Limits...
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.

Show MeSH

Related in: MedlinePlus

Kinetic distribution plot. The kinetic parameters of purified scFvs were determined by SPR spectroscopy (see Materials and Methods and Table 3). The wild type (open circle); Group 1 mutants 2-12 (filled circle), 7-753 (open square), 3-23 (filled square), 7-308 (open triangle) and 7-464 (filled triangle); and Group 2 mutant 2-10 (filled diamond). The dissociation constants (Kd = koff/kon) are represented as gray lines (0.1–10 nM). Group 1 mutants with highest affinity were clustered in the upper right-hand corner of the plot.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC1636464&req=5

fig4: Kinetic distribution plot. The kinetic parameters of purified scFvs were determined by SPR spectroscopy (see Materials and Methods and Table 3). The wild type (open circle); Group 1 mutants 2-12 (filled circle), 7-753 (open square), 3-23 (filled square), 7-308 (open triangle) and 7-464 (filled triangle); and Group 2 mutant 2-10 (filled diamond). The dissociation constants (Kd = koff/kon) are represented as gray lines (0.1–10 nM). Group 1 mutants with highest affinity were clustered in the upper right-hand corner of the plot.

Mentions: The binding affinities of purified scFvs (five Group 1 mutants and a Group 2 mutant, 2-10) were measured by SPR spectroscopy (Table 3 and Figure 4). Although the on-rates of the mutants were similar to that of the wild type, the off-rates were decreased by more than one order of magnitude, suggesting that the application of selection pressure by off-rate selection had been successful. The dissociation constants of Group 1 mutants were improved by ∼30-fold, which is comparable with that obtained by ribosome display (14), probably because the selection conditions were similar to each other.


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)

Kinetic distribution plot. The kinetic parameters of purified scFvs were determined by SPR spectroscopy (see Materials and Methods and Table 3). The wild type (open circle); Group 1 mutants 2-12 (filled circle), 7-753 (open square), 3-23 (filled square), 7-308 (open triangle) and 7-464 (filled triangle); and Group 2 mutant 2-10 (filled diamond). The dissociation constants (Kd = koff/kon) are represented as gray lines (0.1–10 nM). Group 1 mutants with highest affinity were clustered in the upper right-hand corner of the plot.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Kinetic distribution plot. The kinetic parameters of purified scFvs were determined by SPR spectroscopy (see Materials and Methods and Table 3). The wild type (open circle); Group 1 mutants 2-12 (filled circle), 7-753 (open square), 3-23 (filled square), 7-308 (open triangle) and 7-464 (filled triangle); and Group 2 mutant 2-10 (filled diamond). The dissociation constants (Kd = koff/kon) are represented as gray lines (0.1–10 nM). Group 1 mutants with highest affinity were clustered in the upper right-hand corner of the plot.
Mentions: The binding affinities of purified scFvs (five Group 1 mutants and a Group 2 mutant, 2-10) were measured by SPR spectroscopy (Table 3 and Figure 4). Although the on-rates of the mutants were similar to that of the wild type, the off-rates were decreased by more than one order of magnitude, suggesting that the application of selection pressure by off-rate selection had been successful. The dissociation constants of Group 1 mutants were improved by ∼30-fold, which is comparable with that obtained by ribosome display (14), probably because the selection conditions were similar to each other.

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