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Gene-directed enzyme prodrug therapy with a mustard prodrug/carboxypeptidase G2 combination
R Marais1, R A Spooner, Y Light
1CRC Centre for Cancer Therapeutics at the Institute of Cancer Research, Sutton, Surrey, United Kingdom.
Abstract:
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.
Insights
Gene-directed enzyme prodrug therapy using carboxypeptidase G2 (CPG2) and CMDA demonstrated significant cancer cell killing. This approach shows a potent bystander effect, enhancing therapeutic efficacy in preclinical models.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Carboxypeptidase G2 (CPG2) is a bacterial enzyme with potential therapeutic applications.
- Gene-directed enzyme prodrug therapy (GDEPT) utilizes engineered cells to activate cytotoxic drugs.
Purpose of the Study:
- To evaluate the efficacy of expressing bacterial CPG2 in mammalian cells for GDEPT.
- To assess the sensitivity of engineered human tumor cell lines to the prodrug CMDA.
- To investigate the bystander effect of the CPG2-CMDA system.
Main Methods:
- Mammalian cells were engineered to express bacterial CPG2.
- Engineered and control cell lines were treated with the prodrug CMDA.
- Sensitivity was measured by IC50 values.
- Bystander effect was assessed by mixing CPG2-expressing and control cells.
Main Results:
- Mammalian-expressed CPG2 exhibited similar kinetic properties to its bacterial counterpart.
- CPG2-expressing tumor cell lines showed significantly enhanced sensitivity to CMDA (11-95-fold increase in sensitivity).
- A substantial bystander effect was observed, with low percentages of CPG2-expressing cells leading to total tumor cell kill.
Conclusions:
- The CPG2 enzyme and CMDA prodrug combination is effective for GDEPT.
- The observed bystander effect enhances the therapeutic potential of this GDEPT system.
- This strategy holds promise for targeted cancer therapy.