The ability of antibodies to acquire affinity-enhancing mutations while maintaining high thermodynamic stability is a hallmark of natural affinity maturation. However, little is known about how remodeling the complementarity-determining regions (CDRs) for high-affinity binding impacts stability and to what extent additional compensatory mutations are required for maintaining antibody thermodynamic stability. To better understand antibody affinity/stability trade-offs, we have evaluated the impact of mutations acquired during affinity maturation of an antibody variable domain specific for the Alzheimer’s Aβ42 peptide. We find that selecting mutations that enhance Aβ binding in the absence of co-selection for stability leads to antibody domains with increased affinity that are strongly destabilized. To overcome such trade-offs, we have developed a methodology for simultaneously co-selecting sets of mutations that enhance antibody affinity while maintaining antibody thermodynamic stability. This approach yields highly evolved, stable antibody domains with increased affinity. Interesting, dissecting such highly evolved antibody domains reveals that several of the key affinity mutations in the CDRs are strongly destabilizing. Computational analysis reveals that some of these mutations increase the hydrophobicity and positive charge of the most important CDR loop (CDR3). Moreover, the destabilizing effects of these affinity mutations are compensated for by distinct sets of stability mutations. Our results demonstrate that the accumulation of unique sets of affinity and stability mutations is necessary to maintain antibody thermodynamic stability during extensive mutagenesis and affinity maturation <i>in vitro</i>, which is similar to findings for natural antibodies that are subjected to somatic hypermutation <i>in vivo</i>. These findings for diverse antibodies and antibody fragments specific for unrelated antigens collectively suggest that the formation of the antigen-binding site is generally a destabilizing process and that co-enrichment for compensatory mutations is critical for maintaining thermodynamic stability.

Efficient antibody affinity maturation requires co-selection of compensatory mutations to maintain thermodynamic stability

253rd National Meeting (2017)

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