Enhancing the Protein Stability of an Anticancer VHH-Fc Heavy Chain Antibody through Computational Modeling and
Yuan Fang1,2, Menghua Song2, Tianning Pu2
1State Key Laboratory of Genetics and Development of Complex Phenotypes, Shanghai Engineering Research Center of Industrial Microorganisms, MOE Engineering Research Center of Gene Technology, School of Life Sciences, Fudan University, Shanghai, 200438, China.
Advanced Science (Weinheim, Baden-Wurttemberg, Germany)
|April 24, 2025
Summary
This study introduces a computational pipeline to enhance the stability of therapeutic VHH-Fc fusion antibodies. A designed variant showed significantly improved stability, antigen binding, and production yield.
Area of Science:
- Biotechnology
- Computational Biology
- Protein Engineering
Background:
- Therapeutic VHHs (nanobodies) are often fused to Fc fragments to extend bloodstream half-life.
- Protein stability remains a critical challenge for the commercialization of VHH-Fc fusion antibodies.
- Current methods for enhancing VHH-Fc stability are limited.
Purpose of the Study:
- To develop and validate an in silico pipeline for analyzing and improving the stability of VHH-Fc fusion antibodies.
- To identify key residues impacting the stability of an anticancer VHH-Fc fusion antibody (VFA01).
- To design and characterize stable VFA01 variants with enhanced therapeutic potential.
Main Methods:
- Utilized computational modeling to assess VFA01's conformational stability, disulfide bond reduction, aggregation, and degradation.
- Developed mechanistic models to pinpoint hotspot residues influencing protein stability.
- Designed and generated VFA01 variants based on identified hotspot residues.
Main Results:
- Identified hotspot residues C130, F57, Y106, L120, and W111 affecting VFA01 stability.
- A designed variant, M11 (C130S/W111F/F57K), exhibited significantly enhanced stability compared to VFA01.
- M11 demonstrated a 6.2-fold improvement in DLS size stability, 3.4-fold in SEC HMW%, and 1.5-fold in CE-SDS purity.
- Antigen-binding activity and production yield were also improved approximately 1.5-fold for M11.
Conclusions:
- The developed computational pipeline is a promising approach for enhancing the stability of therapeutic VHH-Fc fusion antibodies.
- Protein engineering strategies guided by computational analysis can effectively improve VHH-Fc stability and function.
- This method holds potential for advancing the development of next-generation antibody therapeutics.
