A trivalent TNF-R2 as a new tumor necrosis factor alpha-blocking molecule

Proteins. 2021 Nov;89(11):1557-1564. doi: 10.1002/prot.26177. Epub 2021 Jul 17.

Abstract

The neutralization of tumor necrosis factor alpha (TNFα) with biopharmaceuticals is a successful therapy for inflammatory diseases. Currently, one of the main TNFα-antagonists is Etanercept, a dimeric TNF-R2 ectodomain. Considering that TNFα and its receptors are homotrimers, we proposed that a trimeric TNF-R2 ectodomain could be an innovative TNFα-antagonist. Here, the 3cTNFR2 protein was designed by the fusion of the TNF-R2 ectodomain with the collagen XV trimerization domain. 3cTNFR2 was produced in HEK293 cells and purified by immobilized metal affinity chromatography. Monomers, dimers, and trimers of 3cTNFR2 were detected. The interaction 3cTNFR2-TNFα was assessed. By microscale thermophoresis, the KD value for the interaction was 4.17 ± 0.88 nM, and complexes with different molecular weights were detected by size exclusion chromatography-high performance liquid chromatography. Moreover, 3cTNFR2 neutralized the TNFα-induced cytotoxicity totally in vitro. Although more studies are required to evaluate the anti-inflammatory effect, the results suggest that 3cTNFR2 could be a TNFα-antagonist agent.

Keywords: protein engineering; protein-protein interaction; tumor necrosis factor alpha.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-Inflammatory Agents / chemistry
  • Anti-Inflammatory Agents / metabolism
  • Anti-Inflammatory Agents / pharmacology*
  • Cell Survival / drug effects
  • Collagen / genetics*
  • Collagen / metabolism
  • Endotoxins / antagonists & inhibitors*
  • Endotoxins / metabolism
  • Endotoxins / toxicity
  • Etanercept / chemistry
  • Etanercept / metabolism
  • Etanercept / pharmacology*
  • Gene Expression
  • HEK293 Cells
  • Humans
  • Models, Molecular
  • Protein Binding
  • Protein Conformation
  • Protein Domains
  • Protein Engineering / methods
  • Protein Multimerization
  • Receptors, Tumor Necrosis Factor, Type II / genetics*
  • Receptors, Tumor Necrosis Factor, Type II / metabolism
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Recombinant Fusion Proteins / pharmacology
  • Tumor Necrosis Factor-alpha / antagonists & inhibitors*
  • Tumor Necrosis Factor-alpha / metabolism
  • Tumor Necrosis Factor-alpha / toxicity

Substances

  • Anti-Inflammatory Agents
  • COL15A1 protein, human
  • Endotoxins
  • Receptors, Tumor Necrosis Factor, Type II
  • Recombinant Fusion Proteins
  • Tumor Necrosis Factor-alpha
  • Collagen
  • Etanercept