We and others have reported that multiple autoantibodies are unmasked in human polyclonal antibody preparations after exposure to physiological oxidizing agents (hemin) or electromotive force. We now have asked if oxidation unmasks autoantibody reactivities in monoclonal antibodies (mAb). To do this, we have studied 9 FDA approved mAb used therapeutically, including 4 chimeric, 4 humanized and 1 chemically modified chimeric Fab that were exposed to the physiological oxidizing agent hemin at 36°C for 20 hr. These mAb were studied for autoantibody activity to phospholipids and DNA before and after oxidation with hemin and found to develop autoantibody activities after oxidation, while retaining their original specificity as measured by mAb anti-glycophorin A binding of erythrocytes, CD 19 binding to B lymphocytes and anti-HLA-A29 binding to A29-positive lymphocytes. The finding that certain mAb have the potential to unmask autoantibody activities as a consequence of exposure to physiological redox reactions in vitro gives pause to our present understanding of the immunological basis of tolerance and concern for potential autoimmune side effects in patients receiving mAb for diagnosis or treatment.Genetic engineering is a growth strategy in the design and development of monoclonal antibodies (mAb) for clinical therapeutics, such as drug targeting in cancer chemotherapy.1,2 However, the clinical use of mAb has been associated with serious side effects, 3 presumably due to collateral damage from reactions with antigens of unintended targets. 4 Inasmuch as a tenant of the scientific basis for the clinical use of mAb is that they react only with one or a small number of closely related epitopes, 5 we asked if their specificities might be altered by exposure to physiological concentrations of physiological oxidants known to participate in physiological redox reactions. We posed this query due to our earlier reports of the unmasking of autoantibody activities in human blood as well as in purified IgG that had been exposed to such physiological redox reactions. 6 One might then ask why such redox-reactive antibodies exist. We have proposed that redox-reactive antibodies represent elements of the body's immune defense and surveillance network. 7 Recently, a similar explanation has been put forward by others. 8 In the following paragraphs, we present data indicating that autoimmune activities can be unmasked in solutions of FDA approved mAb of several different specificities and immunochemistries.
Material and MethodsThe experimental design and techniques used in this study are the same as those used in our earlier report of the unmasking of autoantibody activity in human IgG following oxidation with physiological oxidants such as hemin 6 or by using electromotive force (EMF).9 It was shown that hemin was responsible for oxidation of the antibody because (i) none of the antibody changes occurred in the absence of hemin, (ii) none of the antibody changes occurred in the presence of hemopexin, which is a biological inhibitor ...