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

## TL;DR

Researchers designed proteins that can block both human and dog TNFα, a key target in treating inflammation, using structural insights and computational methods.

## Contribution

A novel computational approach to design minibinder proteins that effectively antagonize both canine and human TNFα with high affinity.

## Key findings

- A conserved hydrophobic region on TNFα was identified as a target for universal minibinder design.
- Designed minibinders showed sub-nanomolar to nanomolar affinity for both canine and human TNFα.
- Minibinders demonstrated anti-TNFα activity comparable or superior to existing nanobodies in cell-based assays.

## Abstract

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.

De novo computational design of minibinder proteins, which achieve sub-nanomolar affinity to both human and canine TNFα by targeting a conserved hydrophobic surface region, enables potent cross-species TNFα antagonism.