Screening and Engineering of Hetero-Bivalent Nanobody Targeting Interleukin-33 with Enhanced Binding Stability

Area of Science:

• Biochemistry
• Immunology
• Molecular Biology

Background:

• Interleukin-33 (IL-33) is an IL-1 family cytokine acting as an alarmin.
• IL-33 signaling through the IL-33/ST2 axis impacts inflammation, immune regulation, and cancer.
• Targeting IL-33 is a potential therapeutic strategy for various diseases.

Purpose of the Study:

• To isolate and engineer IL-33-specific nanobodies with enhanced binding affinity.
• To evaluate the binding characteristics of monovalent and bivalent nanobody constructs.
• To assess the functional impact of engineered nanobodies on IL-33-mediated cellular processes.

Main Methods:

• Phage display library screening for IL-33-specific nanobodies.
• Engineering of monovalent nanobodies into bivalent tandem formats.
• Surface Plasmon Resonance (SPR) for binding kinetics (K_D, dissociation rate).
• Competitive SPR analysis to assess binding site compatibility.
• In vitro wound-healing assay using HT-29 colorectal cancer cells.

Main Results:

• Five monovalent nanobodies demonstrated concentration-dependent binding to IL-33 (K_D: 3.6 × 10^-8 to 2.81 × 10^-7 M).
• The hetero-bivalent construct Nb1-Nb2 exhibited superior apparent binding affinity, primarily due to a reduced dissociation rate.
• Competitive SPR confirmed distinct or minimally overlapping binding epitopes for Nb1 and Nb2.
• Nb1-Nb2 significantly attenuated IL-33-induced wound closure in HT-29 cells under low-serum conditions.

Conclusions:

• Hetero-bivalent engineering effectively enhances the binding affinity of IL-33-targeting nanobodies.
• Nb1-Nb2 represents a potent molecular tool for studying IL-33 biology.
• Engineered nanobodies hold promise for investigating IL-33-associated inflammatory and tumor processes.