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

Area of Science:

• Immunology
• Molecular Biology
• Biophysics

Background:

• Monoclonal antibodies (mAbs) and natural ligands targeting costimulatory tumor necrosis factor receptors (TNFRs) have diverse agonistic activities and antitumor responses.
• The precise mechanisms behind these differential activities are not fully understood.

Purpose of the Study:

• To investigate the mechanisms underlying differential agonistic activities of molecules targeting human CD40, 4-1BB, and OX40.
• To correlate molecular structure and receptor clustering with functional agonism.

Main Methods:

• Utilized a panel of experimental and clinically-relevant mAbs and ligands.
• Employed confocal and super-resolution microscopy (STORM) to analyze receptor clustering.
• Compared antibody isotypes (hIgG1 vs. hIgG2) and their effects on receptor aggregation.

Main Results:

• Strongly agonistic reagents induced receptor clusters with small areas and high receptor densities.
• Human IgG2 (hIgG2) mediated significantly greater receptor clustering than hIgG1 across CD40, 4-1BB, and OX40.
• Receptor clustering, particularly high density and unique super-structures like rod-shaped assemblies, correlated with enhanced agonism, shielding the complex.

Conclusions:

• Receptor density and super-structure, rather than simply cluster size, are key determinants of agonistic activity.
• Findings challenge the notion that larger receptor clusters equate to greater agonism.
• Isotype selection (e.g., hIgG2) can significantly influence receptor clustering and therapeutic efficacy.