Mutational landscape of antibody variable domains reveals a switch modulating the interdomain conformational dynamics and antigen binding

Abstract

Somatic mutations within the antibody variable domains are critical to the immense capacity of the immune repertoire. Here, via a deep mutational scan, we dissect how mutations at all positions of the variable domains of a high-affinity anti-VEGF antibody G6.31 impact its antigen-binding function. The resulting mutational landscape demonstrates that large portions of antibody variable domain positions are open to mutation, and that beneficial mutations can be found throughout the variable domains. We determine the role of one antigen-distal light chain position 83, demonstrating that mutation at this site optimizes both antigen affinity and thermostability by modulating the interdomain conformational dynamics of the antigen-binding fragment. Furthermore, by analyzing a large number of human antibody sequences and structures, we demonstrate that somatic mutations occur frequently at position 83, with corresponding domain conformations observed for G6.31. Therefore, the modulation of interdomain dynamics represents an important mechanism during antibody maturation in vivo. Deciphering the molecular mechanism of SHMs in improving antibodies is critical for understanding the evolution of the immune repertoire. It is well recognized that mutations at the CDRs or FWR structurally adjacent to the CDRs can modulate and optimize the antigen-binding interface (4-7), and several studies have used saturated mutagenesis of CDR position to explore the effect of various mutations on affinity and specificity (8-11).The role of SHM in antigen-distal FWR is less well understood, however. Previous studies have suggested that the antigen-distal FWR contributes primarily to the variable domain structural integrity; thus, mutations at these positions would be either detrimental to or neutral for antigen-binding function (12)(13)(14). Other authors have characterized the potential of antigen-distal framework mutation as beneficial for antigen-binding function. For example, framework mutations can compensate for the destabilizing effect of mutations at CDRs needed for antigen binding (15). In other cases, non-CDR SHMs have been shown to be required for the neutralization activity of broadly neutralizing anti-HIV antibodies (16).Thus far, the roles of SHM have been studied primarily by examining the functional consequences of reverting the somatic mutation of selected antibodies back to the germline sequences (15-21). Although these studies have demonstrated that antibodies depend on somatic mutations to achieve high-affinity antigen binding, the approach is limited to the small set of mutations present in a given antibody.Here we took a systematic approach to assessing the mutability of the whole variable domain for maintaining or improving folding stability and antigen binding. We used a welloptimized anti-vascular endothelial cell growth factor (VEGF), G6.31, with high affinity (K d = 0.4 nM) and excellent thermostability [fragment antigen-binding (Fab) melting temperature (T m ) = 84°C]. G6.31 derives its HC and LC variable domains ...

High-throughput antibody engineering in mammalian cells by CRISPR/Cas9-mediated homology-directed mutagenesis

“…One of the most exciting applications of HDM is the ease at which we can perform DMS, a new technique in protein engineering that uncovers sequence–function relationships. DMS relies on the construction of saturation mutagenesis libraries, which requires PCR mutagenesis or synthetic genes and cloning into plasmid expression vectors, thus most DMS studies have been performed in bacteria, phage and yeast ( 24 , 37 , 63 , 64 ). One previous example performed DMS on an antibody clone using plasmid transfection in mammalian cells ( 65 ); however, the transient presence and polyclonality of plasmids make this a challenging approach.…”

Insights into the Structural Basis of Antibody Affinity Maturation from Next-Generation Sequencing

“…To systematically dissect the contribution of mutations in FR residues to affinity maturation, deep mutational scanning was applied to the anti-vascular endothelial cell growth factor antibody G6.31 ( 50 ). A number of FR mutations at positions distal to the CDRs were found to improve the affinity and/or thermostability of G6.31.…”

“…A number of FR mutations at positions distal to the CDRs were found to improve the affinity and/or thermostability of G6.31. In particular, the FR mutation V L Phe83→Ala, which is ~25 Å away from the antigen-binding site, increased both affinity and stability by altering the orientation of the constant domains (C L and C H 1) relative to V L and V H , as well as the orientation of V L to V H ( 50 ). As measured by HDX/MS, the V L Phe83→Ala mutation modulated the interdomain conformational dynamics of antibody G6.31.…”

EFab domain substitution as a solution to the light-chain pairing problem of bispecific antibodies

“…23 Mutations in this region distal from the antigen-binding site have been shown to negatively affect thermo-stability, binding affinity, and bispecific activity by modulating the interdomain conformational dynamics. 24,25 In spite of the structural effects of EFab domain substitution, the monovalent binding kinetics of adalimumab EFab was only two-fold worse than adalimumab to its antigen, human TNF.…”