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

Abstract

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.protein engineering | molecular recognition | cancer M onoclonal antibodies (mAbs) are indispensable tools in research laboratories and have become a central component in modern medicine. Thousands of antibodies are routinely used in research to detect and/or label specific proteins in a variety of settings. In recent years, dozens of mAbs that block signaling pathways, sequester growth factors, and/or induce an immune response have been successfully implemented in the clinic to treat cancer and other diseases, with hundreds still in active development (1, 2). Antibodies are being reengineered to best capitalize on their extraordinary ligand specificity to add new functionalities for a broad range of applications, mostly for clinical use, such as antibody-drug conjugates (ADCs), antibodydirected enzyme prodrug therapies (ADEPT), immune system engagement [e.g., Fc modifications (3), chemokine fusion, bispecific T-cell engagers (4)] and disease imaging (e.g., immunoPET and pretargeted radionuclide imaging; refs. 5 and 6).Invariably, these engineering efforts have been achieved either through posttranslational chemical modifications or manipulation of the gene sequence (7-9). Many of the chemical modifications lead to undesirable consequences that are detrimental to therapeutic and imaging applications (10). For example, to create ADCs to target cytotoxins to disease sites with the mAb, the chemical conjugation of the toxin (typically involves lysines, reduced cysteines, or sugars on the mAb) produ...

Mutational landscape of antibody variable domains reveals a switch modulating the interdomain conformational dynamics and antigen binding

“…The ERs of mutations in V H library from anti-gD and protein L panning were closely correlated (r 2 = 0.98). The ERs from the protein A and protein L-panned V H were also correlated (r 2 = 0.611) with the differential enrichment attributed to the subset of V H positions binding to protein A (28). A similar pattern was seen for the V L library panning data.…”

CODV-Ig, a universal bispecific tetravalent and multifunctional immunoglobulin format for medical applications

“…[85][86][87] Superposition of crystal structures of Fc or Fab domains in complex with subdomains of protein A on CODV-Ig models constructed with the type 1 crystal structure of CODV-Fab IL4 x IL13 show that these binding sites are unobstructed in CODV-Ig except for steric interferences observed at the site of Fv1. 81,[88][89][90] Superposition of the Fab/protein A complexes on the initial type 4 model suggests unobstructed protein A binding sites on both Fv1 and Fv2. Thus, choice of linker lengths may influence purification of CODV-Igs, in particular when Fv domains with HC of subgroup III are employed.…”

Activating mutations of STAT5B and STAT3 in lymphomas derived from γδ-T or NK cells

“…His6-SMT fusions of both WT and N642 mutant STAT5B were purified as previously published 44 . Briefly, cells were thawed and lysed by French pressure cell with DNase I and PMSF.…”