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

Interleukin-33 (IL-33) is an IL-1 family cytokine that functions as an alarmin and contributes to inflammatory responses, immune regulation, and tumor-associated processes through the IL-33/ST2 signaling axis. In this study, IL-33-specific nanobodies were isolated from a synthetic phage display library and further engineered into bivalent tandem formats to improve their binding performance. Five representative monovalent nanobodies showed concentration-dependent binding to IL-33, with SPR-derived K_D values ranging from 3.6 × 10^-8 to 2.81 × 10^-7 M. Among the engineered bivalent constructs, Nb1-Nb2 exhibited the strongest apparent binding affinity, mainly due to a markedly reduced dissociation rate. Competitive SPR analysis indicated that Nb1 and Nb2 show largely compatible binding to IL-33, consistent with distinct or minimally overlapping binding regions, supporting their selection as a hetero-bivalent pair. In a preliminary wound-healing assay using HT-29 colorectal cancer cells, Nb1-Nb2 attenuated IL-33-induced wound closure under low-serum conditions. These results indicate that hetero-bivalent engineering can enhance the apparent binding affinity of IL-33-targeting nanobodies and provide a useful molecular tool for further investigation of IL-33-associated biological responses.