MONOCLONAL-ANTIBODY BETA-LACTAMASE CONJUGATES FOR THE ACTIVATION OF A CEPHALOSPORIN MUSTARD PRODRUG

Cephalosporin mustard (CM) was designed as an anticancer prodrug that could be activated in a site-specific manner by monoclonal antibody-beta-lactamase conjugates targeted to antigens present on tumor cell surfaces. Purified beta-lactamases from Bacillus cereus (BC-beta-L) and Escherichia coli (EC-beta-L) catalyzed the release of phenylenediamine mustard (PDM) from CM through a fragmentation reaction which occurs after the beta-lactam ring of CM is hydrolyzed. The K(m) and V(max) values were 5.7-mu-M and 201-mu-mol/min per mg for BC-beta-L and 43-mu-M and 29-mu-mol/min per mg for EC-beta-L, respectively. Conjugates of BC-beta-L were prepared by combining the F(ab')2 fragments of the maleimide-substituted monoclonal antibodies L6 and 1F5 with thiolated BC-beta-L. The conjugates showed little loss in enzymatic activity and bound nearly as well as the unmodified F(ab')2 monoclonal antibodies to antigens expressed on the H2981 human lung adenocarcinoma cell line (L6 positive, 1F5 negative). PDM was approximately 50-fold more cytotoxic than CM to H2981 cells. Treatment of the cells with L6-BC-beta-L followed by CM resulted in a level of cytotoxic activity that was comparable to that of PDM. This was most likely due to activation of CM by conjugate that bound to cell-surface antigens, since pretreatment of H2981 cells with BC-beta-L or 1F5-BC-beta-L enhanced the activity of CM to a lesser extent. Thus, we have shown that CM is a prodrug, and that it can be activated with immunological specificity by a monoclonal antibody-beta-lactamase conjugate.