Area of Science:

• Biotechnology
• Computational Biology
• Immunology

Background:

• Single-chain variable fragment (scFv) antibodies are promising for diagnostics and therapeutics, particularly for cancer.
• Optimizing scFv expression, solubility, and affinity requires strategic design, with variable domain order being a key factor.
• The ideal VL-VH domain orientation can vary significantly between different scFvs.

Purpose of the Study:

• To computationally evaluate how variable domain orientation affects scFv structure, stability, and antigen-binding affinity.
• To investigate the impact of VL-VH order on anti-HER2 and anti-IL-1β scFv-antigen interactions.
• To provide a predictive framework for designing high-affinity scFvs for biotechnological applications.

Main Methods:

• Utilized computer simulations, including molecular dynamics (MD) simulations for 100 ns.
• Employed the Molecular Mechanics-Poisson-Boltzmann Surface Area (MM-PBSA) method to calculate binding free energies.
• Modeled anti-HER2 scFv and anti-IL-1β scFv constructs with different domain orientations (VL-VH and VH-VL).

Main Results:

• MD simulations confirmed the stability and compactness of both scFv constructs.
• Anti-HER2 scFv constructs (VL-VH and VH-VL) exhibited similar binding affinities to HER2.
• The VH-VL orientation of anti-IL-1β scFv demonstrated a more favorable binding free energy, suggesting higher affinity for IL-1β.

Conclusions:

• Variable domain orientation is a critical parameter influencing scFv binding affinity.
• The VH-VL orientation may enhance binding affinity for certain scFvs, such as anti-IL-1β.
• In silico methods can effectively guide experimental design for developing targeted scFv-based biotechnologies.