Novel partial reduction of the humanized anti-cocaine mAb h2E2 for selective cysteine labeling

Abstract

Monoclonal antibodies are utilized for treating many diseases and disorders, as well as for basic research and development. Covalent labeling of mAbs is important for various antibody applications and creating antibody drug conjugates. Labeling at reactive lysine residues using lysine selective reagents is useful, but is non-selective and can interfere with antigen binding and interactions of the Fc antibody region. In this work, using an anti-cocaine mAb (h2E2), we utilized triphenylphosphine-3,3′,3″-trisulfonic acid (TPPTS), and demonstrated for the first time reduction of disulfides in an antibody by TPPTS. More importantly, this reduction was very reproducible, limited, and selective, and permitted selective labeling of the antibody with a cysteine reactive fluorescent reagent, resulting in labeling of a few specific cysteines. Similar results were obtained using TCEP-agarose reduction. We demonstrated that both of these selective partial reduction methods gave rise to approximately two labels per mAb, mostly by selective reduction of the heavy chain to light chain disulfide bond, as demonstrated by non-reducing SDS-PAGE protein band analysis. Thus, convenient, reproducible, and selective mAb disulfide reduction was achieved under mild conditions. These labeled, partially reduced mAbs were characterized by differential scanning fluorimetry (DSF), detecting the incorporated fluorescein instead of an exogenously added dye, and for antigen (cocaine) binding by isothermal titration calorimetry (ITC). Both the structure and antigen binding of the mAb was maintained. This novel selective reduction and labeling is generally relevant to modification of antibodies and to future development of conjugated mAbs for experimental and therapeutic purposes.