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Engineering a high-affinity peptide binding site into the anti-CEA mAb M5A
Journal article

Engineering a high-affinity peptide binding site into the anti-CEA mAb M5A

Cindy Zer, Kendra N. Avery, Kassondra Meyer, Leah Goodstein, Krzysztof P. Bzymek, Gagandeep Singh and John C. Williams
Protein Engineering, Design and Selection, Vol.30(6)
2017
DOI: https://doi.org/10.1093/protein/gzx016

Abstract

Antibodies, Monoclonal Binding Sites Carcinoembryonic Antigen Cell Line Humans Models, Molecular Peptides Protein Binding Protein Engineering Amino acids Antigen-antibody reactions Binding energy Bins Grafting (chemical) Monoclonal antibodies Peptides amino acid asparagine carcinoembryonic antigen cetuximab glycine immunoglobulin F(ab) fragment isoleucine monoclonal antibody monoclonal antibody M5A phenylalanine proline threonine trastuzumab unclassified drug carcinoembryonic antigen monoclonal antibody peptide protein binding Amino acid substitution Antibody engineering Antibody-drug conjugates Antigen recognition Binding affinities High-affinity peptides meditope Noncovalent attachment amino acid substitution antigen antibody complex Article association constant binding affinity crystal structure dissociation constant disulfide bond drug binding site drug protein binding hydrogen bond hydrophobicity priority journal protein engineering binding site cell line chemistry genetics human immunology metabolism molecular model procedures protein engineering Antibodies
We have previously identified a cyclic peptide called meditope which binds to the central cavity of the Fab portion of cetuximab and shown that this peptide binding site can be grafted, or 'meditope- enabled', onto trastuzumab. This peptide has been shown to act as a hitch for the noncovalent attachment of imaging agents to meditope-enabled antibodies. Herein, we explore the process of grafting this peptide binding site onto M5A, an anti-CEA antibody in clinical trials for cancer diagnostics. In order to explore the contributions of the amino acids, we sequentially introduced pairs of amino acid substitutions into the Fab and then we reverse-substituted key residues in the presence of the other substitutions. We demonstrate that Pro40Thr, Gly41Asn, Phe83Ile and Thr85Asp in the light chain are sufficient to recreate the meditope binding site in M5A with singledigit micromolar affinity. We show that Pro40 abrogates peptide binding in the presence of the other 12 residue substitutions, and that the presence of all 13 substitutions does not interfere with antibody:antigen recognition. Collectively, these studies provide detailed insight for defining and fine-tuning the binding affinity of the meditope binding site within an antibody. © The Author 2017.

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