Mutation of the TGN1412 anti-CD28 monoclonal antibody lower hinge confers specific FcγRIIb binding and retention of super-agonist activity

The agonistic action of several immunomodulatory monoclonal antibodies (mAbs) requires both target antigen binding and clustering of this mAb:target complex by the Fcs interacting with Fcγ receptors (FcγRs), in particular FcγRIIb, on neighboring bystander cells. Fc mutations were made in the immunoglobulin G4 (IgG4)-based TGN1412 anti-CD28 mAb to define the role of FcγR interactions in its "super-agonist" activity. The dual mutation, IgG4-ED^269,270 AA, ablated interaction with all human FcγRs 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-stabilizing mutation (IgG4-S²²⁸ P, L²³⁵ E), this mutation increased affinity for FcγRIIb compared with wild-type IgG4. In addition to having FcγRIIb specificity, these engineered TGN1412 antibodies 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 signaling.