Abstract

The neutralization of tumor necrosis factor alpha (TNF alpha) with biopharmaceuticals is a successful therapy for inflammatory diseases. Currently, one of the main TNF alpha-antagonists is Etanercept, a dimeric TNF-R2 ectodomain. Considering that TNF alpha and its receptors are homotrimers, we proposed that a trimeric TNF-R2 ectodomain could be an innovative TNF alpha-antagonist. Here, the 3cTNFR2 protein was designed by the fusion of the TNF-R2 ectodomain with the collagen XV trimerization domain. 3cTNFR2 was produced in HEK293 cells and purified by immobilized metal affinity chromatography. Monomers, dimers, and trimers of 3cTNFR2 were detected. The interaction 3cTNFR2-TNF alpha was assessed. By microscale thermophoresis, the K-D value for the interaction was 4.17 +/- 0.88 nM, and complexes with different molecular weights were detected by size exclusion chromatography-high performance liquid chromatography. Moreover, 3cTNFR2 neutralized the TNF alpha-induced cytotoxicity totally in vitro. Although more studies are required to evaluate the anti-inflammatory effect, the results suggest that 3cTNFR2 could be a TNF alpha-antagonist agent.