Plasticity of BioPhi-driven humanness optimization in ScFv-CD99 binding affinity validated through AlphaFold, HADDOCK, and MD simulations

BioPhi-guided humanization was utilized to enhance the humanness of a humanized single-chain variable fragment targeting CD99, leading to the development of two variants: HuScFvMT99/3^BP and HuScFvMT99/3^HY. The HuScFvMT99/3^BP variant incorporated framework region modifications, leading to modest improvements in humanness, particularly in the VH domain, although the VL domain remained suboptimal. To address this limitation, HuScFvMT99/3^HY was designed by combining the VL domain of wild-type with the VH domain of HuScFvMT99/3^BP. Molecular dynamics simulations employing AlphaFold2, AlphaFold3, and HADDOCK were performed to evaluate the HuScFv-CD99 peptide complexes. AF2-based simulations demonstrated enhanced binding free energy (ΔG_binding) for both variants compared to HuScFvMT99/3^WT. However, ΔG_binding values obtained from AF3 and HD simulations were inconsistent, with HuScFvMT99/3^BP exhibiting the weakest binding affinity. While ΔG_binding patterns derived from AlphaFold3 and HADDOCK simulations aligned, amino acid decomposition analysis revealed variations in the interaction coordinates of the predicted complexes. Root-mean-square deviation analysis indicated improved structural stability for HuScFvMT99/3^BP (0.975 Å) and HuScFvMT99/3^HY (1.075 Å) relative to HuScFvMT99/3^WT (1.225 Å). Biolayer interferometry further confirmed that HuScFvMT99/3^WT exhibited the highest binding affinity (K_D = 1.35 × 10⁻⁷ M) compared to HuScFvMT99/3^BP (K_D = 2.64 × 10⁻⁷ M) and HuScFvMT99/3^HY (K_D = 3.95 × 10⁻⁷ M). Supporting evidence was provided by ELISA and flow cytometry experiments. PITHA analysis revealed a high immunogenicity risk for all variants, despite HuScFvMT99/3^HY displaying improved humanness, a larger complementarity-determining region (CDR) cavity, and a more hydrophobic CDR-H3 loop. These findings highlight the delicate balance between enhancing humanness and preserving the structural and functional integrity critical for therapeutic antibody development.