Abstract
Antibody affinity is critically important in therapeutic applications, as well as steady state diagnostic assays. Picomolar affinity antibodies, approaching the association limit of protein-protein interactions, have been discovered for highly potent antigens, but even such high-affinity binders have off-rates sufficient to negate therapeutic efficacy. To cross this affinity threshold, antibodies that tether their targets in a manner other than reversible non-covalent interaction will be required. Here we report the design and construction of an antibody that forms an irreversible complex with a protein antigen in a metal-dependent reaction. The complex resists thermal and chemical denaturation, as well as attempts to remove the coordinating metal ion. Such irreversibly binding antibodies could facilitate the development of next generation "reactive antibody" therapeutics and diagnostics.
Highlights
Library Design and Characterization—Irreversible modification of a target protein by an antibody requires placing reactive elements within the combining site without significantly disrupting antigen binding. Such perturbation was successful in the case of a metal chelate-binding antibody by placing nucleophilic cysteine residues in the antibody light chain at positions that were not expected to contact the small molecule antigen based on its co-crystal structure [12]
Homology modeling of the RA7 antibody and its complex with the tumor necrosis factor (TNF)␣ trimer suggested that the VH N terminus was within 10 Å of the target
Because 44 kDa is the molecular mass of a TNF-scFv fusion, and because these reactions were analyzed in the presence of SDS, reducing agent, and following boiling, we reasoned that the 44-kDa band could be a covalent complex that contained both scFv and TNF␣
Summary
Binding antibodies have been raised against electrophilically modified targets [18] and have been shown to form an effectively covalent complex with their antigen. We tested whether the HEXXH phage could bind a metal affinity column and elute with the histidine side chain competitor imidazole (Fig. 1C).
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