Engineering of conserved residues near antibody heavy chain complementary determining region 3 (HCDR3) improves both affinity and stability.

Affinity and stability are crucial parameters in antibody development and engineering approaches. Although improvement in both metrics is desirable, trade-offs are almost unavoidable. Heavy chain complementarity determining region 3 (HCDR3) is the best-known region for antibody affinity but its impact on stability is often neglected. Here, we present a mutagenesis study of conserved residues near HCDR3 to elicit the role of this region in the affinity-stability trade-off. These key residues are positioned around the conserved salt bridge between V-K94 and V-D101 which is crucial for HCDR3 integrity. We show that the additional salt bridge at the stem of HCDR3 (V-K94:V-D101:V-D102) has an extensive impact on this loop's conformation, therefore simultaneous improvement in both affinity and stability. We find that the disruption of π-π stacking near HCDR3 (V-Y100E:V-Y49) at the V-V interface cause an irrecoverable loss in stability even if it improves the affinity. Molecular simulations of putative rescue mutants exhibit complex and often non-additive effects. We confirm that our experimental measurements agree with the molecular dynamic simulations providing detailed insights for the spatial orientation of HCDR3. V-V102 right next to HCDR3 salt bridge might be an ideal candidate to overcome affinity-stability trade-off.