RESEARCH ARTICLE Effective Optimization of Antibody Affinity by Phage Display Integrated with High- Throughput DNA Synthesis and Sequencing Technologies

Phage display technology has been widely used for antibody affinity maturation for de-cades. The limited library sequence diversity together with excessive redundancy and labour-consuming procedure for candidate identification are two major obstacles to wide-spread adoption of this technology. We hereby describe a novel library generation and screening approach to address the problems. The approach started with the targeted diver-sification 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 oli-gonucleotides were synthesized on two microchips and released to construct five single-chain antibody fragment (scFv) gene libraries with 4 x 106 DNA sequences. Deep sequenc-ing 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 sub-stitutions. Potent candidates were identified according to their high frequencies using NGS analysis, by-passing the need for the primary screening of target-binding clones. Further-more, 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