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In vitro evolution of single-chain antibodies using mRNA display.

Fukuda I, Kojoh K, Tabata N, Doi N, Takashima H, Miyamoto-Sato E, Yanagawa H - Nucleic Acids Res. (2006)

Bottom Line: 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.Thus, mRNA display is expected to be useful for rapid artificial evolution of high-affinity diagnostic and therapeutic antibodies by optimizing their CDRs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences and Informatics, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan.

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.

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Competitive ELISA for monitoring the fraction of the mutant scFv library that bound to fluorescein at each round of selection. The wheat germ cell-free translated products containing mutant scFv libraries were pre-incubated with a competitor (0–1000 nM free fluorescein) and allowed to bind to fluorescein-immobilized plates. After washing of the plates, remaining scFvs were detected by HRP-conjugated anti-FLAG M2 antibody and TMB substrate with absorbance measurements at 450 nm (reference at 655 nm). All measurements were performed in duplicate.
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fig2: Competitive ELISA for monitoring the fraction of the mutant scFv library that bound to fluorescein at each round of selection. The wheat germ cell-free translated products containing mutant scFv libraries were pre-incubated with a competitor (0–1000 nM free fluorescein) and allowed to bind to fluorescein-immobilized plates. After washing of the plates, remaining scFvs were detected by HRP-conjugated anti-FLAG M2 antibody and TMB substrate with absorbance measurements at 450 nm (reference at 655 nm). All measurements were performed in duplicate.

Mentions: After four rounds of off-rate selection, the total binding activity of in vitro-translated products of the library DNA at each round was analyzed by competitive ELISA. As shown in Figure 2, the binding activity gradually increased in successive rounds of selection (Figure 2; 0 nM), whereas little or no activity was observed in the presence of competitor (Figure 2; 10 nM, 100 nM and 1 µM), indicating the enrichment of specific binders with high affinity in the library.


In vitro evolution of single-chain antibodies using mRNA display.

Fukuda I, Kojoh K, Tabata N, Doi N, Takashima H, Miyamoto-Sato E, Yanagawa H - Nucleic Acids Res. (2006)

Competitive ELISA for monitoring the fraction of the mutant scFv library that bound to fluorescein at each round of selection. The wheat germ cell-free translated products containing mutant scFv libraries were pre-incubated with a competitor (0–1000 nM free fluorescein) and allowed to bind to fluorescein-immobilized plates. After washing of the plates, remaining scFvs were detected by HRP-conjugated anti-FLAG M2 antibody and TMB substrate with absorbance measurements at 450 nm (reference at 655 nm). All measurements were performed in duplicate.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1636464&req=5

fig2: Competitive ELISA for monitoring the fraction of the mutant scFv library that bound to fluorescein at each round of selection. The wheat germ cell-free translated products containing mutant scFv libraries were pre-incubated with a competitor (0–1000 nM free fluorescein) and allowed to bind to fluorescein-immobilized plates. After washing of the plates, remaining scFvs were detected by HRP-conjugated anti-FLAG M2 antibody and TMB substrate with absorbance measurements at 450 nm (reference at 655 nm). All measurements were performed in duplicate.
Mentions: After four rounds of off-rate selection, the total binding activity of in vitro-translated products of the library DNA at each round was analyzed by competitive ELISA. As shown in Figure 2, the binding activity gradually increased in successive rounds of selection (Figure 2; 0 nM), whereas little or no activity was observed in the presence of competitor (Figure 2; 10 nM, 100 nM and 1 µM), indicating the enrichment of specific binders with high affinity in the library.

Bottom Line: 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.Thus, mRNA display is expected to be useful for rapid artificial evolution of high-affinity diagnostic and therapeutic antibodies by optimizing their CDRs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences and Informatics, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan.

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.

Show MeSH
Related in: MedlinePlus