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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

Schematic representation of the in vitro selection of scFv antibodies by the IVV method. Step 1: a DNA library of scFv mutants is in vitro-transcribed into mRNA. Step 2: the mRNA is enzymatically ligated with puromycin (Pu) through a PEG spacer. Step 3: the ligated product is in vitro-translated by a cell-free translation system, resulting in covalent bonding of puromycin with the full-length protein at the C-terminus. Step 4: the mRNA-displayed scFv library is captured on antigen-immobilized beads and unbound molecules are washed away. Step 5: the bound molecules are eluted by protease digestion. Step 6: the mRNA portion of eluted molecules is reverse-transcribed into cDNA. Step 7: the cDNA is amplified by PCR. Step 8: the DNA can be used for the next round of selection after mutagenesis or can be analyzed by cloning and sequencing after several rounds of selection.
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fig1: Schematic representation of the in vitro selection of scFv antibodies by the IVV method. Step 1: a DNA library of scFv mutants is in vitro-transcribed into mRNA. Step 2: the mRNA is enzymatically ligated with puromycin (Pu) through a PEG spacer. Step 3: the ligated product is in vitro-translated by a cell-free translation system, resulting in covalent bonding of puromycin with the full-length protein at the C-terminus. Step 4: the mRNA-displayed scFv library is captured on antigen-immobilized beads and unbound molecules are washed away. Step 5: the bound molecules are eluted by protease digestion. Step 6: the mRNA portion of eluted molecules is reverse-transcribed into cDNA. Step 7: the cDNA is amplified by PCR. Step 8: the DNA can be used for the next round of selection after mutagenesis or can be analyzed by cloning and sequencing after several rounds of selection.

Mentions: The scheme for in vitro selection of anti-fluorescein scFv using the IVV method is shown in Figure 1. First we constructed the template DNA for IVV, containing an SP6 promoter and Ω enhancer upstream of the anti-fluorescein scFv gene (VH-linker-VL), and the FLAG-tag and poly(A) sequence in the downstream region. The scFv region was subjected to random mutagenesis by error-prone PCR (the error rate was 0.8%; 6–7 nt per scFv gene). The transcribed mRNA (1.2 × 1012) molecules were attached to a PEG spacer containing puromycin at the 3′ end and in vitro-translated in 10 µl of wheat germ cell-free translation system, resulting in the covalent bonding of the C-terminus of the full-length scFv protein (phenotype) with its coding mRNA (genotype) through the puromycin-PEG spacer. We estimated that the number of resultant mRNA-displayed scFv molecules in the initial library was 6 × 1010 (ligation efficiency of mRNA with the puromycin-PEG spacer and efficiency of IVV formation were 50 and 10%, respectively). The library was captured on fluorescein-immobilized beads and the mRNA portion of selected molecules was amplified by RT–PCR. In each round of selection, the recovered DNA was further mutagenized by error-prone DNA shuffling (26,29).


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)

Schematic representation of the in vitro selection of scFv antibodies by the IVV method. Step 1: a DNA library of scFv mutants is in vitro-transcribed into mRNA. Step 2: the mRNA is enzymatically ligated with puromycin (Pu) through a PEG spacer. Step 3: the ligated product is in vitro-translated by a cell-free translation system, resulting in covalent bonding of puromycin with the full-length protein at the C-terminus. Step 4: the mRNA-displayed scFv library is captured on antigen-immobilized beads and unbound molecules are washed away. Step 5: the bound molecules are eluted by protease digestion. Step 6: the mRNA portion of eluted molecules is reverse-transcribed into cDNA. Step 7: the cDNA is amplified by PCR. Step 8: the DNA can be used for the next round of selection after mutagenesis or can be analyzed by cloning and sequencing after several rounds of selection.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Schematic representation of the in vitro selection of scFv antibodies by the IVV method. Step 1: a DNA library of scFv mutants is in vitro-transcribed into mRNA. Step 2: the mRNA is enzymatically ligated with puromycin (Pu) through a PEG spacer. Step 3: the ligated product is in vitro-translated by a cell-free translation system, resulting in covalent bonding of puromycin with the full-length protein at the C-terminus. Step 4: the mRNA-displayed scFv library is captured on antigen-immobilized beads and unbound molecules are washed away. Step 5: the bound molecules are eluted by protease digestion. Step 6: the mRNA portion of eluted molecules is reverse-transcribed into cDNA. Step 7: the cDNA is amplified by PCR. Step 8: the DNA can be used for the next round of selection after mutagenesis or can be analyzed by cloning and sequencing after several rounds of selection.
Mentions: The scheme for in vitro selection of anti-fluorescein scFv using the IVV method is shown in Figure 1. First we constructed the template DNA for IVV, containing an SP6 promoter and Ω enhancer upstream of the anti-fluorescein scFv gene (VH-linker-VL), and the FLAG-tag and poly(A) sequence in the downstream region. The scFv region was subjected to random mutagenesis by error-prone PCR (the error rate was 0.8%; 6–7 nt per scFv gene). The transcribed mRNA (1.2 × 1012) molecules were attached to a PEG spacer containing puromycin at the 3′ end and in vitro-translated in 10 µl of wheat germ cell-free translation system, resulting in the covalent bonding of the C-terminus of the full-length scFv protein (phenotype) with its coding mRNA (genotype) through the puromycin-PEG spacer. We estimated that the number of resultant mRNA-displayed scFv molecules in the initial library was 6 × 1010 (ligation efficiency of mRNA with the puromycin-PEG spacer and efficiency of IVV formation were 50 and 10%, respectively). The library was captured on fluorescein-immobilized beads and the mRNA portion of selected molecules was amplified by RT–PCR. In each round of selection, the recovered DNA was further mutagenized by error-prone DNA shuffling (26,29).

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