Integrated mimicry of B cell antibody mutagenesis using yeast homologous recombination.
Antibody affinity maturation proceeds in vivo via a combination of point mutations, insertions, deletions, and combinatorial shuffling of light chains or portions of the heavy chain, thereby reducing the probability of trapping in local affinity optima in sequence space. In vivo homologous recombination in yeast can be exploited to mimic the broad spectrum of mutational types deployed by B cells, incorporating both receptor revision and receptor editing together with polymerase-directed point mutagenesis. This method was used to effect a 10,000-fold affinity improvement in an anti-peptide single-chain antibody in three rounds of mutagenesis and screening, and a 1,000-fold affinity improvement in an anti-protein single-chain antibody in a single round. When recombinational mutagenesis (CDR or chain shuffling) was directly compared to error-prone PCR, the recombinational approach yielded greater affinity improvement with substantially reduced divergence from germline sequences, demonstrating an advantage of simultaneously testing a broad range of mutational strategies.
Swers
Jeffrey S
JS
Department of Chemical Engineering, Massachusetts Institute of Technology, Building E19-551, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
Yeung
Yik A
YA
Department of Chemical Engineering, Massachusetts Institute of Technology, Building E19-551, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
Wittrup
K Dane
KD
Department of Chemical Engineering and Biological Engineering Division, Massachusetts Institute of Technology, Building E19-551, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
D000595:Amino Acid Sequence; D000906:Antibodies; D000915:Antibody Affinity / Q000235:genetics; D001402:B-Lymphocytes / Q000276:immunology; D032621:DNA Shuffling / Q000379:methods*; D006801:Humans; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D011995:Recombination, Genetic; D017386:Sequence Homology, Amino Acid; D057127:Single-Chain Antibodies / Q000096:biosynthesis* / Q000235:genetics; D015003:Yeasts / Q000235:genetics
D000906:Antibodies; D057127:Single-Chain Antibodies