title: In vitro evolution of single-chain antibodies using mRNA display.
abstract: Here we describe the application of the in vitro virus mRNA display method, which involves covalent linkage of an in vitro-synthesized antibody (phenotype) to its encoding mRNA (genotype) through puromycin, for in vitro evolution of single-chain Fv (scFv) antibody fragments. To establish the validity of this approach to directed antibody evolution, we used random mutagenesis by error-prone DNA shuffling and off-rate selection to improve the affinity of an anti-fluorescein scFv as a model system. After four rounds of selection of the library of mRNA-displayed scFv mutants, we obtained six different sequences encoding affinity-matured mutants with five consensus mutations. Kinetic analysis of the mutant scFvs revealed that the off-rates have been decreased by more than one order of magnitude and the dissociation constants were improved approximately 30-fold. The antigen-specificity was not improved by affinity maturation, but remained similar to that of the wild type. Although the five consensus mutations of the high-affinity mutants were scattered over the scFv sequence, analysis by site-directed mutagenesis demonstrated that the critical mutations for improving affinity were the two that lay within the complementarity determining regions (CDRs). Thus, mRNA display is expected to be useful for rapid artificial evolution of high-affinity diagnostic and therapeutic antibodies by optimizing their CDRs.
authors.affiliation: Department of Biosciences and Informatics, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan.
authors.forename: Isao
authors.initials: I
authors.lastname: Fukuda
authors.forename: Kanehisa
authors.initials: K
authors.lastname: Kojoh
authors.forename: Noriko
authors.initials: N
authors.lastname: Tabata
authors.forename: Nobuhide
authors.initials: N
authors.lastname: Doi
authors.forename: Hideaki
authors.initials: H
authors.lastname: Takashima
authors.forename: Etsuko
authors.initials: E
authors.lastname: Miyamoto-Sato
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Yanagawa
mesh_terms: D000915:Antibody Affinity; D000918:Antibody Specificity; D001667:Binding, Competitive; D032621:DNA Shuffling; D019020:Directed Molecular Evolution / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D019793:Fluorescein / Q000378:metabolism; D015723:Gene Library; D007135:Immunoglobulin Variable Region / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D016296:Mutagenesis; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000737:chemistry*; D020349:Surface Plasmon Resonance; D014158:Transcription, Genetic
chemical_list: D007135:Immunoglobulin Variable Region; D012333:RNA, Messenger; D019793:Fluorescein