Enhancing the Affinity of a Novel Selective scFv for Soluble ST2 through Computational Design

Suppression of Tumorigenicity 2 (ST2) is a member of
the IL-1 receptor
family, which includes transmembrane (ST2L) and soluble (sST2) isoforms.
sST2 functions as a decoy receptor for Interleukin-33 (IL-33), thereby
blocking the activation of the IL-33/ST2L signaling axis, which is
essential for tissue repair and immune regulation. Clinical evidence
indicates that elevated sST2 levels are associated with increased
disease severity in conditions such as ulcerative colitis (UC), cardiovascular
disease, and asthma. However, current antibodies cannot reliably distinguish
between sST2 and its membrane-bound isoform ST2L, limiting their effectiveness
for diagnostic and therapeutic use. To address this limitation, we
developed an antibody that selectively targets sST2. Using a phage
display library, we identified a single-chain variable fragment (scFv)
with high specificity for a unique five amino acid sequence (SKECF)
located at the C-terminus of sST2. Our parental scFv showed high selectivity
for sST2 with minimal cross-reactivity to ST2L, as demonstrated by
both flow cytometry and immunoprecipitation. Molecular simulations
identified key binding residues, allowing the design of four scFv
mutants, three of which displayed improved binding in surface plasmon
resonance (SPR) analyses. The A183YL2 mutant exhibited
a 3.4-fold increase in binding affinity, while G100WH3 demonstrated
reduced binding due to unfavorable conformations. This study presents
an anti-sST2 scFv with enhanced specificity and affinity, offering
a promising tool for the diagnosis and treatment of inflammatory diseases,
in which sST2 interferes with IL-33-mediated tissue repair.