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

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.

• Authors: Joseph Tintelnot, Natalie Baum, Christoph Schultheiß, Friederike Braig, Marie Trentmann, Johannes Finter, William Fumey, Peter Bannas, Boris Fehse, Kristoffer Riecken, Kerstin Schuetze, Carsten Bokemeyer, Thies Rösner, Thomas Valerius, Matthias Peipp, Friedrich Koch-Nolte, Mascha Binder
• Journal: Molecular cancer therapeutics (Mol Cancer Ther) Vol. 18 Issue 4 Pg. 823-833 (04 2019) ISSN: 1538-8514 [Electronic] United States
• PMID: 30824613 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: ©2019 American Association for Cancer Research.

Chemical References

• Epitopes
• Immunoglobulin Fc Fragments
• Immunoglobulin G
• Single-Domain Antibodies
• Panitumumab
• EGFR protein, human
• ErbB Receptors
• Cetuximab

Topics

• Binding Sites
• Cell Line, Tumor
• Cell Proliferation (drug effects)
• Cell Survival (drug effects)
• Cetuximab (immunology, pharmacology, therapeutic use)
• Drug Resistance, Neoplasm (drug effects)
• Epitopes (chemistry, immunology)
• ErbB Receptors (chemistry, genetics, immunology)
• Humans
• Immunoglobulin Fc Fragments (chemistry, genetics)
• Immunoglobulin G (chemistry, genetics)
• Mutation
• Panitumumab (immunology, pharmacology, therapeutic use)
• Polymorphism, Single Nucleotide (genetics)
• Protein Domains (genetics, immunology)
• Single-Domain Antibodies (chemistry, genetics, pharmacology)
• Transduction, Genetic