Remodeling domain interfaces to enhance heterodimer formation

Protein Sci. 1997 Apr;6(4):781-8. doi: 10.1002/pro.5560060404.


An anti-p185HER2/anti-CD3 humanized bispecific diabody was previously constructed from two cross-over single-chain Fv in which YH and VL domains of the parent antibodies are present on different polypeptides. Here this diabody is used to evaluate domain interface engineering strategies for enhancing the formation of functional heterodimers over inactive homodimers. A disulfide-stabilized diabody was obtained by introducing two cysteine mutations, VL L46C and VH D101C, at the anti-p185HER2.VL/VH interface. The fraction of recovered diabody that was functional following expression in Escherichia coli was improved for the disulfide-stabilized compared to the parent diabody (> 96% versus 72%), whereas the overall yield was > 60-fold lower. Eleven "knob-into-hole" diabodies were designed by molecular modeling of sterically complementary mutations at the two VL/VH interfaces. Replacements at either interface are sufficient to improve the fraction of functional heterodimer, while maintaining overall recoverable yields and affinity for both antigens close to that of the parent diabody. For example, diabody variant v5 containing the mutations VL Y87A:F98M and VH V37F:L45W at the anti-p185HER2 VL/VH interface was recovered as 92% functional heterodimer while maintaining overall recovered yield within twofold of the parent diabody. The binding affinity of v5 for p185HER2 extracellular domain and T cells is eightfold weaker and twofold stronger than for the parent diabody, respectively. Domain interface remodeling based upon either sterically complementary mutations or interchain disulfide bonds can facilitate the production of a functional diabody heterodimer. This study expands the scope of domain interface engineering by demonstrating the enhanced assembly of proteins interacting via two domain interfaces.

MeSH terms

  • Antibodies, Bispecific / chemistry*
  • Antibodies, Bispecific / genetics
  • Antibodies, Bispecific / isolation & purification
  • Crystallography, X-Ray
  • Dimerization
  • Humans
  • Mutagenesis
  • Protein Engineering
  • Receptor, ErbB-2 / immunology*


  • Antibodies, Bispecific
  • Receptor, ErbB-2