The structural basis for the selective antagonism of soluble TNF-alpha by shark variable new antigen receptors

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