Modular pentameric protein scaffold based on glutaraldehyde-crosslinked Stx1B for superior multivalent therapeutics.

Multivalent therapeutics harness nature's design principles to achieve enhanced binding avidity, facilitate receptor clustering-mediated signaling modulation, and enable coordinated multi-epitope targeting. These combined properties make them particularly transformative in treating viral infections and cancers. The pentameric B subunit of Shiga toxin 1 (Stx1B) represents an effective multivalent scaffold, yet its noncovalent assembly intrinsically restricts both structural stability and functional efficacy. To overcome this limitation, we developed a glutaraldehyde-crosslinked Stx1B platform that enhances both conformational stability and multivalent effect to improve overall pharmaceutical properties. As a proof-of-concept for SARS-CoV-2 neutralization, we fused this scaffold with the viral spike-targeting mini-protein LCB1 to generate the pentameric construct 1B-LCB1. The crosslinked variant, C1B-LCB1, retained pentameric integrity under denaturing conditions, improved target binding durability and structural stability, and demonstrated potent pseudovirus inhibition along with exceptional physiological stability under multiple pharmaceutically relevant stress conditions. Furthermore, when the Stx1B scaffold was functionalized with a death receptor (DR5)-targeting peptide agonist (DR5TP) to achieve a pentameric apoptotic agonist, the crosslinked C1B-DR5TP significantly enhanced DR5-mediated apoptotic signaling and cancer cell cytotoxicity, underscoring its broad utility. Taken together, this work establishes crosslinked Stx1B as a modular platform capable of presenting diverse therapeutic payloads, including viral inhibitors and tumor-targeting domains, while maintaining the structural integrity and multivalency crucial for clinical applications.