Abstact

The pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is synthesized as transmembrane TNF-alpha (tmTNF-alpha) where proteolytic processing releases soluble TNF-alpha (sTNF-alpha). tmTNF-alpha can act as either a ligand by activating TNF receptors, or a receptor that transmits reverse (outside-to-inside) signalling after binding to native receptors. All TNF-alpha therapies bind tmTNF-alpha and induce reverse signalling which can result in immunosuppression leading to infection. We present crystal structures of two anti-TNF-alpha Variable New Antigen Receptors (VNARs) in complex with sTNF-alpha via two distinct epitopes. The VNAR-D1 recognizes an epitope that selectively engages sTNF-alpha while VNAR-C4 binds an epitope that partially overlaps with other biologic therapies. In activated CD4(+) T cells, our VNARs do not induce reverse signalling in contrast to currently available therapies. Our findings suggest that neutralization through a unique mechanism may lead to anti-TNF-alpha agents with an improved safety profile that will benefit high-risk patients.