Design of minibinder proteins as universal antagonists against canine and human TNFα.

In this study, we resolve the complex structure of canine TNFα (cTNFα) trimer bound to nanobody molecules at a resolution of 3.1 Å using cryo-electron microscopy. Structural comparison between cTNFα and human TNFα (hTNFα) reveals that the non-conserved residue Phe83 in cTNFα induces a conformational change in a nearby loop, which significantly reduces the binding affinity of cTNFα to nanobody TNF30. By analyzing the simulated complex structures of cTNFα with its receptors and resolved structures of hTNFα in complex with its receptors, we identify a conserved hydrophobic region involved in ligand-receptor interaction on both TNFαs. Five hydrophobic residues within this region are determined as target hotspots to allow de novo computational design of universal minibinders against both canine and human TNFα. Purified top-ranked designs exhibit sub-nanomolar to nanomolar affinity toward cTNFα as well as hTNFα, and show anti-TNFα activities to both TNFαs in cell-based assays that are comparable or superior to the anti-hTNFα activity of TNF30. This work provides a practical approach to generate universal TNFα antagonists as promising lead molecules for the development of potent cross-species biologics applicable in both humans and canines.