Design and Construction of a Novel Humanized Single-Chain Variable-Fragment Antibody Against the Tumor Necrosis Factor Alpha

The pro-inflammatory cytokine, tnf-α, which plays a major role in the development and persistence of diseases such as crohn’s disease, psoriasis, psoriatic arthritis, and rheumatoid arthritis, 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 inflammatory 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 single chain fragment variable mab against human tnf-α (hd2). the subsequent 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 gsthd2 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.