Three new
prodrugs, [
prodrug 1: 4-[bis(2-iodoethyl)amino]-phenyloxycarbonyl-
L-glutamic acid;
prodrug 2: 3-fluoro-4-[bis(2-chlorethyl)amino]benzoyl-
L-glutamic acid; and
prodrug 3: 3,5-difluoro-4-[bis(2-iodoethyl)amino]benzoyl-
L-glutamic acid] have been assessed for use with a mutant of
carboxypeptidase G2 (CPG2,
glutamate carboxypeptidase, EC 3.4.17.11,) engineered to be tethered to the outer
tumor cell surface (stCPG2(Q)3) as the activating
enzyme in suicide gene therapy systems. All three of the
prodrugs produce much greater cytotoxicity differentials between stCPG2(Q)3- and control
beta-galactosidase (beta-gal)-expressing
breast carcinoma MDA MB 361 and colon
carcinoma WiDr cells (70- to 450-fold) than was previously observed (19- to 27-fold) with 4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl-
L-glutamic acid (
CMDA).
Prodrug 1 is the most effective
antitumor agent in xenografts in mice inoculated with 100% stCPG2(Q)3-expressing MDA MB 361 cells, whereas
prodrugs 2 and 3 are most effective when the percentage of stCPG2(Q)3-expressing cells is 50% or 10%. In nude mice bearing xenografts arising from inocula of 100% stCPG2(Q)3-expressing WiDr cells,
prodrug 2 is the most effective
antitumor agent. All three of the
prodrugs produced histological evidence of substantial bystander cell killing in WiDr xenografts in which only 10% or 50% of the cells inoculated were expressing stCPG2(Q)3. We conclude that all three of the
prodrugs are more effective therapeutically with stCPG2(Q)3 than is the previously described
prodrug CMDA and, also, that the optimal choice of
prodrug varies among different
tumor types and that
prodrugs, optimized for their bystander effect, are effective when only low percentages of cells in a
tumor express CPG2.