The human combinatorial antibody library HuCAL GOLD combines diversification of all six CDRs according to the natural immune system with a novel display method for efficient selection of high-affinity antibodies

J Mol Biol. 2008 Feb 29;376(4):1182-200. doi: 10.1016/j.jmb.2007.12.018. Epub 2007 Dec 15.

Abstract

This article describes the generation of the Human Combinatorial Antibody Library HuCAL GOLD. HuCAL GOLD is a synthetic human Fab library based on the HuCAL concept with all six complementarity-determining regions (CDRs) diversified according to the sequence and length variability of naturally rearranged human antibodies. The human antibody repertoire was analyzed in-depth, and individual CDR libraries were designed and generated for each CDR and each antibody family. Trinucleotide mixtures were used to synthesize the CDR libraries in order to ensure a high quality within HuCAL GOLD, and a beta-lactamase selection system was employed to eliminate frame-shifted clones after successive cloning of the CDR libraries. With these methods, a large, high-quality library with more than 10 billion functional Fab fragments was achieved. By using CysDisplay, the antibody fragments are displayed on the tip of the phage via a disulfide bridge between the phage coat protein pIII and the heavy chain of the antibody fragment. Efficient elution of specific phages is possible by adding reducing agents. HuCAL GOLD was challenged with a variety of different antigens and proved to be a reliable source of high-affinity human antibodies with best affinities in the picomolar range, thus functioning as an excellent source of antibodies for research, diagnostic, and therapeutic applications. Furthermore, the data presented in this article demonstrate that CysDisplay is a robust and broadly applicable display technology even for high-throughput applications.

MeSH terms

  • Amino Acid Sequence
  • Antibodies / chemistry
  • Antibodies / immunology*
  • Antibody Affinity / immunology*
  • Bacteriophages
  • Blotting, Western
  • Cloning, Molecular
  • Combinatorial Chemistry Techniques / methods*
  • Complementarity Determining Regions / chemistry
  • Complementarity Determining Regions / immunology*
  • Genes
  • Genetic Vectors
  • Humans
  • Immune System / immunology*
  • Immunoglobulin Fab Fragments / chemistry
  • Immunoglobulin Fab Fragments / genetics
  • Immunoglobulin Heavy Chains / chemistry
  • Immunoglobulin Heavy Chains / genetics
  • Immunoglobulin Light Chains / chemistry
  • Immunoglobulin Light Chains / genetics
  • Immunoglobulin Variable Region / chemistry
  • Immunoglobulin Variable Region / immunology
  • Molecular Sequence Data
  • Peptide Library*
  • Protein Conformation
  • beta-Lactamases / genetics

Substances

  • Antibodies
  • Complementarity Determining Regions
  • Immunoglobulin Fab Fragments
  • Immunoglobulin Heavy Chains
  • Immunoglobulin Light Chains
  • Immunoglobulin Variable Region
  • Peptide Library
  • beta-Lactamases