Significant antitumor effect after systemic administration of a urokinase activation-dependent modified anthrax toxin.

3801 Urokinase plasminogen activator (uPA) and its receptor (uPAR) are overexpressed in many cancer types compared to normal tissue. The presence of both uPA and uPAR in cancer tissue may potentially be exploited in cancer therapy for specific and targeted activation of prodrugs. We have constructed a modified form of anthrax protective antigen, PrAg-U2, in which a furin activation site is shifted with an uPA cleavage site. PrAg-U2 administered together with the fusion protein FP59, consisting of lethal factor residues 1–254 and the ADP ribosylation domain of Pseudomonas exotoxin A, constitutes a potent and uPA-activity dependent cytotoxic drug (OTP-001). In vitro binding of pro-uPA to uPAR is necessary for the cytotoxicity of OTP-001 (Liu et al., J. Biol. Chem, 276:17976–84, 2001) and local administration of OTP-001 in mice with transplanted tumors had a potent antitumor effect (Liu et al., PNAS, 100:657–62, 2003). The aim of the present studies was i) To examine the antitumor effect of OTP-001 after systemic administration, ii) To find the optimal ratio between the two components PrAg-U2 and FP59, iii) To define the “therapeutic window” of OTP-001. Five separate in vivo experiments were performed with three different transplanted tumors, Lewis lung carcinoma, T241 fibrosarcoma and B16 melanoma. In all studies, C57BI/6J mice where transplanted with 10 6 tumor cells. When the tumors had reached a volume of approximately 50 mm 3 , a dose range of OTP-001 was administered systemically to the mice at day 0, 3, and 6. Different ratios of PrAg-U2 and FP59 were tested. Tumor sizes were obtained by daily recording of two orthogonal diameters during the growth phase. All experiments unequivocally demonstrated that OTP-001 has significant antitumor effect after systemic administration in mice with transplanted B16, T241 or Lewis lung tumors. These studies showed that OTP-001 displays a clear dose-response relationship with regard to anti-tumor effect and systemic toxicity. The dose-limiting toxicity appears to be due to mucosal reactions in the gut. The anti-tumor effect was most pronounced with a ratio of the two components PrAg-U2 and FP59 of 25:1. Intraperitoneal administration of 30 μg PrAg-U2 and 1.2 μg FP59 day 0, day 3 and day 6 was found to have minimal toxic effects and still had a significant antitumor effect.