REMODELING DOMAIN INTERFACES TO ENHANCE HETERODIMER FORMATION

An anti-p185(HER2)/anti-CD3 humanized bispecific diabody was previousl y constructed from two cross-over single-chain Fv in which V-H and V-L domains of the parent antibodies are present on different polypeptide s. 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 i ntroducing two cysteine mutations, V-L L46C and V-H D101C, at the anti -p185(HER2) V-L/V-H interface. The fraction of recovered diabody that was functional following expression in Escherichia coli was improved f or the disulfide-stabilized compared to the parent diabody (>96% versu s 72%), whereas the overall yield was >60-fold lower. Eleven ''knob-in to-hole'' diabodies were designed by molecular modeling of sterically complementary mutations at the two V-L/V-H interfaces. Replacements at either interface are sufficient to improve the fraction of functional heterodimer, while maintaining overall recoverable yields and affinit y for both antigens close to that of the parent diabody. For example, diabody variant v5 containing the mutations V-L Y87A:F98M and V-H V37F :L45W at the anti-p185(HER2) V-L/V-H interface was recovered as 92% fu nctional heterodimer while maintaining overall recovered yield within twofold of the parent diabody. The binding affinity of v5 for p185(HER 2) extracellular domain and T cells is eightfold weaker and twofold st ronger than for the parent diabody, respectively. Domain interface rem odeling based upon either sterically complementary mutations or interc hain disulfide bonds can facilitate the production of a functional dia body heterodimer. This study expands the scope of domain interface eng ineering by demonstrating the enhanced assembly of proteins interactin g via two domain interfaces.