Identification and grafting of a unique peptide-binding site in the Fab framework of monoclonal antibodies Joshua M. Donaldsona,b,1, Cindy Zera,1, Kendra N. Averya, Krzysztof P. Bzymeka, David A. Hornea, and John C. Williamsa,2 aDepartment of Molecular Medicine, Beckman Research Institute of City of Hope, Duarte, CA 91010; and bDepartment of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, PA 19107 Edited* by Douglas C. Rees, Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA, and approved September 19, 2013 (received for review April 18, 2013) Capitalizing on their extraordinary specificity, monoclonal anti- enhance imaging reduces the valency and, thus, affects both the bodies (mAbs) have become one of the most reengineered classes affinity and the tissue specificity compared with the original of biological molecules. A major goal in many of these engineering mAb. In other cases, as part of a pretargeted imaging protocol to efforts is to add new functionality to the parental mAb, including enhance detection, mAbs conjugated with streptavidin or multi- the addition of cytotoxins and imaging agents for medical applica- ple Fabs/scFvs stitched together through a scaffold (e.g., “lock- tions. Herein, we present a unique peptide-binding site within the and-dock”; ref. 12) can be immunogenic (13), unstable in serum central cavity of the fragment antigen binding framework region of (14), and technically difficult and expensive to produce, especially the chimeric, anti-epidermal growth factor receptor mAb cetuximab. at scales necessary for clinical use (15, 16). Although significant We demonstrate through diffraction methods, biophysical studies, advances in antibody engineering have been achieved, practical and sequence analysis that this peptide, a meditope, has moderate challenges remain, many requiring alternative approaches. affinity for the Fab, is specific to cetuximab (i.e., does not bind to Herein, we present a unique interaction that has the potential fi human IgGs), and has no significant effect on antigen binding. We to overcome some of the dif culties in mAb engineering. We fi further demonstrate by diffraction studies and biophysical methods identi ed a binding site for a small peptide in the center of the that the meditope binding site can be grafted onto the anti-human Fab cavity of cetuximab via diffraction studies. Because of the position of the binding interface, we have named the peptide epidermal growth factor receptor 2 mAb trastuzumab, and that the “ ” medius topos antigen binding affinity of the grafted trastuzumab is indistinguish- meditope ( , middle; , place). We demonstrated that the meditope does not interfere with antigen binding. Based on able from the parental mAb. Finally, we demonstrate a bivalent fi meditope variant binds specifically and stably to antigen-bearing structural and biophysical analysis, we identi ed key residues cells only in the presence of the meditope-enabled mAbs. Collec- within the meditope binding site of the Fab and observed that the fi combination of these residues is absent in other clinically relevant tively, this nding and the subsequent characterization and engi- fi neering efforts indicate that this unique interface could serve as mAbs, including a humanized mAb framework. To con rm the structural model and determine whether we could enable medi- a noncovalent “linker” for any meditope-enabled mAb with applica- tope binding in other mAbs, we grafted selected residues onto tions in multiple mAb-based technologies including diagnostics, im- trastuzumab, a humanized anti-HER2 mAb, and demonstrated aging, and therapeutic delivery. by diffraction and biophysical studies that the meditope binds to this meditope-enabled trastuzumab in a similar manner and af- protein engineering | molecular recognition | cancer finity compared with cetuximab. Finally, we created a bivalent analog of the meditope to demonstrate that this interaction can onoclonal antibodies (mAbs) are indispensable tools in be used to specifically target antigen-bearing cells in the presence Mresearch laboratories and have become a central compo- of a meditope-enabled mAb that binds to the specific antigen. nent in modern medicine. Thousands of antibodies are routinely Overall, these results establish the basis for an alternative mAb used in research to detect and/or label specific proteins in a va- platform that offers unique opportunities to improve mAb-based riety of settings. In recent years, dozens of mAbs that block delivery systems for cancer and other diseases. signaling pathways, sequester growth factors, and/or induce an immune response have been successfully implemented in the Significance clinic to treat cancer and other diseases, with hundreds still in active development (1, 2). Antibodies are being reengineered to fi fi The identi cation and subsequent grafting of a unique peptide best capitalize on their extraordinary ligand speci city to add binding site within the Fab domain offers a unique