We have reported that E6F6, a mouse monoclonal antibody, is a promising treatment option for patients with chronic hepatitis B (CHB). A humanized E6F6 antibody B11 with affinity loss was obtained by CDR-grafting approach. To address this issue, in silico affinity maturation through scanning mutagenesis using CHARMM force field methods was performed on an predicted immune complex model of the B11:HBsAg. We chose four variants with top increased interaction energy for further characterization. The antibody huE6F6-1 within two point mutations (Heavy Chain: Asp65Val; His66Leu) was identified to restore the parental antibody's high binding affinity, neutralization activity, and potent efficacy of viral suppression in vivo. Crystal structure (1.8 Å resolution) based molecular docking proved more stabilized and compact hydrogen bond interactions formed in huE6F6-1.The smaller and dispersed HBV immune complexes of huE6F6-1 by electron microscopy suggested it will have the same therapeutic efficacy as the parental E6F6 mAb. Preclinical study and pharmacokinetics of huE6F6-1 demonstrated that it is a stable and desirable lead candidate to improve the clinical management of CHB. Notably, our structure guided approach may facilitate the humanization and affinity maturation of other rodent antibody candidates during drug development.

我们已经报道，小鼠单克隆抗体E6F6对于慢性乙型肝炎（CHB）患者是一种有前途的治疗选择。通过CDR移植法获得了具有亲和力丧失的人源化E6F6抗体B11。为了解决此问题，对B11：HBsAg的预测免疫复合物模型通过使用CHARMM力场方法进行扫描诱变，进行了计算机亲和力成熟。我们选择了四种具有最大相互作用能量的变体进行进一步表征。鉴定出两个点突变（重链：Asp65Val； His66Leu）内的抗体huE6F6-1可恢复亲本抗体的高结合亲和力，中和活性和体内病毒抑制的有效功效。基于晶体结构（1.8Å分辨率）的分子对接证明在huE6F6-1中形成更稳定和紧密的氢键相互作用。huE6F6-1较小且分散的HBV免疫复合物表明，它将具有与亲本E6F6相同的治疗功效单抗 huE6F6-1的临床前研究和药代动力学证明，它是改善CHB临床管理的稳定且理想的潜在候选药物。值得注意的是，我们的结构指导方法可能会促进药物开发过程中其他啮齿动物抗体候选物的人源化和亲和力成熟。