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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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Related in: MedlinePlus

Competitive ELISA for estimating the antigen-specificity of the affinity-matured mutants. (A) The structures of fluorescein, Oregon green 488 and rhodamine green. For immobilization on the ELISA plates, biotin is attached to the benzene moiety of fluorescein; thus, differences of the xanthene moiety would be critical for scFv binding. The periplasmic extracts of the wild type (B) and a typical mutant 2-12 (C) were pre-incubated in the presence of various concentrations of fluorescein (filled squares), Oregon green 488 (open circles) or rhodamine green (filled triangles). The other five mutants in Group 1 gave similar curves. For experimental details, see the legend of Figure 3 and Materials and Methods.
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fig5: Competitive ELISA for estimating the antigen-specificity of the affinity-matured mutants. (A) The structures of fluorescein, Oregon green 488 and rhodamine green. For immobilization on the ELISA plates, biotin is attached to the benzene moiety of fluorescein; thus, differences of the xanthene moiety would be critical for scFv binding. The periplasmic extracts of the wild type (B) and a typical mutant 2-12 (C) were pre-incubated in the presence of various concentrations of fluorescein (filled squares), Oregon green 488 (open circles) or rhodamine green (filled triangles). The other five mutants in Group 1 gave similar curves. For experimental details, see the legend of Figure 3 and Materials and Methods.

Mentions: Next, we examined the antigen-specificity of the affinity-matured mutants by competitive ELISA. Two fluorescein analogs, Oregon green 488 and rhodamine green, as well as fluorescein were used as competitors (Figure 5A). The affinity of Group 1 mutant 2-12 (Figure 5C) was higher than that of wild type (Figure 5B), not only for fluorescein but also for the two fluorescein analogs, with an almost identical ratios of increase. Similar results were obtained for the other five Group 1 mutants (data not shown). The results suggest that the specificity of the affinity-matured mutants was not altered, but remained similar to that of the wild type.


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 estimating the antigen-specificity of the affinity-matured mutants. (A) The structures of fluorescein, Oregon green 488 and rhodamine green. For immobilization on the ELISA plates, biotin is attached to the benzene moiety of fluorescein; thus, differences of the xanthene moiety would be critical for scFv binding. The periplasmic extracts of the wild type (B) and a typical mutant 2-12 (C) were pre-incubated in the presence of various concentrations of fluorescein (filled squares), Oregon green 488 (open circles) or rhodamine green (filled triangles). The other five mutants in Group 1 gave similar curves. For experimental details, see the legend of Figure 3 and Materials and Methods.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Competitive ELISA for estimating the antigen-specificity of the affinity-matured mutants. (A) The structures of fluorescein, Oregon green 488 and rhodamine green. For immobilization on the ELISA plates, biotin is attached to the benzene moiety of fluorescein; thus, differences of the xanthene moiety would be critical for scFv binding. The periplasmic extracts of the wild type (B) and a typical mutant 2-12 (C) were pre-incubated in the presence of various concentrations of fluorescein (filled squares), Oregon green 488 (open circles) or rhodamine green (filled triangles). The other five mutants in Group 1 gave similar curves. For experimental details, see the legend of Figure 3 and Materials and Methods.
Mentions: Next, we examined the antigen-specificity of the affinity-matured mutants by competitive ELISA. Two fluorescein analogs, Oregon green 488 and rhodamine green, as well as fluorescein were used as competitors (Figure 5A). The affinity of Group 1 mutant 2-12 (Figure 5C) was higher than that of wild type (Figure 5B), not only for fluorescein but also for the two fluorescein analogs, with an almost identical ratios of increase. Similar results were obtained for the other five Group 1 mutants (data not shown). The results suggest that the specificity of the affinity-matured mutants was not altered, but remained similar to that of the wild type.

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