Improving antibody affinity by mimicking somatic hypermutation in vitro

Nat Biotechnol. 1999 Jun;17(6):568-72. doi: 10.1038/9872.

Abstract

In vivo affinity maturation of antibodies involves mutation of hot spots in the DNA encoding the variable regions. We have used this information to develop a strategy to improve antibody affinity in vitro using phage display technology. In our experiment with the antimesothelin scFv, SS(scFv), we identified DNA sequences in the variable regions that are naturally prone to hypermutations, selected a few hot spots encoding nonconserved amino acids, and introduced random mutations to make libraries with a size requirement between 10(3) and 10(4) independent clones. Panning of the hot spot libraries yielded several mutants with a 15- to 55-fold increase in affinity compared with a single clone with a fourfold increased affinity from a library in which mutagenesis was done outside the hot spots. The strategy should be generally applicable for the rapid isolation of higher-affinity mutants of Fvs, Fabs, and other recombinant antibodies from antibody phage libraries that are small in size.

MeSH terms

  • Antibody Affinity / genetics*
  • Antineoplastic Agents / isolation & purification
  • Antineoplastic Agents / pharmacology
  • Base Sequence
  • Cloning, Molecular
  • Enzyme-Linked Immunosorbent Assay
  • GPI-Linked Proteins
  • Humans
  • Immunoglobulin Variable Region / genetics*
  • Immunoglobulin Variable Region / metabolism
  • Immunotoxins / genetics
  • Immunotoxins / immunology
  • Immunotoxins / pharmacology
  • Membrane Glycoproteins / metabolism
  • Molecular Sequence Data
  • Mutagenesis*
  • Tumor Cells, Cultured

Substances

  • Antineoplastic Agents
  • GPI-Linked Proteins
  • Immunoglobulin Variable Region
  • Immunotoxins
  • Membrane Glycoproteins
  • mesothelin