means of new functionalities for a broad range of applications, mostly for adding functionality to monoclonal antibodies through a non- clinical use, such as antibody-drug conjugates (ADCs), antibody- covalent interaction including improved pretargeted imaging, directed enzyme prodrug therapies (ADEPT), immune system alternative payload delivery, and cross-linking of mAbs on cell fi engagement [e.g., Fc modi cations (3), chemokine fusion, bispe- surfaces to enhance their therapeutic potential. cific T-cell engagers (4)] and disease imaging (e.g., immunoPET and pretargeted radionuclide imaging; refs. 5 and 6). Author contributions: J.M.D., C.Z., K.N.A., and J.C.W. designed research; J.M.D., C.Z., K.N.A., Invariably, these engineering efforts have been achieved either K.P.B., and J.C.W. performed research; J.M.D., C.Z., D.A.H., and J.C.W. contributed new through posttranslational chemical modifications or manipula- reagents/analytic tools; J.M.D., C.Z., K.N.A., K.P.B., and J.C.W. analyzed data; and J.M.D., tion of the gene sequence (7–9). Many of the chemical mod- C.Z., K.N.A., K.P.B., and J.C.W. wrote the paper. ifications lead to undesirable consequences that are detrimental Conflict of interest statement: D.A.H. and J.C.W. have founded Meditope Biosciences, to therapeutic and imaging applications (10). For example, to a company based on some of the observations presented here. create ADCs to target cytotoxins to disease sites with the mAb, *This Direct Submission article had a prearranged editor. the chemical conjugation of the toxin (typically involves lysines, Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, reduced cysteines, or sugars on the mAb) produces a heteroge- www.pdb.org (PDB ID code 4GW1, 4GW5, 4HKZ, 4HJG, and 4IOI). neous mixture, which can adversely affect the specificity and 1J.M.D. and C.Z. contributed equally to this work. stability of the mAb and alter its biodistribution (11). Reducing 2To whom correspondence should be addressed. E-mail: [email protected]. the size of the mAb to Fab fragments [e.g., single-chain Fab This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. variable domain (scFvs)] to facilitate tumor penetration and 1073/pnas.1307309110/-/DCSupplemental. 17456–17461 | PNAS | October 22, 2013 | vol. 110 | no. 43 www.pnas.org/cgi/doi/10.1073/pnas.1307309110 Downloaded by guest on September 27, 2021 meditope-Fab interface, ∼850 Å, is similar to the that of protein A, G, and L. (23) (SI Appendix, Table S2). As an additional confirmation of the structural model, we identified key residues on the meditopes that enabled the in- teraction with the Fab based on the superposition of the cQFD-Fab and cQYN-Fab structures. We observed that the hydrophobic side chains of Phe/Tyr3, Leu5, and Leu10 of both meditopes were positioned nearly identically, suggesting that they make important contacts to the Fab (SI Appendix,Fig.S1). The importance of these residues is confirmed by SPR experiments showing alanine mutations at Phe3, Leu5, or Leu10 of the cQFD meditope resulted in a 5- to 140-fold decrease in affinity (SI Ap- pendix,TableS3). We also observed that the guanidinium group of Arg8 of cQFD forms a hydrogen bond to the backbone carbonyl of Gln111 of the Fab heavy chain. Accordingly, Arg8Ala substitution decreased the affinity by 325-fold (SI Appendix, Table S3). The extended conformation of Arg8 is sterically blocked by the hy- droxyl group of Tyr3 of the cQYN meditope, which causes a backbone rotation and eliminates the Arg8–Gln111 interaction (SI Appendix, Fig. S1). The loss of this interaction partially Fig. 1. Meditope binds to cetuximab Fab framework. (A) Ribbon repre- accounts for the nearly 10-fold difference in the binding affinity sentation of cetuximab Fab (light chain, blue-white; heavy chain, white) with between the two meditopes (Fig. 2A and SI Appendix,TableS3) a stick representation of the cyclic CQFDLSTRRLKC meditope (green). (B) Thus, although the point mutations agree with the structural Electron density maps of cQFD meditope, in stereo and contoured at 1σ.(C) Superposition of the cetuximab-EGFR (1YY9) structure to Fab framework ligands: protein L (1HEZ), protein A (1DEE), protein G (1QKZ), and cQFD meditope (4GW1). Results Identification of the Meditope Binding Site on Cetuximab Fab Framework. In our efforts to create tumor activated antibodies (17), we cocrystallized two peptides, CQFDLSTRRLKC (cQFD) and CQYNLSSRALKC (cQYN), individually with the Fab do- main of cetuximab. These peptides were originally identified by phage display (18) and thought to mimic the tumor epitope EGFR domain III. The initial diffraction data indicated that the unit cell dimensions of each cocrystal were the same as that of the apo cetuximab Fab [Protein Data Bank (PDB) ID code 1YY8; ref. 19]. Because the complementarity determining region (CDR) loops in the published structure make extensive lattice contacts, the data suggested that the peptides were either absent or bound to a different region of the Fab.