New insights on the interaction mechanism of rhTNF with its antagonists Adalimumab and Etanercept

TNF is a pro-inflammatory cytokine that is a therapeutic target for inflammatory autoimmune disorders. Thus, TNF antagonists are successfully used for the treatment of these disorders. Here, new association patterns of rhTNF and its antagonists Adalimumab and Etanercept are disclosed. Active rhTNF was purified by IMAC from the soluble fraction of transformed E. coli. Protein detection was assessed by SDS-PAGE and western blot. The KD values for rhTNF interactions with their antagonists were obtained by non-competitive ELISA and by microscale thermophoresis. Molecular sizes of the complexes were characterized by SEC-HPLC. Surprisingly, both antagonists recognized the monomeric form of rhTNF under reducing and non-reducing conditions, indicating unexpected bindings of the antagonists to lineal epitopes and to one protomer of rhTNF. Binding curves of two phases with low and high KD values (10-8 M) were observed during thermophoresis experiments, suggesting the generation of complexes with different stoichiometry, which were confirmed by SEC-HPLC. This pioneer investigation revealed interactions of rhTNF with Adalimumab and Etanercept never described before, which constitute valuable data for future approaches into the study of their interaction mechanism.