Antitumor activities of a cephalosporin prodrug in combination with monoclonal antibody-.beta.-lactamase conjugates

Abstract

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)

Efficient Targeting of Conserved Cryptic Epitopes of Infectious Agents by Single Domain Antibodies

“…Cytotoxicity Assays-The LD 50 of the phenylenediamine mustard parental drug (PDM) and the 7-(4-carboxibutamido)-cephalosporin mustard prodrug (CCM) were determined by incubating 10 6 T. brucei brucei AnTat 1.1 parasites (2.5 h, 30°C) in phosphate-saline-glucose buffer with PDM or CCM (0 -150 M) (26).…”

“…Thus, both the binding of the VHH and the enzymatic activity of the ␤-lactamase are well preserved in the fusion protein. The ␤-lactamase can also convert the mustard (CCM) into the highly toxic PDM (26). A 2.5 h incubation of 0 to 150 M CCM to bloodstream form T. brucei AnTat 1.1 parasites in PSG buffer revealed an LD 50 value of 20 M for this prodrug, which is 20ϫ higher than the parental drug PDM (Fig.…”

“…nitrocefin) as well as cell-permeable fluorescent substrates (coumarin cephalosporin fluorescein) allowing a sensitive screening (40). Moreover, the enzyme can convert the CCM prodrug into a potent toxic PDM (26), so that the fusion can be used in an antibody directed antibody prodrug therapy. Fourth, the lactamase activity is absent in eukaryotic host cells, and finally, sensitive complementation tests are under development (41).…”

Enzyme-Catalyzed Activation of Anticancer Prodrugs

“…CM was 50-fold less cytotoxic toward human lung adenocarcinoma cells than phenylenediamine mustard, whereas after activation by a monoclonal antibody-␤-lactamase conjugate, cytotoxicity was comparable (Svensson et al, 1992). The CM analog 7-(4-carboxybutanamido)-cephalosporin mustard (CCM) was also shown to be activated to phenylenediamine mustard by ␤-lactamases with K m values in the micromolar range (Vrudhula et al, 1993b). CCM was less toxic than the parent drug phenylenediamine mustard toward human lung cancer cells, and prodrug activation was observed after coadministration with an enzyme immunoconjugate consisting of ␤-lactamase and the monoclonal antibody L6 that binds to antigens on lung adenocarcinoma cells.…”

“…CCM was less toxic than the parent drug phenylenediamine mustard toward human lung cancer cells, and prodrug activation was observed after coadministration with an enzyme immunoconjugate consisting of ␤-lactamase and the monoclonal antibody L6 that binds to antigens on lung adenocarcinoma cells. In mice bearing lung adenocarcinoma cells, phenylenediamine mustard was ineffective, whereas a significant antitumor activity was observed using CCM and an enzyme immunoconjugate containing ␤-lactamase (Vrudhula et al, 1993b). In a subsequent study, it was shown that CCM was even more effective than C-DOX in mice bearing human melanoma tumor cells (Kerr et al, 1995), and CCM was more recently shown to be effective in renal cell carcinoma and human lung carcinomas using ADEPT approaches (Svensson et al, 1998;.…”