) clustering by increasing the valency of DR agonist is efficient to induce apoptosis of
tumor cells. As an attractive DR agonist with superior biosafety, the trimeric
tumor necrosis factor-related apoptosis-inducing
ligand (TRAIL) exerts limited antitumor effect in patients, which is predominantly attributed to its low DR clustering ability and short serum half-life. Previous antibody scaffolds-based engineering strategies to increase the valency and/or prolong the serum half-life of TRAIL improve apoptosis induction, however, often produce large
proteins with poor
tumor penetration. Covalent
protein ligation mediated by small molecular superglues such as SpyTag/SpyCatcher might be a novel strategy to assemble higher-order TRAIL variants. Upon fusion to TRAIL promotor, SpyTag/SpyCatcher molecular superglue preferentially ligated two trimeric TRAIL to produce a hexameric TRAIL variant, HexaTR, exhibiting a significantly increased apoptosis induction. In addition, an
albumin-binding HexaTR, ABD-HexaTR, with a prolonged serum half-life by binding to endogenous
albumin was also produced using the same strategy. Compared to the trimeric TRAIL, the hexameric HexaTR and ABD-HexaTR showed 20-50 times greater in vivo antitumor effect, resulting in eradication of several types of large (150-300 mm3)
tumor xenografts. Combination with
bortezomib carried by
liposome further improved the antitumor effects of the hexavalent HexaTR and ABD-HexaTR in refractory
cancer. Our results indicate that the superglue-mediated higher-order assembly is promising to improve the DR clustering and proapoptotic signaling of TRAIL, showing great advantages in constructing the next generation of DR agonists for
cancer therapy.