TNF receptor agonists induce distinct receptor clusters to mediate differential agonistic activity

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.

Agonistic CD27 antibody potency is determined by epitope-dependent receptor clustering augmented through Fc-engineering

“…In fact, in the PBMC assay, anti-hCD27 h1 treatment reduces CD8 + T-cell proliferation, suggesting possible depletion of CD27 + cells by NK cells in the culture. Further, enhanced clustering of CD27 by h2 compared to h1 mAb was observed in the absence of FcγR-expressing cells, supporting the ability of this isotype to cluster TNFR irrespective of FcγR binding and this is similar to OX40, 4-1BB and CD40 mAb 52 . Based on these findings, hCD27 h2, h1 V11 or SE/LF mAb deliver stronger agonism than h1 counterparts.…”

Effect of Smoking and Its Cessation on the Transcript Profile of Peripheral Monocytes in COPD Patients

“…67 Interactions of TNFA and TNFR1 leads to clustering of TRADD (TNF (Tumor Necrosis Factor) R1 (Receptor Type 1)-Associated Death Domain) results in activation of caspases leading to "Caspase Cascade" and apoptosis. 68,69 It is notable that the differential display in the expression of two (2) candidate genes, CASP9 and TNFRSF1A that were integral to these pathways were externally validated by quantitative RT-PCR experiments in the present study.…”

Optimal target saturation of ligand-blocking anti-GITR antibody IBI37G5 dictates FcγR-independent GITR agonism and antitumor activity

“…Recent studies have shown that by simply switching the IgG isotypes (hIgG1→hIgG2), the CD40 antagonists can be converted into strong agonists, 54 possibly due to the hIgG2-mediated higher binding avidity and strong self-association tendency upon antigen binding. 55 Although such self-association has been previously described for other human IgG isotypes, 56 , 57 it is less likely that IBI37G5 exerts its agonistic activity through similar mechanism due to the much weaker self-association capability of hIgG1. In addition, the assembly of IgG1 oligomers is dependent on Fc-Fc interactions, 58 but the Fc-absent F(ab') 2 of IBI37G5 is fully functional, therefore excluding the effects of IgG self-assembly on agonist activity.…”