Research output: Contribution to journal › Article › peer-review
Development of a stable eukaryotic strain producing fully human monoclonal antibody on the basis of the human antibody against ectromelia virus. / Matveev, A. L.; Khlusevich, Ya A.; Baykov, I. K. et al.
In: Вавиловский журнал генетики и селекции, Vol. 21, No. 8, 01.01.2017, p. 993-1000.Research output: Contribution to journal › Article › peer-review
}
TY - JOUR
T1 - Development of a stable eukaryotic strain producing fully human monoclonal antibody on the basis of the human antibody against ectromelia virus
AU - Matveev, A. L.
AU - Khlusevich, Ya A.
AU - Baykov, I. K.
AU - Babkin, I. V.
AU - Goncharova, E. P.
AU - Morozova, V. V.
AU - Tikunova, N. V.
PY - 2017/1/1
Y1 - 2017/1/1
N2 - Fully-human antibodies have a great therapeutic importance; however, the development of stable strains providing a high level of production of full-size antibodies is a challenging task, as antibody molecules contain two types of polypeptide chains. To develop the producing strain, random integration of the plasmid containing the gene encoding the target protein into the genome of the host cells is commonly used. The aim of this study was the development of an original expression system, using gene targeting to integrate the gene encoding the fully-human antibody into the transcriptionally active region of the genome of eukaryotic suspension cells CHO-S. To develop a stable strain, the cassette vector plasmid pCDNA5/FRTDHFR- CH-CL containing the site of homologous recombination and the genes encoding heavy and light chains of the fully human antibody of the IgG1/kappa class was constructed at the first step. Notably, DNA of the plasmid pCDNA5/FRT-DHFR-CH-CL was organized in such a way that the restriction sites for rapid cloning of DNA fragments encoding the variable domains of heavy and light chains were inserted upstream of the sequences encoding constant domains of the heavy and light chains of the antibody. Secondly, DNA fragments encoding the variable domains of the heavy and light chains of antibody against orthopoxvirus protein p35 were inserted into the pCDNA5/FRT-DHFRCH- CL cassette plasmid. Then, CHO-S/FRT cells, which contain the FRT-site for homologous recombination and are able to produce green fluorescence protein GFP, were transfected with the constructed plasmid. After the insertion of the target genes into the FRT-site, GFP production was supposed to stop. Using this selection system, a stable clone producing target antibody fh8E was selected with the level of production of about 100 μg/ml. The binding affinity of purified antibody fh8E with the targeted protein, measured by surface plasmon resonance, was 12 nM. In addition, antibody fh8E demonstrated anti-vaccinia virus activity in the plaque reduction neutralization test in vitro.
AB - Fully-human antibodies have a great therapeutic importance; however, the development of stable strains providing a high level of production of full-size antibodies is a challenging task, as antibody molecules contain two types of polypeptide chains. To develop the producing strain, random integration of the plasmid containing the gene encoding the target protein into the genome of the host cells is commonly used. The aim of this study was the development of an original expression system, using gene targeting to integrate the gene encoding the fully-human antibody into the transcriptionally active region of the genome of eukaryotic suspension cells CHO-S. To develop a stable strain, the cassette vector plasmid pCDNA5/FRTDHFR- CH-CL containing the site of homologous recombination and the genes encoding heavy and light chains of the fully human antibody of the IgG1/kappa class was constructed at the first step. Notably, DNA of the plasmid pCDNA5/FRT-DHFR-CH-CL was organized in such a way that the restriction sites for rapid cloning of DNA fragments encoding the variable domains of heavy and light chains were inserted upstream of the sequences encoding constant domains of the heavy and light chains of the antibody. Secondly, DNA fragments encoding the variable domains of the heavy and light chains of antibody against orthopoxvirus protein p35 were inserted into the pCDNA5/FRT-DHFRCH- CL cassette plasmid. Then, CHO-S/FRT cells, which contain the FRT-site for homologous recombination and are able to produce green fluorescence protein GFP, were transfected with the constructed plasmid. After the insertion of the target genes into the FRT-site, GFP production was supposed to stop. Using this selection system, a stable clone producing target antibody fh8E was selected with the level of production of about 100 μg/ml. The binding affinity of purified antibody fh8E with the targeted protein, measured by surface plasmon resonance, was 12 nM. In addition, antibody fh8E demonstrated anti-vaccinia virus activity in the plaque reduction neutralization test in vitro.
KW - CHO cells
KW - Ectromelia virus
KW - Fully human antibody
KW - Genomic amplification
KW - Plasmid
KW - Producing strain
UR - http://www.scopus.com/inward/record.url?scp=85040969188&partnerID=8YFLogxK
U2 - 10.18699/VJ17.324
DO - 10.18699/VJ17.324
M3 - Article
AN - SCOPUS:85040969188
VL - 21
SP - 993
EP - 1000
JO - Вавиловский журнал генетики и селекции
JF - Вавиловский журнал генетики и селекции
SN - 2500-0462
IS - 8
ER -
ID: 10455527