Generation and iterative affinity maturation of antibodies in vitro using hypermutating B-cell lines

Nat Biotechnol. 2002 Nov;20(11):1129-34. doi: 10.1038/nbt752. Epub 2002 Oct 15.


We show that iterative antigen-mediated selection of B-cell lines that constitutively hypermutate their immunoglobulin V genes during culture can be exploited to generate antibodies in vitro. From Ramos, a hypermutating human B-cell line expressing IgM of unknown specificity, we derived descendants that exhibit stepwise improved binding to streptavidin. Binding is initially conferred by mutations in complementarity-determining regions (CDRs), but maturation is due to strategic framework mutations. A more powerful system is provided by a hypermutating chicken B-lymphoma line, owing to its rapid proliferation, high rate of mutation accumulation, and genetic tractability. Starting from a single cell, we selected parallel lineages of derivatives, making mutated antibodies of increasing affinity to independent test antigens. Selection is initiated at an exceedingly low affinity threshold, but antibodies can be delivered with nanomolar affinities. The strategy could prove useful for in vitro generation of antigen-specific monoclonal antibodies and may be extendable to the maturation of other protein-ligand interactions.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Monoclonal / biosynthesis
  • Antibodies, Monoclonal / genetics*
  • Antibodies, Monoclonal / immunology
  • Antibody Affinity / genetics
  • Antibody Affinity / immunology
  • Antibody Formation / drug effects
  • Antibody Formation / genetics
  • Antibody Formation / immunology
  • Binding Sites, Antibody / drug effects
  • Binding Sites, Antibody / genetics
  • Burkitt Lymphoma / genetics*
  • Burkitt Lymphoma / immunology*
  • Burkitt Lymphoma / metabolism
  • Chickens
  • Cloning, Molecular
  • Directed Molecular Evolution / methods*
  • Gene Expression Regulation, Neoplastic
  • Humans
  • Immunoglobulins, Intravenous / biosynthesis
  • Immunoglobulins, Intravenous / genetics*
  • Immunoglobulins, Intravenous / immunology*
  • Mutagenesis
  • Rats
  • Reference Values
  • Selection, Genetic
  • Sensitivity and Specificity
  • Species Specificity
  • Staphylococcal Protein A / immunology
  • Staphylococcal Protein A / metabolism
  • Streptavidin / administration & dosage
  • Streptavidin / immunology
  • Tumor Cells, Cultured


  • Antibodies, Monoclonal
  • Immunoglobulins, Intravenous
  • Staphylococcal Protein A
  • Streptavidin