Functional optimization of agonistic antibodies to OX40 receptor with novel Fc mutations to promote antibody multimerization

Abstract

Immunostimulatory receptors belonging to the tumor necrosis factor receptor (TNFR) superfamily are emerging as promising targets for cancer immunotherapies. To optimize the agonism of therapeutic antibodies to these receptors, Fc engineering of antibodies was applied to facilitate the clustering of cell surface TNFRs to activate downstream signaling pathways. One engineering strategy is to identify Fc mutations that facilitate antibody multimerization on the cell surface directly. From the analyses of the crystal packing of IgG1 structures, we identified a novel set of Fc mutations, T437R and K248E, that facilitated antibody multimerization upon binding to antigens on cell surface. In a NF-κB reporter assay, the engineered T437R/K248E mutations could facilitate enhanced agonism of an anti-OX40 antibody without the dependence on FcγRIIB crosslinking. Nonetheless, the presence of cells expressing FcγRIIB could facilitate a boost of the agonism of the engineered antibody with mutations on IgG1 Fc, but not on the silent IgG2σ Fc. The Fc engineered antibody also showed enhanced effector functions, including antibody-dependent cell-meditated cytotoxicity, antibody-dependent cellular phagocytosis, and complement-dependent cytotoxicity, depending on the IgG subtypes. Also, the engineered antibodies showed normal FcRn binding and pharmacokinetic profiles in mice. In summary, this study elucidated a novel Fc engineering approach to promote antibody multimerization on a cell surface, which could enhance agonism and improve effector function for anti-TNFR antibodies as well as other therapeutic antibodies.

Tetravalent biepitopic targeting enables intrinsic antibody agonism of tumor necrosis factor receptor superfamily members

“…demonstrated that different Fc-engineered variants that do not form solution hexamers can retain intrinsic agonist activity, yet possess more normal clearance properties in SCID mice. 19 While we have generated similar results with a broader panel of Fc-engineered variants that behave as monomeric or hexameric antibody species, we have found the in vivo activity and disposition properties of Fcengineered variants that promote Fc hexamerization to be complex (data not shown), and have therefore limited the scope of the current report. Overall, while further investigation is needed to understand the basis of these results, together our data suggest that multiple Fc regions can present PK or immunogenicity liabilities for multivalent antibody formats.…”

“…Increasing valency to 12 using the noncovalent Fc variant RGY resulted in intrinsic activation, consistent with previous work. 16,17,19 While tetravalent monoepitopic targeting (red diamonds) provided a modest level of agonism independent of extrinsic crosslinking, tetravalent biepitopic targeting (blue circles) provided a more profound level of intrinsic activity (Figure 2(b-e)). The same trend in tetravalent biepitopic superiority was observed across the four distinct antibody formats, and either Fv orientation of the r:Fv-IgG and r:Fab-IgG formats demonstrated comparable enhanced activity (Figure 2(b,c)).…”

Antibody Structure and Function: The Basis for Engineering Therapeutics

“…This cross-linking to FcγRIIB can lead to a further boost of the agonism of the anti-OX40 antibody with an IgG1 Fc but not with the silent IgG2σ Fc region, which lacks binding to FcγRs. The ADCC and CDC activities of the anti-OX40 antibody with the E345R mutation were affected by the choice of IgG subtypes [362]. With so many oligomeric Ab targets, there are continuing applications of hexameric therapeutic Abs that can affect downstream signaling events [328,329,363].…”

FcγRII-binding Centyrins mediate agonism and antibody-dependent cellular phagocytosis when fused to an anti-OX40 antibody

“… 17 Several Fc mutations have subsequently been identified that can facilitate antibody multimerization upon antigen binding. 19 , 20 Such mutations could facilitate FcγRIIB-independent agonism enhancement with clustered anti-OX40 antibody. 17 , 20 Furthermore, likely due to increased binding of Fcγ receptors to clustered antibodies with IgG1 Fc, the engineered anti-OX40 antibody showed an additional boost of agonism and elevated effector functions.…”

“… 17 , 20 Furthermore, likely due to increased binding of Fcγ receptors to clustered antibodies with IgG1 Fc, the engineered anti-OX40 antibody showed an additional boost of agonism and elevated effector functions. 17 , 20 …”