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

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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: HuScFvMT99/3BP and HuScFvMT99/3HY. The HuScFvMT99/3BP 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, HuScFvMT99/3HY was designed by combining the VL domain of wild-type with the VH domain of HuScFvMT99/3BP. 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 (ΔGbinding) for both variants compared to HuScFvMT99/3WT. However, ΔGbinding values obtained from AF3 and HD simulations were inconsistent, with HuScFvMT99/3BP exhibiting the weakest binding affinity. While ΔGbinding 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 HuScFvMT99/3BP (0.975 Å) and HuScFvMT99/3HY (1.075 Å) relative to HuScFvMT99/3WT (1.225 Å). Biolayer interferometry further confirmed that HuScFvMT99/3WT exhibited the highest binding affinity (KD = 1.35 × 10⁻⁷ M) compared to HuScFvMT99/3BP (KD = 2.64 × 10⁻⁷ M) and HuScFvMT99/3HY (KD = 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 HuScFvMT99/3HY 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.