Abstract
Monoclonal antibodies (mAb) and natural ligands targeting costimulatory tumor necrosis factor receptors (TNFR) exhibit a wide range of agonistic activities and antitumor responses. The mechanisms underlying these differential agonistic activities remain poorly understood. Here, we employ a panel of experimental and clinically-relevant molecules targeting human CD40, 4-1BB and OX40 to examine this issue. Confocal and STORM microscopy reveal that strongly agonistic reagents induce clusters characterized by small area and high receptor density. Using antibody pairs differing only in isotype we show that hIgG2 confers significantly more receptor clustering than hIgG1 across all three receptors, explaining its greater agonistic activity, with receptor clustering shielding the receptor-agonist complex from further molecular access. Nevertheless, discrete receptor clustering patterns are observed with different hIgG2 mAb, with a unique rod-shaped assembly observed with the most agonistic mAb. These findings dispel the notion that larger receptor clusters elicit greater agonism, and instead point to receptor density and subsequent super-structure as key determinants.
Highlights
Monoclonal antibodies and natural ligands targeting costimulatory tumor necrosis factor receptors (TNFR) exhibit a wide range of agonistic activities and antitumor responses
Chemical crosslinking of the cell surface indicates that TNFR1, TNFR2, Fas, TRAILR2 and CD40 can selfassociate into non-covalent dimers or trimers, mediated by the pre-ligand assembly domain (PLAD) within CRD1 via homotypic interaction[24,25,26,27,28]
We previously demonstrated that a complex interplay between epitope and isotype determines anti-TNFR Monoclonal antibodies (mAb) agonism, with the human IgG2 isotype able to impart superior agonistic activity and convert CD40 antagonism to agonism[8,9,31]
Summary
Monoclonal antibodies (mAb) and natural ligands targeting costimulatory tumor necrosis factor receptors (TNFR) exhibit a wide range of agonistic activities and antitumor responses. Selective TNFR activation has been shown to augment antitumor immunity and confer robust therapeutic benefits in animal models[1] with TNFR-targeting mAbs and recombinant ligands showing promising antitumor activities in early phase trials[1,4,5], they remain behind checkpointblocking reagents in their development, likely due to a poorer understanding of how the activity is achieved and their multiple potential mechanisms of action[6,7] These clinical candidates display a range of agonistic activities resulting from differential TNFR activation, with their level of agonism correlated positively with their pharmacodynamic properties in clinical trials[8,9,10,11,12,13]. We previously demonstrated that a complex interplay between epitope and isotype determines anti-TNFR mAb agonism, with the human IgG2 (hIgG2) isotype able to impart superior agonistic activity and convert CD40 antagonism to agonism[8,9,31]
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