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Impact of antibody framework residue V(H)-71 on the stability of a humanised anti-MUC1 scFv and derived immunoenzyme

Anti-MUC1 single-chain Fv (scFv) fragments generated from the humanised antibody huHMFG1 had adequate antigen-binding properties but very poor stability irrespective of the applied linker or domain orientation. Mutagenesis of heavy-chain framework residue V(H)-71, previously described as a key resid...

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Detalles Bibliográficos
Autores principales: Krauss, J, Arndt, M A E, Zhu, Z, Newton, D L, Vu, B K, Choudhry, V, Darbha, R, Ji, X, Courtenay-Luck, N S, Deonarain, M P, Richards, J, Rybak, S M
Formato: Texto
Lenguaje:English
Publicado: Nature Publishing Group 2004
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2409732/
https://www.ncbi.nlm.nih.gov/pubmed/15150594
http://dx.doi.org/10.1038/sj.bjc.6601759
Descripción
Sumario:Anti-MUC1 single-chain Fv (scFv) fragments generated from the humanised antibody huHMFG1 had adequate antigen-binding properties but very poor stability irrespective of the applied linker or domain orientation. Mutagenesis of heavy-chain framework residue V(H)-71, 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 MUC1(+) tumour cells with an IC(50) of 80 nM. We expect this novel immunoenzyme to become a promising tool for the treatment of MUC1(+) malignancies.