An antibody targeting the proximal extracellular domain of OSMRβ inhibits IL-31 signaling: mechanism and structural insights.

Oncostatin M receptor beta (OSMRβ) is an essential signal-transducing subunit in the IL-31/IL-31 receptor alpha (IL-31RA)/OSMRβ complex and is implicated in inflammatory and pruritic diseases. We previously developed a high-affinity monoclonal antibody (2O2) that targets the proximal extracellular domain (amino acids 619-734) of canine OSMRβ (cOSMRβ) and shows no cross-reactivity with human OSMRβ (hOSMRβ). Here, we elucidate its functional mechanism of action and determine the co-crystal structure of the 2O2 Fab fragment in complex with this domain at a resolution of 2.76 Å. Consistent with the epitope location far away from the cytokine-binding domain (CBD) of cOSMRβ, 2O2 does not compete with canine IL-31 (cIL-31) for cOSMRβ binding. However, the in vitro functional assay demonstrated that 2O2 inhibits cIL-31-induced phosphorylation of STAT5 in DH-82 cells. Mechanistically, this inhibition is achieved through antibody-induced internalization of cOSMRβ from the cell surface. Structural analysis reveals that 2O2 binds at an oblique angle relative to the membrane, creating steric hindrance that induces localized lipid bilayer invagination. The bivalent IgG exhibits ∼34-fold higher potency than its monovalent Fab, reflecting not only avidity but also mechanical consequences of dual receptor engagement: crosslinking two receptor complexes exerts a stretching force that amplifies membrane invagination and promotes efficient endocytic uptake. This mechanism enables clearance of the entire signaling complex, including the cytokine, from the cell surface-a functional advantage over conventional cytokine-blocking antibodies. These findings establish 2O2 as a novel canine therapeutic candidate with a distinct internalization-driven mechanism of action.