Generation of a Panel of Related Human scFv Antibodies With High Affinities for Human CEA

Immunotechnology. 1996 Sep;2(3):181-96. doi: 10.1016/s1380-2933(96)00046-2.


Background: A human single chain Fv (scFv) specific for human carcinoembryonic antigen (CEA) has been isolated from a 2.0 x 10(9) phage display library from unimmunised human donors. The dissociation constant of the scFv has been measured by surface plasmon resonance (SPR) and found to be 7.7 x 10(-9) M, with an off-rate component of 6.2 x 10(-3) s-1. In order to investigate directly whether increased affinity leads to improved targeting of CEA-positive tumours, this scFv has been affinity matured by both targeted mutagenesis of the CDRs of heavy and light chains, and by light chain shuffling.

Study design: A partial randomisation scheme, biased towards amino acids commonly found as somatic mutations of germline antibody sequences, was used for directed diversification of VH and VL CDR3s. Diversification of the entire VL region was also introduced by light chain shuffling of the parental anti-CEA scFv. Selection of the mutagenised repertoires was carried out to enrich for antibodies with a reduced koff.

Results: Sequencing the selected clones identified a number of amino acid changes in the VH CDR3, one of which gave a four-fold reduction in koff. Stringent selection of the light chain shuffled library resulted in several clones with a two- to three-fold reduction in koff. It has been possible to combine the selected changes from both mutagenesis approaches by using the mutagenised heavy chain and a light chain derived by shuffling to give a human scFv with a dissociation constant for human CEA of 6.0 x 10(-10) M.

Conclusion: A panel of human anti-CEA scFvs has been generated with differing dissociation constants for antigen, which will allow the correlation between tumour targeting efficiency in relation to binding affinity to be assessed directly. The scFv panel will be valuable in the optimisation of human antibodies for immunotherapy.

MeSH terms

  • Amino Acid Sequence
  • Antigen-Antibody Reactions / physiology
  • Base Sequence
  • Carcinoembryonic Antigen / metabolism*
  • HeLa Cells
  • Humans
  • Immunoglobulin Fragments / genetics*
  • Immunoglobulin Fragments / isolation & purification
  • Immunoglobulin Fragments / metabolism*
  • Immunoglobulin Variable Region / genetics*
  • Immunoglobulin Variable Region / isolation & purification
  • Immunoglobulin Variable Region / metabolism*
  • Kinetics
  • Molecular Sequence Data
  • Mutagenesis


  • Carcinoembryonic Antigen
  • Immunoglobulin Fragments
  • Immunoglobulin Variable Region