Fischer et al. accepted for publication in Scientific Reports

Novel strategies to mimic transmembrane tumor necrosis factor-dependent activation of tumor necrosis factor receptor 2

• Roman Fischer ^1,2, Jessica Marsal ¹, Cristiano Guttà ¹, Stephan A. Eisler ³, Nathalie Peters ¹, John R. Bethea ², Klaus Pfizenmaier ¹ & Roland E. Kontermann ¹
• ¹Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, 70569, Stuttgart, Germany.
• ²Department of Biology, Drexel University, 3245 Chestnut Street, Philadelphia, PA, 19104, USA.
• ³Stuttgart Research Center Systems Biology, Nobelstraße 15, University of Stuttgart, Stuttgart, Germany.

Abstract

Tumor necrosis factor receptor 2 (TNFR2) is known to mediate immune suppression and tissue
regeneration. Interestingly, the transmembrane form of tumor necrosis factor (tmTNF) is necessary to
robustly activate TNFR2. To characterize the stoichiometry and composition of tmTNF during TNFR2
activation, we constructed differently oligomerized single chain TNF ligands (scTNF) comprised of
three TNF homology domain (THD) protomers that mimic tmTNF. Using a variety of cellular and
in vivo assays, we can show that higher oligomerization of the scTNF trimers results in more efficient TNF/
TNFR2 clustering and subsequent signal transduction. Importantly, the three-dimensional orientation
of the scTNF trimers impacts the bioactivity of the oligomerized scTNF ligands. Our data unravel the
organization of tmTNF-mimetic scTNF ligands capable of robustly activating TNFR2 and introduce
novel TNFR2 agonists that hold promise as therapeutics to treat a variety of diseases.