Comparing Antibody Interfaces to Inform Rational Design of New Antibody Formats

Abstract

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 (CH3-CH3), with different mutations on each domain, the engineered Fc fragments form rather heterodimers than homodimers. The first constant domain (CH1-CL) shares a very similar fold and interdomain orientation with the CH3-CH3 dimer. Thus, numerous well-established design efforts for CH3-CH3 interfaces, have also been applied to CH1-CL dimers to reduce the number of mispairings in the Fabs. Given the high structural similarity of the CH3-CH3 and CH1-CL 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 CH3-CH3 and CH1-CL interfaces and already find unexpected features of these interfaces shedding new light on possible design variations. Apart from identifying clear differences between the similar CH3-CH3 and CH1-CL dimers, we structurally characterize the effects of point-mutations in the CH3-CH3 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.

Bispecific antibodies—effects of point mutations on CH3-CH3 interface stability

“…2 ). This is in line with previous studies, which show that the C H 3-C H 3 interface is mainly characterized by salt bridge interactions, while the hydrophobic contacts are dominantly present in the structurally similar C H 1-C L domains ( Fernández-Quintero et al. , 2022 ).…”

Evolution of the Immunoglobulin Isotypes—Variations of Biophysical Properties among Animal Classes

“…On the other side, few charged residues are present in the G strand of IgG C H 1 domains, but no other modification is noticeable. In fact, as literature confirms, while the IgG C H 3-C H 3 interface is mainly characterized by salt bridges, the C H 1-C L interface is mostly hydrophobic with the only salt bridge present between the G strand and the AB-turn [98,99]. The light chain isotypes are quite similar, with only few distinctions observed in the BC and DE strands.…”

Structural mechanism of Fab domain dissociation as a measure of interface stability

“…For this reason, several studies have been carried out to mutate the interface residues and, on one side, ensure the correct chain pairing, but also increase the overall protein stability [ 4 , 19 ]. It is therefore important to characterize the interactions that take place in the antibodies’ interface, to avoid functionality or developability issues [ 69 ].…”

“…Interdomain contacts are key determinants for stability. Therefore, we studied the constant domain interface, showing that it is mainly characterized by hydrophobic interactions and H-bonds [ 69 ]. The β-strands A, B, D, E and the loops AB and DE in both chains are mainly responsible for the interdomain interactions, since they are in the center of the interface (Fig.…”