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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 analysis of the contributions of the five consensus mutations in Group 1 mutants. The periplasmic extracts of the wild-type and mutant scFvs were pre-incubated in the presence of various concentrations of fluorescein. For experimental details, see the legend of Figure 3 and Materials and Methods. (A) The wild type (open circles, dashed line) and the mutant CM5 (filled triangles, dotted line). (B) The single mutants W47C (filled squares), V21I (open diamonds) and S15F (open triangles); the wild type (a dashed line) and CM5 (a dotted line). (C) The single mutants D101N (filled circles) and H94Y (open squares); the double mutant D101N/H94Y (filled diamonds); the wild type (dashed line) and CM5 (dotted line).
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fig6: Competitive ELISA for analysis of the contributions of the five consensus mutations in Group 1 mutants. The periplasmic extracts of the wild-type and mutant scFvs were pre-incubated in the presence of various concentrations of fluorescein. For experimental details, see the legend of Figure 3 and Materials and Methods. (A) The wild type (open circles, dashed line) and the mutant CM5 (filled triangles, dotted line). (B) The single mutants W47C (filled squares), V21I (open diamonds) and S15F (open triangles); the wild type (a dashed line) and CM5 (a dotted line). (C) The single mutants D101N (filled circles) and H94Y (open squares); the double mutant D101N/H94Y (filled diamonds); the wild type (dashed line) and CM5 (dotted line).

Mentions: To determine the most important mutations for affinity improvement, we first constructed an scFv mutant that contains only the five consensus mutations in Group 1 mutants (named CM5). As shown in Figure 6A (filled triangles), the activity of the CM5 mutant was similar to that of Group 1 mutants (such as 2-12 shown in Figure 5C, filled squares), indicating that only the five mutations in Group 1 contribute significantly to the increased affinity. Subsequently, we constructed and examined five scFv mutants, each with a single amino acid mutation, W47C, D101N, S15F, V21I or H94Y. Three mutations, W47C and V21I in the framework and S15F in the linker, did not influence binding activity (Figure 6B), whereas two, D101N or H94Y in the CDR, resulted in intermediate binding activity between CM5 and wild type (Figure 6C). Moreover, the double mutant containing both D101N and H94Y showed binding activity similar to that of CM5 (Figure 6C, filled diamonds). These results clearly demonstrated that the critical mutations for improving affinity are D101N and H94Y in CDR3 of VH and VL, respectively. This result is consistent with the previous findings in laboratory evolution of high-affinity scFv mutants with CDR mutations (11,14). The locations of the CDR mutations on the three-dimensional structure were very close to the fluorescein site (14). It has been argued that the mutation H94Y in VL directly affects the interaction with the antigen in the antigen-binding site, and that mutations D101G, D101A and D101S in VH break the salt bridge between D101 and R94 in VH and increase the flexibility of CDR H3 (14). Our results suggest that the mutation D101N in VH also affects the affinity via a mechanism similar to that of the mutations D101G, D101A and D101S. The framework mutations W47C in VH and V21I in VL, as well as the linker mutation S15F, may be neutral mutations fixed in an early round of selection by random genetic drift.


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 analysis of the contributions of the five consensus mutations in Group 1 mutants. The periplasmic extracts of the wild-type and mutant scFvs were pre-incubated in the presence of various concentrations of fluorescein. For experimental details, see the legend of Figure 3 and Materials and Methods. (A) The wild type (open circles, dashed line) and the mutant CM5 (filled triangles, dotted line). (B) The single mutants W47C (filled squares), V21I (open diamonds) and S15F (open triangles); the wild type (a dashed line) and CM5 (a dotted line). (C) The single mutants D101N (filled circles) and H94Y (open squares); the double mutant D101N/H94Y (filled diamonds); the wild type (dashed line) and CM5 (dotted line).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1636464&req=5

fig6: Competitive ELISA for analysis of the contributions of the five consensus mutations in Group 1 mutants. The periplasmic extracts of the wild-type and mutant scFvs were pre-incubated in the presence of various concentrations of fluorescein. For experimental details, see the legend of Figure 3 and Materials and Methods. (A) The wild type (open circles, dashed line) and the mutant CM5 (filled triangles, dotted line). (B) The single mutants W47C (filled squares), V21I (open diamonds) and S15F (open triangles); the wild type (a dashed line) and CM5 (a dotted line). (C) The single mutants D101N (filled circles) and H94Y (open squares); the double mutant D101N/H94Y (filled diamonds); the wild type (dashed line) and CM5 (dotted line).
Mentions: To determine the most important mutations for affinity improvement, we first constructed an scFv mutant that contains only the five consensus mutations in Group 1 mutants (named CM5). As shown in Figure 6A (filled triangles), the activity of the CM5 mutant was similar to that of Group 1 mutants (such as 2-12 shown in Figure 5C, filled squares), indicating that only the five mutations in Group 1 contribute significantly to the increased affinity. Subsequently, we constructed and examined five scFv mutants, each with a single amino acid mutation, W47C, D101N, S15F, V21I or H94Y. Three mutations, W47C and V21I in the framework and S15F in the linker, did not influence binding activity (Figure 6B), whereas two, D101N or H94Y in the CDR, resulted in intermediate binding activity between CM5 and wild type (Figure 6C). Moreover, the double mutant containing both D101N and H94Y showed binding activity similar to that of CM5 (Figure 6C, filled diamonds). These results clearly demonstrated that the critical mutations for improving affinity are D101N and H94Y in CDR3 of VH and VL, respectively. This result is consistent with the previous findings in laboratory evolution of high-affinity scFv mutants with CDR mutations (11,14). The locations of the CDR mutations on the three-dimensional structure were very close to the fluorescein site (14). It has been argued that the mutation H94Y in VL directly affects the interaction with the antigen in the antigen-binding site, and that mutations D101G, D101A and D101S in VH break the salt bridge between D101 and R94 in VH and increase the flexibility of CDR H3 (14). Our results suggest that the mutation D101N in VH also affects the affinity via a mechanism similar to that of the mutations D101G, D101A and D101S. The framework mutations W47C in VH and V21I in VL, as well as the linker mutation S15F, may be neutral mutations fixed in an early round of selection by random genetic drift.

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