In suicide gene therapy, the site of subcellular localization of the activating enzyme is more important than the rate at which it activates prodrug

Abstract

The bacterial enzyme carboxypeptidase G2 (CPG2) can be expressed both intracellularly (CPG2*) or tethered to the outer surface (stCPG2(Q)3) of mammalian cells, where it is able to activate mustard prodrugs for use in suicide gene therapy protocols. Here we compare the properties of CPG2 expressed in these two locations. CPG2 is active as a dimer, and one of the mutations required to block glycosylation of stCPG2(Q)3 destabilizes the dimers. Some of the mutations to this site partially correct the dimerization defect and recover a proportion of the activity. Surface tethering also recovers some enzyme activity, but through an unknown mechanism. The efficacy of CPG2 in these two locations is compared with the tumor cell lines A2780, SK-OV-3, and WiDr, which are sensitized to the prodrug 4-([2-chloroethyl][2-mesyloxyethyl]amino)benzoyl-L-glutamic acid (CMDA) by both CPG2* and stCPG2(Q)3 expression in suicide gene therapy protocols in vitro. We find that stCPG2(Q)3 is a more efficient mediator of CMDA-dependent cell killing than CPG2*. Lower levels of stCPG2(Q)3 activity are required to give cell killing that can only be achieved by higher levels of CPG2*. In bystander effect assays, low levels of stCPG2(Q)3 are required for efficient killing, whereas relatively high levels of CPG2* activity are required. Also, shorter exposures to prodrug are required for cell killing when stCPG2(Q)3 is expressed compared with when CPG2* is expressed. These data demonstrate that the location of the enzyme in the cell is more important than the enzyme activity as the determinant in mediating cytotoxicity.