Regula Joerg Thomas, Imhof-Jung Sabine, Mølhøj Michael, Benz Joerg, Ehler Andreas, Bujotzek Alexander, Schaefer Wolfgang, Klein Christian
Roche Pharmaceutical Research and Early Development, Large Molecule Research, Roche Innovation Center Munich, Penzberg, Germany.
Roche Pharmaceutical Research and Early Development, Chemical Biology, Roche Innovation Center Basel, Basel, Switzerland.
Protein Eng Des Sel. 2018 Jul 1;31(7-8):289-299. doi: 10.1093/protein/gzy021.
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.
双特异性抗体的生产技术需要克服两个主要挑战。第一个是正确的重链组装，这通过“旋钮入孔”技术或CH3结构域中的电荷相互作用得以解决。第二个挑战是正确的轻链组装。这可以通过设计Fab臂界面或应用免疫球蛋白结构域交叉方法来解决。有三种不同的交叉方式是可行的，即Fab臂、恒定结构域和可变结构域交叉。CrossMabCH1-CL交换不会导致意外副产物的形成，而CrossMabFab和CrossMabVH-VL形式会导致典型副产物的形成。因此，CrossMabCH1-CL最初在治疗性抗体开发中受到青睐。在此，我们报告一种新的改进的CrossMab设计原则，该原则利用这些CrossMab恒定结构域中带电荷氨基酸对特异性位置交换，以实现CrossMabVH-VL中正确的轻链组装，并对CrossMabFab设计进行改进。
