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

Journal Article · Mon Oct 07 00:00:00 EDT 2013 · Proceedings of the National Academy of Sciences of the United States of America

DOI:https://doi.org/10.1073/pnas.1307309110· OSTI ID:1135749

Donaldson, Joshua M. ^[1]; Zer, Cindy ^[2]; Avery, Kendra N. ^[2]; Bzymek, Krzysztof P. ^[2]; Horne, David A. ^[2]; Williams, John C. ^[2]

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1. Beckman Research Institute of City of Hope, Duarte, CA (United States); Thomas Jefferson Univ., Philadelphia, PA (United States)
2. Beckman Research Institute of City of Hope, Duarte, CA (United States)

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. Lastly, 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.

• Research Organization: SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States)
• Sponsoring Organization: USDOE Office of Science (SC)
• Grant/Contract Number: AC02-76SF00515
• OSTI ID: 1135749
• Report Number(s): SLAC-REPRINT--2014-168
• Journal Information: Proceedings of the National Academy of Sciences of the United States of America, Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Issue: 43 Vol. 110; ISSN 0027-8424
• Publisher: National Academy of Sciences, Washington, DC (United States)
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
cancer
molecular recognition
protein engineering