Analysis of FcγR-Dependent Agonism of Antibodies Specific for Receptors of the Tumor Necrosis Factor (TNF) Receptor Superfamily (TNFRSF).

Abstract

In vivo research of the last decade revealed that the anchoring of antitumor necrosis factor (TNF) receptor superfamily (TNFRSF) receptor antibodies to cell-expressed Fcγ receptors (FcγR) can be of decisive relevance for their receptor-stimulatory activity. Indeed, FcγR anchoring may even result in the conversion of antagonistic to agonistic anti-TNFR antibody activity. The knowledge on this issue is obviously not only relevant to understand the in vivo effects of anti-TNFR antibodies but also of overwhelming importance for the rational clinical development of antibodies and antibody derivatives. Based on the fact that with exception of the decoy TNFRSF receptors (TNFRs) all TNFRs are able to trigger proinflammatory NFκB signaling, resulting in the production of chemokines and cytokines, we established an easy and broadly applicable coculture assay for the evaluation of the FcγR-dependency of the agonism of anti-TNFR antibodies. In this assay, TNFR responder cells, which produce high amounts of IL8 in response to TNFR stimulation, were pairwise incubated with empty vector- and FcγR-transfected HEK293 cells, which produce only very low amounts of IL8. This cocultures were then comparatively analyzed with respect to anti-TNFR antibody-induced IL8 production as a readout for TNFR activation to uncover proagonistic effects of FcγR binding.