In Vivo Directed Evolution Of Antibody Affinity And The Implementation Of A Continuous Directed Evolution System

Over the past decades, laboratory-directed evolution has emerged as a powerful technology platform in protein engineering. Currently, directed evolution of a target molecule involves repeated rounds of (i) random gene library construction,(ii) expression of genes in a suitable host (transform) and (iii) screening the library variants (mutants) for the function of interest until desirable functional variant(s) acquired. We have effectively optimized an anti-ErbB2antibody, HuA21by the traditional directed evolution phage display technology.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 CDRs of the humanized anti-ErbB2antibody, 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,7,749degenerate oligonucleotides were synthesized on two microchips and released to construct five single-chain antibody fragment (scFv) gene libraries with4x106DNA 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 the10most abundant variants from four CDRs was constructed and screened, and a mutant with158-fold increased affinity (Kd=25.5pM) was obtained. These results suggest the potential application of the developed methodology for optimizing the binding properties of other antibodies and biomoleculesWhile a powerful tool, traditional directed evolution is inherently limited by the size of the library can be constructed, the rate at which selection of screening can be carried out and the requirement for significant and time-consuming intervention by researches at every cycle of library preparation and screening/selection. Nevertheless, recent advances from lab David R Liu have helped to mitigate, even solve these limitations through their system of phage-assisted continuous directed evolution (PACE). The PACE system contains a fixed-volume vessel to which uninfected Escherichia coli cells are continuously supplied and a mixture of infected and uninfected E. coli cells are continuously removed. At the same time, a phage population encoding the target gene is replicating in the same vessel. The infection ability of phage is linked to the desired function of the target gene, which means the maintenance or enrichment of a particular phage in the vessel is dependent on the improved function of the target gene. Therefore, one phage replication/infection cycle equals to one cycle of traditional mutation/selection, and those cycles happen continuously without human intervention. Nevertheless, the PACE system has been limited to the Lab of David R Liu in recent years, and more efforts should be made for the widespread use of it. Herein, by identifying the bacterial sigma70(rpoD gene) mutants that can specifically recognize an irrelevant DNA sequence through PACE system, we tried to set up the scaffold and apply it to the function endowing for the artificially synthesized non-function proteins. However, some more exploring experiments should be carried out in order to construct the stable directed evolution approach.