Document Type: Research article
Department of Cellular and Molecular Biology, Faculty of Basic Sciences, Azarbaijan Shahid Madani University, Tabriz, Iran.
Biotechnology Research Center and Department of Medicinal Chemistry, School of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran.
Faculty of Pharmacy, Near East University, Nicosia, North Cyprus, Mersin 10, Turkey
The pro-inflammatory cytokine, TNF-α, which plays a major role in the development and persistence of inflammatory diseases, is the basis for the use of anti-TNF-α therapies. The neutralization of TNF-α or blockage of its binding to the corresponding receptor has mainly served as a therapeutic strategy against some diseases. This study aimed to investigate the production of a humanized single chain antibody (scFv) against TNF-α. Therefore, a murine monoclonal antibody, D2 mAb, was selected for humanizing by the complementarity determining region (CDR)-grafting method. Briefly, the replacement of the CDRs from D2 mAb with the specific human single chain scaffold led to the production of a novel humanized scFv mAb against human TNF-α (hD2). Cloning of hD2 into a suitable expression vector, pGEX-6P-1, resulted in the expression of a 52-kDa GST-fusion protein in E. coli, mostly in the form of inclusion bodies. The solubilization and refolding of GST-hD2 inclusion bodies was achieved with the addition of 4 M urea and subsequent dialysis to recover the fusion protein in soluble form. Then the soluble GST-hD2 was purified by affinity chromatography through immobilized glutathione. The GST pull-down experiment showed a positive interaction between GST-hD2 and TNF-α protein. Moreover, the results of an MTT assay showed that the purified GST-hD2 has TNF-α neutralizing activity (Kd of 1.03 nM) and hence hD2 has the potential to be developed into a therapeutic agent. However, more investigation is needed to elucidate the potential of in vivo TNF-α neutralizing activity of hD2 in comparison to other anti-TNF-α antibodies.