Enforced covalent trimerization increases the activity of the TNF ligand family members TRAIL and CD95L.

Variants of human TRAIL (hTRAIL) and human CD95L (hCD95L), encompassing the TNF homology domain (THD), interact with the corresponding receptors and stimulate CD95 and TRAILR2 signaling after cross-linking. The murine counterparts (mTRAIL, mCD95L) showed no or only low receptor binding and were inactive/poorly active after cross-linking. The stalk region preceding the THD of mCD95L conferred secondary aggregation and restored CD95 activation in the absence of cross-linking. A corresponding variant of mTRAIL, however, was still not able to activate TRAIL death receptors, but gained good activity after cross-linking. Notably, disulfide-bonded fusion proteins of the THD of mTRAIL and mCD95L with a subdomain of the tenascin-C (TNC) oligomerization domain, which still assembled into trimers, efficiently interacted with their cognate cellular receptors and robustly stimulated CD95 and TRAILR2 signaling after secondary cross-linking. Introduction of the TNC domain also further enhanced the activity of THD encompassing variants of hTRAIL and hCD95L. Thus, spatial fixation of the N-terminus of the THD appears necessary in some TNF ligands to ensure proper receptor binding. This points to yet unanticipated functions of the stalk and/or transmembrane region of TNF ligands for the functionality of these molecules and offers a broadly applicable option to generate recombinant soluble ligands of the TNF family with superior activity.