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.
Highlights
Antibodies play a central role in the adaptive immune system, as they can recognize and neutralize foreign antigens (Chiu et al, 2019)
We observe structural differences in the overall architecture between the CH1-CL/CH3-CH3 and the VH-VL domains, as the VH-VL domains differ in their number of strands (9 β-strands arranged in two sheets of 4 and 5 strands), and in their relative orientation between the VH and VL monomers with respect to each other
By using molecular dynamics simulations, we find substantial differences in interaction patterns of the structurally highly similar CH1-CL and CH3-CH3 interfaces
Summary
Antibodies play a central role in the adaptive immune system, as they can recognize and neutralize foreign antigens (Chiu et al, 2019). The immunoglobulin heavy and light chains are composed of various discrete protein domains. Interesting is that these domains all have a similar folded structure, which is known as the immunoglobulin fold (Chiu et al, 2019). Even though they share a similar fold, there are distinct structural differences between these domains (Figure 1). The CH3 domains bind tightly with each other by hydrophobic interactions at the center, surrounded by salt bridges and thereby forming the foundation for the heavy chain dimer association (Teplyakov et al, 2013). Mutations in the CH3CH3 interface have been shown to strongly influence the stability and the association of the two domains (Rose et al, 2013)
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