Production and characterization of bispecific single-chain antibody fragments

Mol Immunol. 1995 Dec;32(17-18):1405-12. doi: 10.1016/0161-5890(95)00089-5.


We report the construction, expression and purification of a bispecific single-chain Fv antibody fragment produced in Escherichia coli. The protein possesses a dual specificity: the single-chain FvB1 portion is directed to the Idiotype of BCL1 lymphoma cells, the single-chain Fv2C11 moiety binds to the CD3 marker on T cells. The two domains are joined by a flexible peptide linker. Using Immobilized Metal Affinity Chromatography, the recombinant protein was purified from bacterial insoluble membrane fractions. After refolding of the bispecific protein, it was affinity-purified. As demonstrated by flow cytometry, both binding sites are retained in the refolded protein. Retargeted cytotoxicity and T cell proliferation assays further prove the biological activity and specificity of the bispecific single-chain Fv. Thus, these bispecific molecules show a potential anti-tumor activity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Bispecific / biosynthesis*
  • Antibodies, Bispecific / chemistry*
  • Antibodies, Bispecific / genetics
  • Base Sequence
  • Cytotoxicity Tests, Immunologic
  • Flow Cytometry
  • Hybridomas / chemistry
  • Immunoglobulin Fab Fragments / biosynthesis*
  • Immunoglobulin Fab Fragments / chemistry*
  • Immunoglobulin Fab Fragments / genetics
  • Immunoglobulin Variable Region / biosynthesis*
  • Immunoglobulin Variable Region / chemistry*
  • Immunoglobulin Variable Region / genetics
  • Male
  • Mice
  • Mice, Inbred BALB C
  • Mice, Inbred C57BL
  • Molecular Sequence Data
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / isolation & purification


  • Antibodies, Bispecific
  • Immunoglobulin Fab Fragments
  • Immunoglobulin Variable Region
  • Recombinant Fusion Proteins