LAUSR

The agonistic action of several immunomodulatory monoclonal antibodies (mAb) requires both target antigen binding and clustering of this mAb:target complex by the Fcs interacting with Fcγ receptors (FcγR), in particular FcγRIIb, on neighbouring bystander cells. Fc mutations were made in the IgG4-based TGN1412 anti-CD28 mAb to define the role of FcγR interactions in its "super-agonist" activity. The dual mutation, IgG4-ED269,270 AA, ablated interaction with all human FcγR and agonistic action was consequentially lost, confirming the FcγR-dependence on the action of TGN1412. The IgG4 lower hinge region (F234 L235 G236 G237 ) was modified by L235 E mutation (F234 E235 G236 G237 ), a mutation commonly used to ablate FcγR binding, including in approved therapeutic mAbs. However, rather than ablating all FcγR binding, IgG4-L235 E conferred specific binding to FcγRIIb, the inhibitory Fc receptor. Furthermore, in combination with the core hinge-stabilising mutation (IgG4-S228 P, L235 E), this mutation increased affinity for FcγRIIb compared to wild type IgG4. In addition to having FcγRIIb specificity, these engineered TGN1412 antibodies also retained their super-agonistic ability, demonstrating that CD28 and FcγRIIb-specific binding are together sufficient for agonistic function. The FcγRIIb-specific nature of IgG4-L235 E has utility for mAb-mediated immune agonism therapies that are dependent on FcγRIIb interaction and of anti-inflammatory mAbs in allergy and autoimmunity that harness FcγRIIb inhibitory signalling.