Vrudhula V M, Svensson H P, Kennedy K A, Senter P D, Wallace P M
Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, Washington 98121.
Bioconjug Chem. 1993 Sep-Oct;4(5):334-40. doi: 10.1021/bc00023a005.
7-(Phenylacetamido)cephalosporin mustard (CM) and 7-(4-carboxybutanamido)cephalosporin 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 (EC beta L), Bacillus cereus (BC beta L), and Enterobacter cloacae (ECl beta L). This resulted in the release of phenylenediamine mustard (PDM), a potent cytotoxic drug. The Km and kcat values of the reactions were determined, and it was found that ECl beta L effected the hydrolysis of CM and CCM more rapidly than the other enzymes. Conjugates of ECl beta L were prepared by reacting maleimide-substituted F(ab')2 fragments of the monoclonal antibodies L6 and P1.17 to ECl beta L that had been modified with sulfhydryl groups. In vitro experiments indicated that CCM (IC50 = 25-45 microM) was less toxic than PDM (IC50 = 1.5 microM) 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-ECl beta L. 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-ECl beta L 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-ECl beta L prior to CCM.(ABSTRACT TRUNCATED AT 250 WORDS)
7-（苯乙酰胺基）头孢菌素氮芥（CM）和7-（4-羧基丁酰胺基）头孢菌素氮芥（CCM）被开发为抗癌前药，它们可被靶向肿瘤细胞表面抗原的单克隆抗体-β-内酰胺酶缀合物位点选择性激活。CM和CCM均被来自大肠杆菌（EC βL）、蜡样芽孢杆菌（BC βL）和阴沟肠杆菌（ECl βL）的纯化β-内酰胺酶水解。这导致强效细胞毒性药物苯二胺氮芥（PDM）的释放。测定了反应的Km和kcat值，发现ECl βL比其他酶更快速地催化CM和CCM的水解。通过使单克隆抗体L6和P1.17的马来酰亚胺取代的F(ab')2片段与用巯基修饰的ECl βL反应，制备了ECl βL缀合物。体外实验表明，CCM（IC50 = 25 - 45 μM）对H2981肺腺癌细胞（L6抗原阳性，P1.17抗原阴性）的毒性低于PDM（IC50 = 1.5 μM），并且当用L6-ECl βL处理细胞时发生了免疫特异性前药激活。裸鼠体内实验表明，CCM的毒性低于CM，并且两种前药的毒性均远低于PDM。CCM和PDM在体内均未对皮下H2981肿瘤发挥抗肿瘤活性。然而，在给予CCM前96小时接受L6-ECl βL的小鼠中获得了显著的抗肿瘤效果。该效果具有免疫特异性（P < 0.05），因为在给予CCM前接受非结合对照缀合物P1.17-ECl βL的小鼠中获得的抗肿瘤活性程度较小。（摘要截短于250字）
