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

Arslan M., Uluçay T., Kale S., Kalyoncu S.

Biochimica et Biophysica Acta - Proteins and Proteomics, vol.1871, no.4, 2023 (SCI-Expanded) identifier identifier

  • Publication Type: Article / Article
  • Volume: 1871 Issue: 4
  • Publication Date: 2023
  • Doi Number: 10.1016/j.bbapap.2023.140915
  • Journal Name: Biochimica et Biophysica Acta - Proteins and Proteomics
  • Journal Indexes: Science Citation Index Expanded (SCI-EXPANDED), Scopus, Academic Search Premier, BIOSIS, Biotechnology Research Abstracts, CAB Abstracts, Chemical Abstracts Core, EMBASE, Food Science & Technology Abstracts, MEDLINE, Veterinary Science Database
  • Keywords: Affinity-stability trade-offs, Antibody, Antibody engineering, Molecular dynamics, Vernier zone
  • Dokuz Eylül University Affiliated: No


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 VH-K94 and VH-D101 which is crucial for HCDR3 integrity. We show that the additional salt bridge at the stem of HCDR3 (VH-K94:VH-D101:VH-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 (VH-Y100E:VL-Y49) at the VH-VL 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. VH-V102 right next to HCDR3 salt bridge might be an ideal candidate to overcome affinity-stability trade-off.