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

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Development

Background:

• Multivalent therapeutics offer enhanced binding avidity and signaling modulation for treating viral infections and cancers.
• The pentameric Shiga toxin B subunit (Stx1B) is a promising multivalent scaffold, but its noncovalent assembly limits stability and efficacy.

Purpose of the Study:

• To develop a stabilized Stx1B platform to overcome limitations in structural stability and functional efficacy.
• To demonstrate the utility of the crosslinked Stx1B scaffold for SARS-CoV-2 neutralization and cancer therapy.

Main Methods:

• Glutaraldehyde crosslinking was used to enhance the conformational stability of the Stx1B scaffold.
• The modified scaffold was fused with LCB1 for SARS-CoV-2 spike targeting (C1B-LCB1) and a DR5-targeting peptide agonist (C1B-DR5TP).
• Stability, binding, neutralization, and therapeutic signaling were assessed under various conditions.

Main Results:

• The crosslinked C1B-LCB1 construct maintained pentameric integrity, enhanced binding durability, and showed potent pseudovirus inhibition.
• The crosslinked C1B-DR5TP construct significantly enhanced DR5-mediated apoptosis and cancer cell cytotoxicity.
• The crosslinked Stx1B platform demonstrated exceptional physiological stability.

Conclusions:

• Crosslinked Stx1B serves as a modular and stable platform for presenting diverse therapeutic payloads.
• This approach enhances structural integrity and multivalency, crucial for clinical applications in virology and oncology.
• The stabilized scaffold broadens therapeutic potential for viral inhibitors and tumor-targeting agents.