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Structural evaluation of EGFR inhibition mechanisms for nanobodies/VHH domains
Karl R Schmitz1, Atrish Bagchi, Rob C Roovers
1Department of Physiology and Graduate Group in Biochemistry and Molecular Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Abstract:
The epidermal growth factor receptor (EGFR) is implicated in human cancers and is the target of several classes of therapeutic agents, including antibody-based drugs. Here, we describe X-ray crystal structures of the extracellular region of EGFR in complex with three inhibitory nanobodies, the variable domains of heavy chain only antibodies (VHH). VHH domains, the smallest natural antigen-binding modules, are readily engineered for diagnostic and therapeutic applications. All three VHH domains prevent ligand-induced EGFR activation, but use two distinct mechanisms. 7D12 sterically blocks ligand binding to EGFR in a manner similar to that of cetuximab. EgA1 and 9G8 bind an epitope near the EGFR domain II/III junction, preventing receptor conformational changes required for high-affinity ligand binding and dimerization. This epitope is accessible to the convex VHH paratope but inaccessible to the flatter paratope of monoclonal antibodies. Appreciating the modes of binding and inhibition of these VHH domains will aid in developing them for tumor imaging and/or cancer therapy.
Insights
Three nanobodies (VHH) inhibit epidermal growth factor receptor (EGFR) activation through distinct mechanisms. These VHH domains offer potential for cancer therapy and imaging by targeting EGFR.
Area of Science:
- Structural Biology
- Immunology
- Oncology
Background:
- Epidermal growth factor receptor (EGFR) is a key target in cancer therapy.
- Antibody-based drugs are used to treat EGFR-driven cancers.
- Nanobodies (VHH) are small, engineered antibody fragments with therapeutic potential.
Purpose of the Study:
- To determine the X-ray crystal structures of EGFR's extracellular region complexed with inhibitory nanobodies.
- To elucidate the distinct mechanisms by which VHH domains inhibit EGFR activation.
- To explore the therapeutic and diagnostic applications of these VHH domains.
Main Methods:
- X-ray crystallography to obtain high-resolution structures.
- Biophysical and biochemical assays to assess EGFR inhibition.
- Structural analysis to compare VHH binding modes with monoclonal antibodies.
Main Results:
- Three VHH domains (7D12, EgA1, 9G8) were crystallized with the EGFR extracellular region.
- 7D12 inhibits EGFR by sterically blocking ligand binding, similar to cetuximab.
- EgA1 and 9G8 bind a unique epitope, preventing receptor activation and dimerization by altering conformation.
Conclusions:
- VHH domains inhibit EGFR through two distinct mechanisms: direct ligand-binding blockade or allosteric inhibition.
- The unique binding epitope of EgA1 and 9G8 is accessible to VHH but not conventional antibodies.
- These VHH domains show promise for developing novel cancer therapeutics and imaging agents targeting EGFR.
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