Isolation of a high affinity scFv from a monoclonal antibody recognising the oncofoetal antigen 5T4

Biochim Biophys Acta. 2000 Dec 15;1524(2-3):238-46. doi: 10.1016/s0304-4165(00)00165-3.


The oncofoetal antigen 5T4 is a 72 kDa glycoprotein expressed at the cell surface. It is defined by a monoclonal antibody, mAb5T4, that recognises a conformational extracellular epitope in the molecule. Overexpression of 5T4 antigen by tumours of several types has been linked with disease progression and poor clinical outcome. Its restricted expression in non-malignant tissue makes 5T4 antigen a suitable target for the development of antibody directed therapies. The use of murine monoclonal antibodies for targeted therapy allows the tumour specific delivery of therapeutic agents. However, their use has several drawbacks, including a strong human anti-mouse immune (HAMA) response and limited tumour penetration due to the size of the molecules. The use of antibody fragments leads to improved targeting, pharmacokinetics and a reduced HAMA. A single chain antibody (scFv) comprising the variable regions of the mAb5T4 heavy and light chains has been expressed in Escherichia coli. The addition of a eukaryotic leader sequence allowed production in mammalian cells. The two 5T4 single chain antibodies, scFv5T4WT19 and LscFv5T4, described the same pattern of 5T4 antigen expression as mAb5T4 in normal human placenta and by FACS. Construction of a 5T4 extracellular domain-IgGFc fusion protein and its expression in COS-7 cells allowed the relative affinities of the antibodies to be compared by ELISA and measured in real time using a biosensor based assay. MAb5T4 has a high affinity, K(D)=1.8x10(-11) M, as did both single chain antibodies, scFv5T4WT19 K(D)=2.3x10(-9) M and LscFv5T4 K(D)=7.9x10(-10) M. The small size of this 5T4 specific scFv should allow construction of fusion proteins with a range of biological response modifiers to be prepared whilst retaining the improved pharmacokinetic properties of scFvs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Antibodies, Monoclonal / biosynthesis
  • Antibodies, Monoclonal / genetics
  • Antibodies, Monoclonal / immunology*
  • Antigens, Neoplasm / immunology
  • Base Sequence
  • Cloning, Molecular
  • Enzyme-Linked Immunosorbent Assay
  • Female
  • Genetic Therapy
  • Humans
  • Immunoglobulin Fragments / genetics
  • Immunoglobulin Fragments / immunology
  • Immunoglobulin Fragments / isolation & purification*
  • Immunoglobulin Variable Region / genetics
  • Immunoglobulin Variable Region / immunology
  • Immunoglobulin Variable Region / isolation & purification*
  • Immunohistochemistry
  • Membrane Glycoproteins / analysis
  • Membrane Glycoproteins / immunology*
  • Molecular Sequence Data
  • Mutation
  • Placenta / immunology
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / genetics
  • Stomach Neoplasms / immunology
  • Surface Plasmon Resonance


  • Antibodies, Monoclonal
  • Antigens, Neoplasm
  • Immunoglobulin Fragments
  • Immunoglobulin Variable Region
  • Membrane Glycoproteins
  • Recombinant Fusion Proteins
  • trophoblastic glycoprotein 5T4, human