Technologies for the production of bispecific antibodies need to overcome two major challenges. The first one is correct heavy chain assembly, which was solved by knobs-into-holes technology or charge interactions in the CH3 domains. The second challenge is correct light chain assembly. This can be solved by engineering the Fab-arm interfaces or applying the immunoglobulin domain crossover approach. There are three different crossovers possible, namely Fab-arm, constant domain and variable domain crossovers. The CrossMabCH1-CL exchange does not lead to the formation of unexpected side products, whereas the CrossMabFab and the CrossMabVH-VL formats result in the formation of typical side products. Thus, CrossMabCH1-CL was initially favored for therapeutic antibody development. Here, we report a novel improved CrossMab design principle making use of site-specific positional exchanges of charged amino acid pairs in the constant domain of these CrossMabs to enable the correct light chain assembly in the CrossMabVH-VL and improvements for the CrossMabFab design.

中文翻译：

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可变重链-可变轻域和 Fab 臂 CrossMab，具有带电残基交换，以强制正确的轻链组装。


双特异性抗体的生产技术需要克服两大挑战。第一个是正确的重链组装，这是通过旋钮入孔技术或 CH3 结构域中的电荷相互作用解决的。第二个挑战是正确的轻链组装。这可以通过设计 Fab-arm 接口或应用免疫球蛋白结构域交叉方法来解决。存在三种可能的不同交叉，即Fab臂交叉、恒定域交叉和可变域交叉。 CrossMabCH1-CL 交换不会导致意外副产物的形成，而 CrossMabFab 和 CrossMabVH-VL 格式会导致典型副产物的形成。因此，CrossMabCH1-CL 最初受到治疗性抗体开发的青睐。在这里，我们报告了一种新的改进的 CrossMab 设计原理，利用这些 CrossMab 恒定域中带电氨基酸对的位点特异性位置交换，以实现 CrossMabVH-VL 中正确的轻链组装，并改进 CrossMabFab 设计。