Variable heavy–variable light domain and Fab-arm CrossMabs with charged residue exchanges to enforce correct light chain assembly

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 CrossMab(CH1–CL) exchange does not lead to the formation of unexpected side products, whereas the CrossMab(Fab) and the CrossMab(VH–VL) formats result in the formation of typical side products. Thus, CrossMab(CH1–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 CrossMab(VH–VL) and improvements for the CrossMab(Fab) design.