title: Impact of antibody framework residue VH-71 on the stability of a humanised anti-MUC1 scFv and derived immunoenzyme.
abstract: 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 degrees 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.
authors.affiliation: SAIC, National Cancer Institute at Frederick, Frederick, MD 21702, USA.
authors.forename: J
authors.initials: J
authors.lastname: Krauss
authors.forename: M A E
authors.initials: MA
authors.lastname: Arndt
authors.forename: Z
authors.initials: Z
authors.lastname: Zhu
authors.forename: D L
authors.initials: DL
authors.lastname: Newton
authors.forename: B K
authors.initials: BK
authors.lastname: Vu
authors.forename: V
authors.initials: V
authors.lastname: Choudhry
authors.forename: R
authors.initials: R
authors.lastname: Darbha
authors.forename: X
authors.initials: X
authors.lastname: Ji
authors.forename: N S
authors.initials: NS
authors.lastname: Courtenay-Luck
authors.forename: M P
authors.initials: MP
authors.lastname: Deonarain
authors.forename: J
authors.initials: J
authors.lastname: Richards
authors.forename: S M
authors.initials: SM
authors.lastname: Rybak
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology*; D000915:Antibody Affinity; D003198:Computer Simulation; D004795:Enzyme Stability; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D008565:Membrane Proteins / Q000276:immunology*; D018448:Models, Immunological; D009154:Mutation; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D008565:Membrane Proteins; D011994:Recombinant Proteins; C469215:pemtumomab