(660e) Affinity-Enhancing Mutations in Different Antibody Complementarity-Determining Regions Display Unique Affinity/Stability Trade-Offs

The natural process by which germline antibodies acquire somatic mutations to mediate specific recognition of target antigens must be balanced by mutations that compensate for the destabilizing effects of remodeling the complementarity-determining regions (CDRs) for high-affinity binding. However, little is known about affinity/stability trade-offs for mutations that accumulate within the CDRs and framework regions of antibodies during affinity maturation. We have evaluated the contribution of mutations that were acquired during directed evolution of an antibody variable (V_H) domain specific for the Alzheimerâ??s Aβ42 peptide that was initially grafted with Aβ residues 33-42 in CDR3. We find that the most important binding mutation is located in the grafted Aβ peptide in CDR3. The other key binding mutations were also located in the CDRs. Importantly, the binding mutations in CDR3 did not significantly impact stability, while those binding mutations in other CDRs reduced stability. The destabilizing effects of the binding mutations were compensated for by multiple stabilizing mutations. Our findings demonstrate that affinity-enhancing mutations in CDR3 of an autonomous human V_H domain typically lack affinity/stability trade-offs, while mutations in the other CDRs and framework regions typically display such trade-offs. We expect that these findings will lead to improved methods for designing antibodies and libraries thereof with reduced affinity/stability trade-offs, which is important for the reliable generation of antibody fragments and full-length antibodies with high stability in addition to high affinity.