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Mol Cancer Ther. 2019 Apr;18(4):823-833. doi: 10.1158/1535-7163.MCT-18-0849. Epub 2019 Mar 1.

Nanobody Targeting of Epidermal Growth Factor Receptor (EGFR) Ectodomain Variants Overcomes Resistance to Therapeutic EGFR Antibodies.

Author information

1
Department of Oncology and Hematology, BMT with section Pneumology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
2
Department of Pediatrics, Center for Obstetrics and Pediatrics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
3
Institute of Immunology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
4
Department of Diagnostic and Interventional Radiology and Nuclear Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
5
Research Department Cell and Gene Therapy, Department of Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
6
Division of Stem Cell Transplantation and Immunotherapy, Department of Internal Medicine ll, Christian-Albrechts-University Kiel, Kiel, Germany.
7
Department of Oncology and Hematology, BMT with section Pneumology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. Mascha.Binder@uk-halle.de.

Abstract

Epidermal growth factor receptor (EGFR) ectodomain variants mediating primary resistance or secondary treatment failure in cancer patients treated with cetuximab or panitumumab support the need for more resistance-preventive or personalized ways of targeting this essential pathway. Here, we tested the hypothesis that the EGFR nanobody 7D12 fused to an IgG1 Fc portion (7D12-hcAb) would overcome EGFR ectodomain-mediated resistance because it targets a very small binding epitope within domain III of EGFR. Indeed, we found that 7D12-hcAb bound and inhibited all tested cell lines expressing common resistance-mediating EGFR ectodomain variants. Moreover, we assessed receptor functionality and binding properties in synthetic mutants of the 7D12-hcAb epitope to model resistance to 7D12-hcAb. Because the 7D12-hcAb epitope almost completely overlaps with the EGF-binding site, only position R377 could be mutated without simultaneous loss of receptor functionality, suggesting a low risk of developing secondary resistance toward 7D12-hcAb. Our binding data indicated that if 7D12-hcAb resistance mutations occurred in position R377, which is located within the cetuximab and panitumumab epitope, cells expressing these receptor variants would retain sensitivity to these antibodies. However, 7D12-hcAb was equally ineffective as cetuximab in killing cells expressing the cetuximab/panitumumab-resistant aberrantly N-glycosylated EGFR R521K variant. Yet, this resistance could be overcome by introducing mutations into the Fc portion of 7D12-hcAb, which enhanced immune effector functions and thereby allowed killing of cells expressing this variant. Taken together, our data demonstrate a broad range of activity of 7D12-hcAb across cells expressing different EGFR variants involved in primary and secondary EGFR antibody resistance.

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