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

Area of Science:

• Biotechnology
• Molecular Biology
• Structural Biology

Background:

• Recombinant antibody fragments like 3D8 single-chain variable fragment (3D8 scFv) have therapeutic potential due to their antiviral activity.
• Instability issues such as low solubility and aggregation limit the clinical application of 3D8 scFv.

Purpose of the Study:

• To enhance the stability and production of 3D8 scFv using structure-based in silico modeling and mutagenesis.
• To assess the antiviral efficacy and cytotoxicity of the engineered variants.

Main Methods:

• In silico modeling to identify vulnerable regions in 3D8 scFv.
• Site-directed mutagenesis to create single and double point mutants.
• Expression in E. coli, SDS-PAGE, and size-exclusion chromatography to evaluate stability and aggregation.
• Nuclease activity assays and antiviral assays against human coronavirus OC43.

Main Results:

• Several mutants, including Y101P and S31W;Y101P, exhibited improved monomeric stability and reduced aggregation.
• Mutants Y101P and S31W;Y101P demonstrated significantly enhanced expression yields.
• The engineered variants retained nuclease activity and effectively suppressed human coronavirus OC43 replication.
• No significant cytotoxicity was observed for the tested mutants.

Conclusions:

• Structure-based mutagenesis is an effective strategy to overcome the instability of single-chain variable fragment molecules.
• Engineered 3D8 scFv variants show promise as stable, highly expressed antiviral agents.
• These findings support the advancement of 3D8 scFv for industrial and therapeutic applications.