Anti-TNF-α [anti-(tumour necrosis factor-α)] therapy is widely considered to be among the most efficient treatments available for rheumatoid arthritis, psoriatic arthritis and inflammatory bowel disease. In the present study a tetravalent mini-antibody, named ‘TNF-TeAb’, was constructed by fusing the tetramerization domain of human p53 to the C-terminus of an anti-TNF-scFv [anti-(TNF-α–single-chain variable fragment)] via a long and flexible linking peptide derived from human serum albumin. TNF-TeAb was overexpressed as inclusion bodies in the cytoplasm of Escherichia coli, purified to homogeneity by immobilized- metal affinity chromtaography under denaturing conditions and produced in functional form by using an in vitro refolding system. In vitro bioactivity assays suggested that tetramerization of TNF-scFv resulted in an enormous gain in avidity, which endowed TNF-TeAb with a stronger ability to inhibit both receptor binding and cytolytic activity of TNF-α. TNF-α targeting therapy in rats with collagen-induced arthritis demonstrated that TNF-TeAb provided a much more significant therapeutic effect than did TNF-scFv in suppressing arthritis progression, attenuating inflammation and destruction in joints, and down-regulating pro-inflammatory cytokines and anti-(type II collagen) antibody. The conclusions are therefore (i) that multimerization of the antibody fragment by a self-association peptide is an efficient way to increase its avidity and (ii) that TNF-TeAb has potential applicability for anti-TNF-α therapy.

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