Affinity maturation of antibodies: optimized methods to generate high-quality ScFv libraries and isolate IgG candidates by high-throughput screening

Methods Mol Biol. 2012:907:451-61. doi: 10.1007/978-1-61779-974-7_26.

Abstract

As a growing number of therapeutic antibodies are developed, robust methods to efficiently improve the affinity and/or specificity of antibody candidates are needed. Here we describe our powerful platform that combines scFv affinity maturation and IgG high-throughput screening. After creating diversity with our random mutagenesis technology (MutaGen™), the scFv libraries are fully cleaned using a fusion system introducing the beta-lactamase gene to select in-frame and stop codon free variants on the basis of ampicillin resistance. The high-quality scFv libraries thereby constructed are then selected on the target in vitro using phage display technology. Contrary to standard procedures, instead of producing a limited number of affinity matured scFv as IgG molecules, we developed a cloning system to directly transfer the entire pool of selected scFv into an IgG expression vector permitting rapid IgG small-scale production (96 wells) in mammalian cells. Our integrated process allows us to generate high-quality scFv libraries and test numerous IgG variants, increasing the chances to select the best therapeutic antibody candidate.

MeSH terms

  • Ampicillin / pharmacology
  • Antibody Affinity / drug effects
  • Antibody Affinity / immunology*
  • Cell Line
  • Cloning, Molecular
  • Genetic Vectors / genetics
  • High-Throughput Screening Assays / methods*
  • Humans
  • Immunoglobulin G / biosynthesis
  • Immunoglobulin G / genetics
  • Immunoglobulin G / isolation & purification*
  • Open Reading Frames / genetics
  • Peptide Library*
  • Single-Chain Antibodies / biosynthesis*

Substances

  • Immunoglobulin G
  • Peptide Library
  • Single-Chain Antibodies
  • Ampicillin