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KMID : 0613820200300111012
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Journal of Life Science
2020 Volume.30 No. 11 p.1012 ~ p.1020
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The Improved Antigen-binding Activity of Biosimilar Remicade ScFv Antibodies by Fusion of the Leucine Zipper Domain
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Kim Jin-Kyoo
Kim Tae-Hwan
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Abstract
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Remicade is a therapeutic biosimilar natural antibody in which the mouse variable domain has been linked to the human constant domain. It is a chimeric monoclonal antibody specific to tumor necrosis factor-alpha (TNF-α) and has been developed for the treatment of rheumatoid arthritis. To investigate the biological activity of the Remicade antibody, we carried out a bioinformatics study using a protein data bank to characterize the TNF-α antigen binding mechanism of the Remicade natural antibody. Because the production of the Remicade antibody is often limited by genetic instability of the natural antibody-producing cell, we generated a Remicade single-chain variable domain fragment antibody (Remicade) in which a heavy chain variable domain (VH) is joined with a light chain variable domain (VL) by a polypeptide linker. Furthermore, Remicade was fused to a leucine zipper (RemicadeScZip) for higher production and higher antigen-binding activity than Remicade. The Remicade and Remicade ScZip were expressed in Escherichia coli and purified by a Ni+-NTA-agarose column. As expected, the purified proteins had migrated as 28.80 kDa and 33.96 kDa in sodium dodecyl sulfate?polyacrylamide electrophoresis. The TNF-α antigen binding activity of Remicade was not observed by ELISA and western blot. In contrast, RemicadeScZip showed antigen-binding activity. Additional bio-layer interferometry analysis confirmed the antigen-binding activity of RemicadeScZip, suggesting that the leucine zipper stabilized the folding of RemicadeScZip in a denatured condition and improved the TNF-α antigen- binding activity.
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KEYWORD
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Bio-layer interferometry, leucine zipper, remicade, remicadescZip, single-chain variable domain fragment
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