Background: Fn14 is a therapeutic target in various diseases. Results: Anti-Fn14 antibodies activate the alternative NFB pathway but not other Fn14-related activities induced by soluble or membrane-bound TWEAK. Fc␥R-bound anti-Fn14 antibodies, however, activate the full spectrum of Fn14-associated activities. Conclusion: Anti-Fn14 antibodies elicit agonistic activities differing from those of the natural Fn14 ligand TWEAK. Significance: These findings influence the rationale of designing Fn14-targeted therapies.

Background: TWEAK and its receptor Fn14 are targets in oncology and autoimmunity. Results: Ligand oligomerization has no major effect on Fn14-TWEAK interaction but strongly enhances TWEAK-induced IL8 production. Conclusion: Avidity is irrelevant for TWEAK trimer binding to Fn14 but required for robust IL8 induction. Significance: Enhanced activity of oligomerized TWEAK trimers is not related to an avidity-related increase in Fn14 occupancy.

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Antibodies specific for TNFRSF receptors that bind soluble ligands without getting properly activated generally act as strong agonists upon FcγR binding. Systematic analyses revealed that the FcγR dependency of such antibodies to act as potent agonists is largely independent from isotype, FcγR type, and of the epitope recognized. This suggests that the sole cellular attachment, achieved by Fc domain-FcγR interaction, dominantly determines the agonistic activity of antibodies recognizing TNFRSF receptors poorly responsive to soluble ligands. In accordance with this hypothesis, we demonstrated that antibody fusion proteins harboring domains allowing FcγR-independent cell surface anchoring also act as strong agonist provided they have access to their target. This finding defines a general possibility to generate anti-TNFRSF receptor antibodies with FcγR-independent agonism. Moreover, anti-TNFRSF receptor antibody fusion proteins with an anchoring domain promise superior applicability to conventional systemically active agonists when an anchoring target with localized disease associated expression can be addressed.

To combine the CD27 stimulation inhibitory effect of blocking CD70 antibodies with an antibody-dependent cellular cytotoxicity (ADCC)-independent, cell death-inducing activity for targeting of CD70-expressing tumors, we evaluated here fusion proteins of the apoptosis-inducing TNF family member TRAIL and a single-chain variable fragment (scFv) derived from a high-affinity llama-derived anti-human CD70 antibody (lαhCD70). A fusion protein of scFv:lαhCD70 with TNC-TRAIL, a stabilized form of TRAIL, showed strongly enhanced apoptosis induction upon CD70 binding and furthermore efficiently interfered with CD70-CD27 interaction. Noteworthy, introduction of recently identified mutations that discriminate between TRAILR1 and TRAILR2 binding into the TRAIL part of scFv:lαhCD70-TNC-TRAIL resulted in TRAIL death receptor-specific fusion proteins with CD70-restricted activity.