Abstract

Tumor necrosis factor-alpha (TNF alpha) inhibitors could prevent neurological disorders systemically, but their design generally relies on molecules unable to cross the blood-brain barrier (BBB). This research was aimed to design and characterize a novel TNF alpha inhibitor based on the angiopeptide-2 as a BBB shuttle molecule fused to the extracellular domain of human TNF alpha receptor 2 and a mutated vascular endothelial growth factor (VEGF) dimerization domain. This new chimeric protein (MTV) would be able to trigger receptor-mediated transcytosis across the BBB via low-density lipoprotein receptor-related protein-1 (LRP-1) and inhibit the cytotoxic effect of TNF alpha more efficiently because of its dimeric structure. Stably transformed CHO cells successfully expressed MTV, and its purification by Immobilized-Metal Affinity Chromatography (IMAC) rendered high purity degree. Mutated VEGF domain included in MTV did not show cell proliferation or angiogenic activities measured by scratch and aortic ring assays, which corroborate that the function of this domain is restricted to dimerization. The pairs MTV-TNF alpha (Kd 279 +/- 40.9 nM) and MTV-LRP1 (Kd 399 +/- 50.5 nM) showed high affinity by microscale thermophoresis, and a significant increase in cell survival was observed after blocking TNF alpha with MTV in a cell cytotoxicity assay. Also, the antibody staining in CHOK1 and bEnd3 cells demonstrated the adhesion of MTV to the LRP1 receptor located in the cell membrane. These results provide compelling evidence for the proper functioning of the three main domains of MTV individually, which encourage us to continue the research with this new molecule as a potential candidate for the systemic treatment of neurological disorders.