Development and Structural Analysis of Novel Bispecific Human IgG1 Antibody platform

Abstract

IgG antibody molecules consist of two heavy chains and two light chains. The distinct feature of natural antibodies is high affinity and specificity for a one molecular target. Antibodies are able to bind antigens with high specificity and affinity. Bispecific antibodies (BsAbs) are artificially modified proteins with two different antigen-binding sites, which enable the antibodies to simultaneously bind to two different epitopes. Due to this advantage, it is currently widely used for therapeutic purposes. There are variety of methods that can be used to create BsAbs (knob-into-hole, electrostatic effect, SEED, IgG4 exchange, CovX-body, Diabodies, etc). These methods usually create BsAbs by creating heterodimerization sites in the CH3 domains of the heavy chains. However, in many cases, due to random assembly of both half antibodies, homodimers are produced, and structural changes by mutations can lead to cause destabilization of the antibodies. Therefore, we designed novel BsAb to not only have high yield but also be similar to structure of the human IgG wild type. We adopted mutation sites to favor heterodimers more than homodimer. In addition, we also considered mutation sites that are predicted not to change the structure of wild-type IgG1. Antibodies could be expressed in both mammalian and prokaryotic expression systems as shown in previous studies. However, mammalian cell system has tendency to produce the antibodies with higher binding activity. Thus, the antibodies were expressed by expiCHO-S cell line, mammalian expression system. In order to increase the purity of the heterodimer, the heterodimeric assembly condition were optimized. The obtained antibody mutants showed purity greater than 98%. In addition, the proteins were crystallized to demonstrate the structure of novel BsAbs. The crystal structure of the novel BsAb protein was compared to wild-type Fc structures to evaluate potential conformational differences with the BsAb Fc structures, and it was compared to the structure of another BsAb.