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

Protein Eng. 1995 Mar;8(3):301-14. doi: 10.1093/protein/8.3.301.


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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibodies, Monoclonal / genetics*
  • Antibodies, Monoclonal / immunology
  • Antibodies, Neoplasm / genetics*
  • Antibodies, Neoplasm / immunology
  • Autoradiography
  • Base Sequence
  • Cysteine / genetics
  • Digoxin / immunology*
  • Disulfides
  • Immunoglobulin Fragments / genetics*
  • Immunoglobulin Fragments / immunology
  • Immunoglobulin Heavy Chains / genetics
  • Immunoglobulin Variable Region / genetics*
  • Immunoglobulin Variable Region / immunology
  • Mice
  • Mice, SCID
  • Molecular Sequence Data
  • Neoplasms, Experimental / immunology
  • Peptide Fragments / immunology*
  • Polymerase Chain Reaction
  • Protein Conformation
  • Protein Engineering / methods*
  • Protein Folding
  • Receptor, ErbB-2 / immunology*
  • Recombinant Proteins / immunology
  • Solubility
  • Sulfhydryl Compounds / chemistry
  • Tissue Distribution


  • Antibodies, Monoclonal
  • Antibodies, Neoplasm
  • Disulfides
  • Immunoglobulin Fragments
  • Immunoglobulin Heavy Chains
  • Immunoglobulin Variable Region
  • Peptide Fragments
  • Recombinant Proteins
  • Sulfhydryl Compounds
  • immunoglobulin Fv
  • Digoxin
  • Receptor, ErbB-2
  • Cysteine