Impact of antibody framework residue VH-7I on the stability of a humanised anti-MUCI scFv and derived immunoenzyme

Abstract

Anti-MUCI single-chain Fv (scFv) fragments generated from the humanised antibody huHMFGI had adequate antigen-binding properties but very poor stability irrespective of the applied linker or domain orientation. Mutagenesis of heavy-chain framework residue VH-7I, previously described as a key residue for maintaining the CDR-H2 main-chain conformation and thus important for antigen binding, markedly stabilised the scFv while having only a minor effect on the binding affinity of the molecule. Because of its improved stability, the engineered fragment exhibited immunoreactivity with tumour cells even after 7 days of incubation in human serum at 37°C. It also showed, in contrast to the wild-type scFv, a concentration-dependent binding to the target antigen when displayed on phage. When fusing the scFv to the recombinant ribonuclease rapLRI, only the fusion protein generated with the stable mutant scFv was able to kill MUCI+ tumour cells with an IC50 of 80 nM. We expect this novel immunoenzyme to become a promising tool for the treatment of MUCI+ malignancies. © 2004 Cancer Research UK.