Identification and grafting of a unique peptide-binding site in the Fab framework of monoclonal antibodies

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 110; no. 43; pp. 17456 - 17461
• Main Authors: Donaldson, Joshua M., Zer, Cindy, Avery, Kendra N., Bzymek, Krzysztof P., Horne, David A., Williams, John C.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 22.10.2013; NATIONAL ACADEMY OF SCIENCES; National Acad Sciences; National Academy of Sciences, Washington, DC (United States)
• Subjects: Amino Acid Sequence; Antibodies; Antibodies, Monoclonal - chemistry; Antibodies, Monoclonal - genetics; Antibodies, Monoclonal - immunology; Antibodies, Monoclonal, Humanized - chemistry; Antibodies, Monoclonal, Humanized - genetics; Antibodies, Monoclonal, Humanized - immunology; Antigens; BASIC BIOLOGICAL SCIENCES; Binding sites; Binding Sites - genetics; Binding Sites - immunology; Biological Sciences; Biophysics; cancer; Carbon; Cell Line, Tumor; Cetuximab; Crystallography, X-Ray; Diffraction; ErbB Receptors - immunology; Humans; Imaging; Immunoglobulin Fab Fragments - chemistry; Immunoglobulin Fab Fragments - genetics; Immunoglobulin Fab Fragments - immunology; Medical treatment; Microscopy, Fluorescence; Models, Molecular; Molecular biology; molecular recognition; Molecular Sequence Data; Monoclonal antibodies; Mutation; Peptides; Peptides - chemistry; Peptides - genetics; Peptides - immunology; Physical Sciences; Protein Binding - immunology; protein engineering; Protein Engineering - methods; Protein Structure, Tertiary; Proteins; Receptor, ErbB-2 - immunology; Sequence Homology, Amino Acid; Surface Plasmon Resonance; Tissue grafting; Trastuzumab; Wave diffraction; molecular recognition; protein engineering; cancer
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1307309110

Capitalizing on their extraordinary specificity, monoclonal antibodies (mAbs) have become one of the most reengineered classes of biological molecules. A major goal in many of these engineering efforts is to add new functionality to the parental mAb, including the addition of cytotoxins and imaging agents for medical applications. Herein, we present a unique peptide-binding site within the central cavity of the fragment antigen binding framework region of the chimeric, anti-epidermal growth factor receptor mAb cetuximab. We demonstrate through diffraction methods, biophysical studies, and sequence analysis that this peptide, a meditope, has moderate affinity for the Fab, is specific to cetuximab (i.e., does not bind to human IgGs), and has no significant effect on antigen binding. We further demonstrate by diffraction studies and biophysical methods that the meditope binding site can be grafted onto the anti-human epidermal growth factor receptor 2 mAb trastuzumab, and that the antigen binding affinity of the grafted trastuzumab is indistinguishable from the parental mAb. Finally, we demonstrate a bivalent meditope variant binds specifically and stably to antigen-bearing cells only in the presence of the meditope-enabled mAbs. Collectively, this finding and the subsequent characterization and engineering efforts indicate that this unique interface could serve as a noncovalent “linker” for any meditope-enabled mAb with applications in multiple mAb-based technologies including diagnostics, imaging, and therapeutic delivery.