MOLECULAR DYNAMICS ANALYSIS REVEALED THE ENHANCED STABILITY AND SPECIFICITY OF SCFV-CYTOKINE COMPLEXES TARGETING PRIMARY TARGETS

Abstract

Single-chain variable fragments (scFvs) are promising alternatives to full-length monoclonal antibodies because of their small size, high specificity, and improved tissue penetration. However, potential cross-reactivity with structurally related proteins may limit their therapeutic applicability. In the present study, an in-silico workflow integrating protein structure prediction, protein–protein docking, and molecular dynamics (MD) simulations were employed to evaluate the binding specificity and structural stability of murine anti-IL-6 scFv and anti-TNF-α scFv toward their primary cytokine targets and related off-target cytokines. Predicted scFv structures were docked with IL-6, IL-11, TNF-α, and TNF-β, followed by 200ns MD simulations of the resulting complexes. Comparative analyses were performed using root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), solvent-accessible surface area (SASA), and hydrogen-bonding profiles. Complexes formed with the primary targets showed lower structural deviation and greater conformational stability than cross-reactive complexes. The anti-IL-6 scFv–IL-6 and anti-TNF-α scFv–TNF-α systems exhibited lower average RMSD values than their corresponding control complexes with IL-11 and TNF-β. In addition, primary target complexes demonstrated more stable intermolecular interactions, improved compactness, and reduced residue-level fluctuations at key binding regions. Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) analysis suggested that the scFv showed more efficient binding with its primary target. Overall, these findings suggest that the studied scFvs displayed preferential binding and enhanced dynamic stability toward their intended cytokine targets. These computational studies may provide a structural basis for future experimental validation and rational engineering of antibody fragments which targets the respective cytokines. Both TNF-α and IL-6 are important cytokines, and their levels are elevated in many chronic diseases. Blocking them looks like a promising way to treat these diseases and blocking their activities by scFv may provide alternative ways to treat these diseases.