Comparing Antibody Interfaces to Inform Rational Design of New Antibody Formats

As the current biotherapeutic market is dominated by antibodies, the design of different antibody formats, like bispecific antibodies and other new formats, represent a key component in advancing antibody therapy. When designing new formats, a targeted modulation of pairing preferences is key. Several existing approaches are successful, but expanding the repertoire of design possibilities would be desirable. Cognate immunoglobulin G antibodies depend on homodimerization of the fragment crystallizable regions of two identical heavy chains. By modifying the dimeric interface of the third constant domain (C 3-C 3), with different mutations on each domain, the engineered Fc fragments form rather heterodimers than homodimers. The first constant domain (C 1-C ) shares a very similar fold and interdomain orientation with the C 3-C 3 dimer. Thus, numerous well-established design efforts for C 3-C 3 interfaces, have also been applied to C 1-C dimers to reduce the number of mispairings in the Fabs. Given the high structural similarity of the C 3-C 3 and C 1-C domains we want to identify additional opportunities in comparing the differences and overlapping interaction profiles. Our vision is to facilitate a toolkit that allows for the interchangeable usage of different design tools from crosslinking the knowledge between these two interface types. As a starting point, here, we use classical molecular dynamics simulations to identify differences of the C 3-C 3 and C 1-C interfaces and already find unexpected features of these interfaces shedding new light on possible design variations. Apart from identifying clear differences between the similar C 3-C 3 and C 1-C dimers, we structurally characterize the effects of point-mutations in the C 3-C 3 interface on the respective dynamics and interface interaction patterns. Thus, this study has broad implications in the field of antibody engineering as it provides a structural and mechanistical understanding of antibody interfaces and thereby presents a crucial aspect for the design of bispecific antibodies.