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

7-(Phenylacetamido)cephalosporin mustard (CM) and 7-(4-carboxybutanami do)cepahalosporin mustard (CCM) were developed as anticancer prodrugs that could be activated site selectively by monoclonal antibody-beta-l actamase conjugates targeted to antigens present on tumor cell surface s. Both CM and CCM were hydrolyzed by purified beta-lactamases from Es cherichia coli (ECbetaL), Bacillus cereus (BCbetaL), and Enterobacter cloacae (EClbetaL). This resulted in the release of phenylenediamine m ustard (PDM), a potent cytotoxic drug. The K(m) and k(cat) values of t he reactions were determined, and it was found that EClbetaL effected the hydrolysis of CM and CCM more rapidly than the other enzymes. Conj ugates of EClbetaL were prepared by reacting maleimide-substituted F(a b')2 fragments of the monoclonal antibodies L6 and P1.17 to EClbetaL t hat had been modified with sulfhydryl groups. In vitro experiments ind icated 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 a ntigen negative) and that immunologically specific prodrug activation took place when the cells were treated with L6-EClbetaL. In vivo exper iments 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. Howev er, a significant antitumor effect was obtained in mice that received L6-EClbetaL 96 h prior to the administration of CCM. The effect was im munologically specific (P < 0.05), since a smaller degree of antitumor activity was obtained in mice that received the nonbinding control co njugate P1.17-EClbetaL prior to CCM. These studies demonstrate the pot ential therapeutic utility of monoclonal antibody-beta-lactamase conju gates for the activation of cephalosporin-containing prodrugs.