Identification of Anti-TNF alpha VNAR Single Domain Antibodies from Whitespotted Bambooshark (Chiloscyllium plagiosum)

Tumor necrosis factor alpha (TNF alpha), an important clinical testing factor and drug target, can trigger serious autoimmune diseases and inflammation. Thus, the TNF alpha antibodies have great potential application in diagnostics and therapy fields. The variable binding domain of IgNAR (VNAR), the shark single domain antibody, has some excellent advantages in terms of size, solubility, and thermal and chemical stability, making them an ideal alternative to conventional antibodies. This study aims to obtain VNARs that are specific for mouse TNF (mTNF) from whitespotted bamboosharks. After immunization of whitespotted bamboosharks, the peripheral blood leukocytes (PBLs) were isolated from the sharks, then the VNAR phage display library was constructed. Through phage display panning against mTNF alpha, positive clones were validated through ELISA assay. The affinity of the VNAR and mTNF alpha was measured using ELISA and Bio-Layer Interferometry. The binding affinity of 3B11 VNAR reached 16.7 nM. Interestingly, one new type of VNAR targeting mTNF was identified that does not belong to any known VNAR type. To understand the binding mechanism of VNARs to mTNF alpha, the models of VNARs-mTNF alpha complexes were predicted by computational modeling combining HawkDock and RosettaDock. Our results showed that four VNARs' epitopes overlapped in part with that of mTNFR. Furthermore, the ELISA assay shows that the 3B11 potently inhibited mTNF alpha binding to mTNFR. This study may provide the basis for the TNF alpha blockers and diagnostics applications.