Site-specific modification of a fragment of a chimeric monoclonal antibody using reverse proteolysis

Abstract

We propose a novel method for the site-specific labeling of antibodies under mild conditions and give as an example the modification of an F(ab')2-like fragment of the chimeric monoclonal antibody B72.3. The F(ab')2-like fragment was produced by the action of the protease lysyl endopeptidase. Reverse proteolysis, catalyzed by the same enzyme, was then used to attach carbohydrazide specifically to the carboxyl termini of the heavy chains of the fragment. Finally, a radiolabeled chelator possessing an aldehyde group was conjugated to the modified fragment through a hydrazone linkage. The resulting site-specifically labeled F(ab')2-like fragment was characterized by gel electrophoresis and by enzymic digestion. It was found to possess immunoreactivity equivalent to that of the unmodified F(ab')2-like fragment as determined by immunofluorescence and ELISA (enzyme-linked immunosorbent assay) techniques. The advantages and disadvantages of this labeling method, which appear to be of quite general applicability, are discussed.