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Updated: Feb 25, 2026
Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody
Published on: May 16, 2020
Functional optimization of agonistic antibodies to OX40 receptor with novel Fc mutations to promote antibody
Di Zhang1, Anthony A Armstrong1, Susan H Tam1
1a Janssen Research and Development, L.L.C. , Spring House , PA , USA.
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.
Insights
Novel Fc mutations (T437R/K248E) enhance cancer immunotherapy by promoting antibody clustering on tumor cells. This improves agonism and effector functions for tumor necrosis factor receptor (TNFR) superfamily targets.
Area of Science:
- Immunology
- Biotechnology
- Oncology
Background:
- Immunostimulatory receptors of the tumor necrosis factor receptor (TNFR) superfamily are key targets for cancer immunotherapy.
- Fc engineering of therapeutic antibodies can optimize receptor agonism by facilitating cell surface clustering and downstream signaling.
- Current strategies aim to enhance antibody multimerization for improved therapeutic efficacy.
Purpose of the Study:
- To identify novel Fc mutations that promote antibody multimerization on cell surfaces for enhanced TNFR agonism.
- To evaluate the impact of these mutations on antibody-dependent effector functions and pharmacokinetics.
- To assess the potential of Fc engineering in developing next-generation cancer immunotherapies.
Main Methods:
- Analysis of IgG1 crystal structures to identify potential Fc mutations (T437R and K248E).
- Assessment of antibody agonism using NF-κB reporter assays with engineered anti-OX40 antibodies.
- Evaluation of FcγRIIB crosslinking effects on antibody agonism.
- Characterization of Fc engineered antibody effector functions (ADCC, ADCP, CDC) and FcRn binding/pharmacokinetics in mice.
Main Results:
- Identified T437R and K248E Fc mutations that facilitate antibody multimerization on cell surfaces.
- Engineered mutations enhanced agonism of an anti-OX40 antibody, independent of FcγRIIB crosslinking.
- FcγRIIB-expressing cells boosted agonism of engineered IgG1 Fc antibodies.
- Enhanced effector functions (ADCC, ADCP, CDC) were observed, dependent on IgG subtypes.
- Fc engineered antibodies maintained normal FcRn binding and pharmacokinetic profiles in vivo.
Conclusions:
- A novel Fc engineering approach using T437R/K248E mutations promotes antibody multimerization on cell surfaces.
- This approach enhances agonism and effector functions for anti-TNFR antibodies and potentially other therapeutic antibodies.
- Fc engineering offers a promising strategy to improve the efficacy of cancer immunotherapies targeting TNFR superfamily receptors.
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