ANTITUMOR ACTIVITIES OF A CEPHALOSPORIN PRODRUG IN COMBINATION WITH MONOCLONAL ANTIBODY-BETA-LACTAMASE CONJUGATES

7-(Phenylacetamido)cephalosporin mustard (CM) and 7-(4-carboxybutanamido)cepahalosporin mustard (CCM) were developed as anticancer prodrugs that could be activated site selectively by monoclonal antibody-beta-lactamase conjugates targeted to antigens present on tumor cell surfaces. Both CM and CCM were hydrolyzed by purified beta-lactamases from Escherichia coli (ECbetaL), Bacillus cereus (BCbetaL), and Enterobacter cloacae (EClbetaL). This resulted in the release of phenylenediamine mustard (PDM), a potent cytotoxic drug. The K(m) and k(cat) values of the reactions were determined, and it was found that EClbetaL effected the hydrolysis of CM and CCM more rapidly than the other enzymes. Conjugates of EClbetaL were prepared by reacting maleimide-substituted F(ab')2 fragments of the monoclonal antibodies L6 and P1.17 to EClbetaL that had been modified with sulfhydryl groups. In vitro experiments indicated that CCM (IC50 = 25-45 muM) was less toxic than PDM (IC50 = 1.5 muM) to H2981 lung adenocarcinoma cells (L6 antigen positive, P1.17 antigen negative) and that immunologically specific prodrug activation took place when the cells were treated with L6-EClbetaL. In vivo experiments in nude mice demonstrated that CCM was less toxic than CM, and that both prodrugs were much less toxic than PDM. Neither CCM nor PDM exerted antitumor activity on subcutaneous H2981 tumors in vivo. However, a significant antitumor effect was obtained in mice that received L6-EClbetaL 96 h prior to the administration of CCM. The effect was immunologically specific (P < 0.05), since a smaller degree of antitumor activity was obtained in mice that received the nonbinding control conjugate P1.17-EClbetaL prior to CCM. These studies demonstrate the potential therapeutic utility of monoclonal antibody-beta-lactamase conjugates for the activation of cephalosporin-containing prodrugs.