A trimeric anti-HER2/neu ScFv and tumor necrosis factor-α fusion protein induces HER2/neu signaling and facilitates repair of injured epithelia

Tumor necrosis factor ( TNF)-alpha genetically fused to the carboxyl terminus of a single-chain Fv ( ScFv) antibody specific for the human HER2/neu ( anti-HER2/neu ScFv-TNF-alpha) forms a homotrimeric structure that retains both TNF-alpha activity and the ability to bind HER2/neu. In contrast to anti-HER2/neu IgG3, anti-HER2/neu ScFv-TNF-alpha induces potent HER2/neu signaling, activating the downstream mitogen-activated protein kinase ( MAPK) and Akt pathways in SKBR3 cells. Activation of MAPK and Akt by anti-HER2/neu ScFv-TNF-alpha inhibited the apoptosis of SKBR3 cells induced by actinomycin D. Remarkably, anti-HER2/neu ScFv-TNF-alpha facilitated the repair of injured epithelia. Accelerated wound healing required binding to HER2/ neu but not TNF-alpha activity since anti-HER2/neu ScFv-TNF-alpha ( S147Y), containing a mutant TNF-alpha with significantly decreased biological activity, demonstrated equivalent ability to facilitate wound healing and soluble HER2/ neu inhibited the effect. These results suggest that trimeric anti-HER2/neu ScFv has the potential to facilitate wound healing. In addition, fusion with TNF-alpha provides a novel approach to producing polymeric antibodies.