title: Antibody engineering: the use of Escherichia coli as an expression host.
abstract: The hypervariable loops of an antibody molecule are supported on the relatively conserved beta-sheeted frameworks of the heavy- and light-chain variable domains (designated VH and VL domains, respectively). Residues within and flanking these loops interact with antigen and confer the specificity and affinity of antigen binding on the immunoglobulin molecule. Thus, the isolation and expression of VH and VL domain genes are of particular interest both for analysis of the determinants of antibody specificity and for generation of fragments with binding affinities for use in therapy and diagnosis. The PCR can now be used to isolate diverse repertoires of antibody VH and VL domain genes from antibody-producing cells from different species, including humans and mice. The genes can be expressed as either secreted or surface-bound Fv or Fab fragments, using Escherichia coli expression systems, and the desired antigen-binding specificity screened for or, preferably, selected. The use of E. coli as an expression host allows the required antigen-binding specificity to be isolated in clonal form in a matter of days. The VH and VL domain genes can also be hypermutated and higher-affinity variants isolated by screening or selection. Thus, the use of this technology should allow the isolation of novel binding specificities or specificities that are difficult to generate by hybridoma technology. It will also facilitate the isolation of human-derived Fv/Fab fragments that may be less immunogenic in therapy. This approach therefore has almost unlimited potential in the generation of therapeutics with binding specificities to order. The fragments can be used either alone or linked to effector functions in the form of antibody-constant domains or toxins. The new technology could prove to be a method of choice for the rapid and convenient production of designer antibodies.
authors.affiliation: Cancer Immunobiology Center, University of Texas Southwestern Medical Center, Dallas 75235-9048.
authors.forename: E S
authors.initials: ES
authors.lastname: Ward
mesh_terms: D000906:Antibodies / Q000235:genetics* / Q000276:immunology / Q000627:therapeutic use; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005803:Genes, Immunoglobulin; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction
chemical_list: D000906:Antibodies; D007128:Immunoglobulin Fragments