As the current biotherapeutic market is dominated by antibodies, the design of different antibody formats, like bispecific antibodies, represent a key component in advancing antibody therapy. Bispecific antibodies contain two different antigen-binding sites and thus can recognize two distinct antigens at the same time. Cognate, immunoglobulin G antibodies depend on homodimerization of the fragment crystallizable regions of two identical heavy chains. By modifying the CH3-CH3 interface, with different mutations on each domain, the engineered Fc fragments form rather heterodimers than homodimers. Here, we use classical molecular dynamics simulations and apply different biophysical analysis techniques to identify key interactions in the CH3-CH3, CH1-CL and VH-VL interfaces, that contribute to their stability and tendency to heterodimerize. Additionally, we compare CH3-CH3 domains, to the structurally similar CH1-CL interfaces in antigen-binding fragments (Fabs). The CH1-CL domain 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. By comparing CH3-CH3 and CH1-CL dimers with each other we find similar interaction patterns. However, we also observe pairing specific interface interactions and show that also different combinations of heavy chains with different light chain isoforms result in characteristic interface contacts. Additionally, we present exemplary dissociation mechanisms for both CH3-CH3 and CH1-CL dimers and identify key interactions that contribute to stability and their tendency to heterodimerize.<br/><br/>While CH1-CL interfaces are dominated by hydrophobic interactions, we find that the CH3-CH3 interfaces are stabilized by numerous salt bridge interactions surrounding the hydrophobic core. Furthermore, we provide quantitative difference contact maps between CH1-CL and CH3-CH3 domains, which highlight which strands are key determinants for their structural integrity.<br/><br/>Thus, these findings have broad implications in the field of antibody engineering as they provide a structural and mechanistic understanding of antibody interfaces and thereby present a crucial aspect for the design of bispecific antibodies.

Biophysics of immunoglobulin fold interfaces in IgGs and new formats

ACS Spring 2022

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