CMDA prodrug system as an effective combination for the gene-directed

The gene for the bacterial enzyme carboxypeptidase G2 (CPG2) was expressed internally in mammalian cells. Mammalian-expressed CPG2 had kinetic properties Indistinguishablefrom bacterially expressed CPG2. Human tumor cell lines A2780, SK-OV-3 (ovarian adenocarcinomas), LS174T, and WLDr(colon carcinomas) were engineered to express con stitutively either CPG2 or bacterial 13-galactosidase. These cell lines were subjected to a gene-directed enzyme prodrug therapy regime, using the prodrug 4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl-L-glutamic acid (CMDA). The lines which expressed CPG2 had enhanced sensitivity to CMDA. Comparing IC@s, WIDr-CPG2 and SK-OV-3-CPG2 were 11-16-fold more sensitive, whereas A2780-CPG2 and LS174T-CPG2 were —95-foldmore sensitive than the corresponding control lines. CPG2expressing cells and control cells were mixed in differing proportions and then treated with prodrug. Total kill occurred when only —12%of cells expressed CPG2 with the W1Dr and SK-OV-3 lines and when only 4-5% of cells expressed CPG2 with the LS174T and A2780 lines, indicating a substantial bystander effect. These results establish this CPG2 enzyme! CMDA prodrug system as an effective combination for the gene-directed enzyme prodrug therapy approach.