Engineering disulfide-linked single-chain Fv dimers [(sFv')2] with improved solution and targeting properties: anti-digoxin 26-10 (sFv')2 and anti-c-erbB-2 741F8 (sFv')2 made by protein folding and bonded through C-terminal cysteinyl peptides.
Single-chain Fv fusions with C-terminal cysteinyl peptides (sFv') have been engineered using model sFv proteins based upon the 26-10 anti-digoxin IgG and 741F8 anti-c-erbB-2 IgG monoclonal antibodies. As part of the 741F8 sFv construction process, the PCR-amplified 741F8 VH gene was modified in an effort to correct possible primer-induced errors. Genetic replacement of the N-terminal beta-strand sequence of 741F8 VH with that from the FR1 of anti-c-erbB-2 520C9 VH resulted in a dramatic improvement of sFv folding yields. Folding in urea-glutathione redox buffers produced active sFv' with a protected C-terminal sulfhydryl, presumably as the mixed disulfide with glutathione. Disulfide-bonded (sFv')2 homodimers were made by disulfide interchange or oxidation after reductive elimination of the blocking group. Both 26-10 (sFv')2 and 741F8 (sFv')2 existed as stable dimers that were well behaved in solution, whereas 741F8 sFv and sFv' exhibited considerable self-association. The 741F8 sFv binds to the extracellular domain (ECD) of the c-erbB-2 oncogene protein, which is often overexpressed in breast cancer and other adenocarcinomas. The recombinant ECD was prepared to facilitate the analysis of 741F8 binding site properties; the cloned ECD gene, modified to encode a C-terminal Ser-Gly-His6 peptide, was transfected into Chinese hamster ovary cells using a vector that also expressed dihydrofolate reductase to facilitate methotrexate amplification. Optimized cell lines expressed ECD-His6 at high levels in a cell bioreactor; after isolation by immobilized metal affinity chromatography, final ECD yields were as high as 47 mg/l. An animal tumor model complemented physicochemical studies of 741F8 species and indicated increased tumor localization of the targeted 741F8 (sFv')2 over other monovalent 741F8 species.
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics* / Q000276:immunology; D000912:Antibodies, Neoplasm / Q000235:genetics* / Q000276:immunology; D001345:Autoradiography; D001483:Base Sequence; D003545:Cysteine / Q000235:genetics; D004077:Digoxin / Q000276:immunology*; D004220:Disulfides; D007128:Immunoglobulin Fragments / Q000235:genetics* / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000276:immunology; D051379:Mice; D016513:Mice, SCID; D008969:Molecular Sequence Data; D009374:Neoplasms, Experimental / Q000276:immunology; D010446:Peptide Fragments / Q000276:immunology*; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology; D012995:Solubility; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014018:Tissue Distribution
D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D003545:Cysteine; D004077:Digoxin; D004220:Disulfides; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D010446:Peptide Fragments; D018719:Erb-b2 Receptor Tyrosine Kinases; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; C055966:immunoglobulin Fv