Usage of a split CH2 domain as building block for new antibody formats

Antibodies, and in particular bi- or multispecific antibodies, are the most promising candidates for new therapy options in cancer or inflammatory diseases. So far, multiple different antibody variants containing only distinct parts of antibodies, e.g. the variable domains (Fv), and a plethora of bi- and multispecific antibody formats have been described. The vast majority of these formats have been created using classical protein engineering strategies, i.e. by combining or changing protein domains or introducing distinct mutations into the protein chains to achieve formation of the envisaged proteins. Antibodies consist of different domains that can fold independently into a three-dimensional structure. Here we have focused on the constant domain 2 of the heavy chain (CH2) that has no direct contacts to other domains in an antibody architecture. As protein complementation is a well-established methodology, we asked whether it is possible to split and reassemble the CH2 domain. Here we demonstrate that it is possible to split the CH2 domain into two parts, fuse the two CH2 domain parts to different antibody domains, and show that the split CH2 parts are able to reassemble into functional proteins. The usage of the split CH2 domain approach as a building block offers new opportunities for the design of bi- or multispecific antibody variants.