Computational design of CAR-scFv linker variants for enhanced CD20 binding against B-cell malignancies

Chimeric Antigen Receptor (CAR)-T cell therapy is a transformative immunotherapy that engineer's T cells to selectively target and eliminate cancer cells. While highly effective in hematological malignancies, challenges such as antigen escape, suboptimal antigen binding, and limited CAR-T cell persistence. Although current FDA-approved CAR-T therapies primarily target CD19 and BCMA, expanding CAR-T technology to target CD20, a key antigen in B-cell malignancies, remains critical. The membrane-proximal location of CD20 and its short extracellular epitopes pose unique binding challenges, potentially reducing CAR-T efficacy. To address this, we employed a molecular modeling strategy using Ofatumumab, a fully human monoclonal antibody, as the framework for engineering CAR-scFvs, focusing on their binding mode, stability, and affinity towards membrane-bound CD20. We investigated the structural and functional impact of incorporating different linker regions, specifically the Whitlow and G₄S₃ linkers, on scFv performance. Molecular dynamics (MD) simulations, free energy landscape analysis and dynamics cross-correlation matrix revealed that scFv-Whitlow maintains a stable dynamic framework with flexible, independently moving V_H and V_L domains, supporting functional robustness, while scFv-G₄S₃ showed dynamics with preserved structural integrity. Molecular docking and MD simulations identified the extracellular loop 2 (ECL2) of CD20 as a key interaction site. Notably, binding energy calculations indicated higher binding affinity of scFv-Whitlow (-37.12 kcal/mol) for CD20 compared to scFv-G₄S₃ (-27.97 kcal/mol), highlighting its superior interaction dynamics. These findings position scFv-Whitlow as a promising candidate for anti-CD20 CAR-T cell therapy and provide valuable insights for the rational design of next-generation CAR constructs targeting CD20.