Humanized monoclonal antibodies (mAbs) are among the most promising modern therapeutics, but defined engineering strategies are still not available. Antibody humanization often leads to a loss of affinity, as it is the case for our model antibody Ab2/3H6 (PDB entry 3BQU). Identifying appropriate back-to-mouse mutations is needed to restore binding affinity, but highly challenging. In order to get more insight, we have applied molecular dynamics simulations and correlated them to antibody binding and expression in wet lab experiments. In this study, we discuss six mAb variants and investigate a tyrosine conglomeration, an isopolar substitution and the improvement of antibody binding towards wildtype affinity. In the 3D structure of the mouse wildtype, residue R94h is surrounded by three tyrosines which form a so-called 'tyrosine cage'. We demonstrate that the tyrosine cage has a supporting function for the CDRh3 loop conformation. The isopolar substitution is not able to mimic the function appropriately. Finally, we show that additional light chain mutations can restore binding to wildtype-comparable level, and also improve the expression of the mAb significantly. We conclude that the variable light chain of Ab2/3H6 is of underestimated importance for the interaction with its antigen mAb 2F5.

中文翻译：

---

将湿式实验室实验与分子模拟相结合以改进单克隆抗体人源化的经验教训。


人源化单克隆抗体 (mAb) 是最有前途的现代疗法之一，但仍然没有明确的工程策略。抗体人源化通常会导致亲和力丧失，我们的模型抗体 Ab2/3H6（PDB 条目 3BQU）就是这种情况。需要识别适当的回鼠突变来恢复结合亲和力，但极具挑战性。为了获得更多见解，我们应用了分子动力学模拟，并将其与湿实验室实验中的抗体结合和表达相关联。在这项研究中，我们讨论了六种 mAb 变体，并研究了酪氨酸聚集、同极取代以及抗体与野生型亲和力结合的改进。在小鼠野生型的 3D 结构中，残基 R94h 被三个酪氨酸包围，形成所谓的“酪氨酸笼”。我们证明酪氨酸笼对 CDRh3 环构象具有支持功能。同极性取代不能适当地模拟该功能。最后，我们表明额外的轻链突变可以将结合恢复到与野生型相当的水平，并且还可以显着提高 mAb 的表达。我们得出的结论是，Ab2/3H6 的可变轻链对于与其抗原 mAb 2F5 的相互作用的重要性被低估了。