Gene-directed enzyme prodrug therapy with a mustard prodrug/carboxypeptidase G2 combination.
The gene for the bacterial enzyme carboxypeptidase G2 (CPG2) was expressed internally in mammalian cells. Mammalian-expressed CPG2 had kinetic properties indistinguishable from bacterially expressed CPG2. Human tumor cell lines A2780, SK-OV-3 (ovarian adenocarcinomas), LS174T, and WiDr (colon carcinomas) were engineered to express constitutively either CPG2 or bacterial beta-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 IC50S, WiDr-CPG2 and SK-OV-3-CPG2 were 11-16-fold more sensitive, whereas A2780-CPG2 and LS174T-CPG2 were approximately 95-fold more sensitive than the corresponding control lines. CPG2-expressing cells and control cells were mixed in differing proportions and then treated with prodrug. Total kill occurred when only approximately 12% of cells expressed CPG2 with the WiDr 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.
Marais
R
R
CRC Centre for Cancer Therapeutics at the Institute of Cancer Research, Sutton, Surrey, United Kingdom.
Spooner
R A
RA
Light
Y
Y
Martin
J
J
Springer
C J
CJ
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000627:therapeutic use*; D019556:COS Cells / Q000201:enzymology; D003110:Colonic Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000628:therapy*; D004354:Drug Screening Assays, Antitumor; D005260:Female; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D005839:Gentamicins / Q000494:pharmacology; D005971:Glutamates / Q000627:therapeutic use*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009588:Nitrogen Mustard Compounds / Q000627:therapeutic use*; D010051:Ovarian Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000628:therapy*; D011355:Prodrugs / Q000627:therapeutic use*; D014162:Transfection; D014407:Tumor Cells, Cultured; D011623:gamma-Glutamyl Hydrolase / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*
D000900:Anti-Bacterial Agents; D000970:Antineoplastic Agents; D005839:Gentamicins; D005971:Glutamates; D009588:Nitrogen Mustard Compounds; D011355:Prodrugs; C092574:4-((2-chloroethyl)(2-mesyloxyethyl)amino)benzoylglutamic acid; C010680:antibiotic G 418; D011623:gamma-Glutamyl Hydrolase