ABSTRACT
Development of monoclonal antibody therapeutics against vascular endothelial growth factor receptor 2 (VEGFR-2) protein, which is the main regulator in angiogenesis, has been a major challenge for years. In the current study, we engineer an inclusion body forming single-chain variable fragment (scFv) against VEGFR-2 by using complementarity determining regions (CDR) grafting technique to improve its solubility and investigate the activity of the engineered molecule. CDR sequences of the target scFv were grafted into the framework of another intrinsically soluble scFv molecule. Based on the computational results, CDR grafting has increased the solubility of the grafted scFv molecule. Results confirmed that the grafting approach increased in vivo folding properties of the target scFv molecule compared with the original scFv molecule. Similar binding affinities to the VEGFR-2 were observed for the original and the grafted scFv by surface plasmon resonance assays. Biological activity assays, including human umbilical vein endothelial cells proliferation and wound healing assays, showed that grafted scFv molecule has an antiangiogenic property. This study suggests that an antiangiogenic scFv fully expressed as an inclusion body can be rescued by grafting its CDR regions to a scFv expressed in a soluble form without any loss in its binding property and its activity.
Subject(s)
Single-Chain Antibodies , Humans , Vascular Endothelial Growth Factor Receptor-2/genetics , Vascular Endothelial Growth Factor Receptor-2/metabolism , Escherichia coli/metabolism , Vascular Endothelial Growth Factor A , Amino Acid Sequence , Human Umbilical Vein Endothelial CellsABSTRACT
In December 2019 a novel coronavirus was detected in Wuhan City of Hubei Province-China. Owing to a high rate of transmission from human to human, the new virus called SARS-CoV-2 differed from others by its unexpectedly rapid spread. The World Health Organization (WHO) described the most recent coronavirus epidemic as a global pandemic in March 2020. The virus spread triggered a health crisis (the COVID-19 disease) within three months, with socioeconomic implications. No approved targeted-therapies are available for COVID-19, yet. However, it is foreseen that antibody-based treatments may provide an immediate cure for patients. Current neutralizing antibody development studies primarily target the S protein among the structural elements of SARS-CoV-2, which mediates the cell entry of the virus through the angiotensin converting enzyme 2 (ACE2) receptor of host cells. This review aims to provide some of the neutralizing antibody development strategies for SARS-CoV-2 and in vitro and in vivo neutralization assays.
