Structure‐Guided Engineering of 3D8 scFv Enhances Stability and Antiviral Potential

The instability of recombinant antibody fragments hinders their therapeutic development. Single-chain variable fragment 3D8 (3D8 scFv), which possesses nucleic acid-hydrolyzing activity, exhibits broad-spectrum antiviral potential; however, its low solubility, rapid aggregation, and inconsistent production restrict its clinical application. Therefore, we used structure-based in silico modeling to identify hydrophobic residues and structurally vulnerable regions within 3D8 scFv, followed by mutagenesis. Nine single-point mutants (Y101P, Y101E, S31W, S31E, Y235V, V33F, V33I, Y174F, and V93A) and one double mutant (S31W;Y101P) were generated and expressed in Escherichia coli. SDS-PAGE and size-exclusion chromatography revealed improved monomeric stability and reduced aggregation of several variants. Y101P and S31W;Y101P showed significantly enhanced expression yields while retaining nuclease activity. These mutants effectively suppress human coronavirus OC43 replication without inducing cytotoxicity. Collectively, mutagenesis can overcome the structural limitations of scFv molecules, and our findings provide a foundation for advancing 3D8 scFv as an antiviral candidate for industrial and therapeutic use.