title: Effective Optimization of Antibody Affinity by Phage Display Integrated with High-Throughput DNA Synthesis and Sequencing Technologies.
abstract: Phage display technology has been widely used for antibody affinity maturation for decades. The limited library sequence diversity together with excessive redundancy and labour-consuming procedure for candidate identification are two major obstacles to widespread adoption of this technology. We hereby describe a novel library generation and screening approach to address the problems. The approach started with the targeted diversification of multiple complementarity determining regions (CDRs) of a humanized anti-ErbB2 antibody, HuA21, with a small perturbation mutagenesis strategy. A combination of three degenerate codons, NWG, NWC, and NSG, were chosen for amino acid saturation mutagenesis without introducing cysteine and stop residues. In total, 7,749 degenerate oligonucleotides were synthesized on two microchips and released to construct five single-chain antibody fragment (scFv) gene libraries with 4 x 10(6) DNA sequences. Deep sequencing of the unselected and selected phage libraries using the Illumina platform allowed for an in-depth evaluation of the enrichment landscapes in CDR sequences and amino acid substitutions. Potent candidates were identified according to their high frequencies using NGS analysis, by-passing the need for the primary screening of target-binding clones. Furthermore, a subsequent library by recombination of the 10 most abundant variants from four CDRs was constructed and screened, and a mutant with 158-fold increased affinity (Kd = 25.5 pM) was obtained. These results suggest the potential application of the developed methodology for optimizing the binding properties of other antibodies and biomolecules.
authors.affiliation: School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, China.
authors.forename: Dongmei
authors.initials: D
authors.lastname: Hu
authors.affiliation: School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, China.
authors.forename: Siyi
authors.initials: S
authors.lastname: Hu
authors.affiliation: School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, China.
authors.forename: Wen
authors.initials: W
authors.lastname: Wan
authors.affiliation: School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, China.
authors.forename: Man
authors.initials: M
authors.lastname: Xu
authors.affiliation: School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, China.
authors.forename: Ruikai
authors.initials: R
authors.lastname: Du
authors.affiliation: School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhao
authors.affiliation: School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, China; Department of Biology and Biochemistry, University of Houston, Houston, Texas, United States of America.
authors.forename: Xiaolian
authors.initials: X
authors.lastname: Gao
authors.affiliation: School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Liu
authors.affiliation: School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, China.
authors.forename: Haiyan
authors.initials: H
authors.lastname: Liu
authors.affiliation: School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, China.
authors.forename: Jiong
authors.initials: J
authors.lastname: Hong
mesh_terms: D061067:Antibodies, Monoclonal, Humanized / Q000235:genetics / Q000276:immunology*; D000915:Antibody Affinity; D001483:Base Sequence; D022801:Complementarity Determining Regions / Q000235:genetics / Q000276:immunology*; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D016296:Mutagenesis; D019151:Peptide Library; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology*; D017422:Sequence Analysis, DNA; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology*
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D022801:Complementarity Determining Regions; D019151:Peptide Library; D018719:Erb-b2 Receptor Tyrosine Kinases; D057127:Single-Chain Antibodies; C508053:ERBB2 protein, human