Optimization of Knobs-into-holes Structure of Bispecific Antibody based on Molecular Dynamics Simulation

Objective: To evaluate the stability and binding efficiency of CH3-CH3 heterodimer assembly generated by knobs-into-holes (KIH) method, and to lay the foundation for designing more stable bispecific antibodies by optimizing the structure. Methods: Molecular dynamics simulations coupled with molecular mechanics Poisson Boltzmann surface area (MM-PBSA) analysis were performed to study the interaction of CH3-CH3 heterodimer produced by KIH method. The mammalian expression system, human embryonic kidney cell-F clone cells (293F), were transfected with polyethylenimine (PEI) to express the KIH and the optimized structure lock-key. Proteins were collected and purified from the cell supernatant, and the binding efficiency of heterologous heavy chains was examined by SDS-PAGE followed by silver staining. Results: Compared with a wild-type (WT) antibody at the temperature of 355K, the thermal stability of CH3-CH3 heterodimer generated by KIH decreased. Root-mean-square deviation (RMSD) of backbone atoms in the CH3 domain of WT and KIH antibody were about 1.0Å and 2.0Å, respectively. Significant root-mean-square fluctuation (RMSF) increases could be observed at 3-27, 45-52, and 70-80 residues of the knob part in KIH. Moreover, CH3-CH3 heterodimer of KIH exhibited higher relative free binding energy than WT (107.0082kJ/mol vs 79.8649kJ/mol). Ten amino acid residues located at the CH3 binding interface of KIH had little positive contribution to the binding of CH3-CH3. The binding efficiency of CH3-CH3 heterodimer of KIH was just about 80%, which was significantly lower than that of WT. Further, we designed a novel structure of bispecific antibodies named lock-key that promoted the formation of CH3-CH3 heterodimer by optimizing the 10 residues with poor interactions of KIH. Conclusions: The thermal stability and binding efficiency of CH3-CH3 heterodimer produced by KIH method were not as good as WT. Ten residues lack of stability and had little contribution to the binding have been found. A lock-key structure with a higher binding efficiency of heterologous heavy chain dimerization of bispecific antibodies was constructed by optimizing these 10 residues of KIH. Our findings will be beneficial to improve the stability of bispecific antibodies.