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Three New Prodrugs for Suicide Gene Therapy Using Carboxypeptidase G2 Elicit Bystander Efficacy in Two Xenograft Models

Cancer Research Campaign Centre for Cancer Therapeutics, Institute of Cancer Research, Sutton, Surrey, SM2 5NG United Kingdom [F. F., L. D., I. S., L. M. O., J. M., S. M. S., R. A. S., I. N-D., C. J. S.], and Cancer Research Campaign Centre for Cell and Molecular Biology, Institute of Cancer Research, London, SW3 6JB United Kingdom [R. A. S., R. M.]

	ABSTRACT

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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 ß-galactosidase (ß-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.

	INTRODUCTION

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Prodrugs have often been proposed as a solution to the major problem of nonspecific toxicity to normal tissues, such as the bone marrow and lining of the gut, often associated with conventional cytotoxic chemotherapy (1) . Theoretically, prodrugs are relatively noncytotoxic molecules, capable of being converted to cytotoxic species only at the site of the tumor, affording enhanced antitumor selectivity. Several approaches to endogenous mechanisms of activation have been considered, including tumor-associated hypoxia (2) , low pH (3) , and enhanced enzyme levels (4) . More recently, the use of exogenous activating enzymes by enzyme-antibody conjugates (5) , fusion proteins (6) , or catalytic antibodies (7) , known as antibody-directed enzyme prodrug therapy (ADEPT) has been attempted and has proceeded to clinical testing (8, 9, 10) . An alternative approach is the genetic manipulation of the tumor cells to express the activating enzyme. This has been variously called virally directed enzyme prodrug therapy (VDEPT), GDEPT,² gene prodrug activation therapy (GPAT), or suicide gene therapy (11, 12, 13) .

Several enzyme/prodrug systems have been designed for GDEPT but by far the most extensively studied are bacterial or yeast cytosine deaminase with 5-fluorocytosine, and herpes simplex virus thymidine kinase with ganciclovir, which have progressed to clinical trial (14 , 15) . Both the enzymes in these systems activate their respective prodrugs to intermediates that require subsequent metabolic processing to yield the drug. Also, the ultimate cytotoxins, being antimetabolites, are mainly cell phase specific, requiring active DNA synthesis before they can exert their cytotoxic effect. This is a disadvantage in tumors containing quiescent cells. We have used the Pseudomonas RS16 enzyme CPG2 (glutamate carboxypeptidase, EC 3.4.17.11), which hydrolyzes aromatic N-substituted glutamates to release benzoic acid, phenol, and aniline mustards (16 , 17) or, in the case of self-immolative prodrugs, can release many conventional drugs, e.g., anthracycline antibiotics (18) . The benefits of the CPG2 system with nitrogen mustard prodrugs are that the cytotoxin is not cell phase specific and that it is a one-step activation, thus avoiding the potential problem of rate limitation in the subsequent metabolism of the product.

A number of improvements are required before GDEPT can become a successful therapy. Many gene-delivery systems are currently in development, including conditionally replicating (19) and nonreplicating viruses (20) , nonviral delivery, (21) and bacterial vectors (22) . However, these systems are unlikely to be able to transduce more than a portion of the cells of the tumor, variously reported to be between 15–50% (23, 24, 25) , requiring the action of a bystander effect. This is the ability of those cells expressing the therapeutic gene to convert an excess of prodrug to active drug that can migrate to, and kill, neighboring non-enzyme-expressing cells, thereby mitigating the limitations of the gene delivery system. Another area of improvement of GDEPT is focused on the optimization of the bystander effect. This can be addressed by engineering the enzymes or by designing better prodrugs. We have shown that engineering CPG2 to be displayed at the outer cell surface conferred on the cells an improved sensitivity to the prodrug CMDA compared with cytoplasmic expression of CPG2 and resulted in a greater bystander effect (26) . Accordingly, in the present study, we have chosen to use only stCPG2(Q)3. CPG2 is particularly amenable to prodrug optimization, having a broad substrate specificity that can be further expanded by the self-immolative strategy, and we have previously exemplified a number of prodrug alternatives (13 , 17 , 18 , 27) . Here, we describe the cytotoxicity properties of three prodrugs 1, 2, and 3 (Fig. 1) for CPG2. One novel prodrug (prodrug 3) is reported here for the first time, and the other two have not been previously assessed in GDEPT. We report on their in vivo antitumor efficacy in the bystander xenograft model with stCPG2(Q)3-transfected MDA MB 361 breast carcinoma cells, described previously in (26) , and one new stCPG2(Q)3-engineered WiDr xenograft of colorectal carcinoma.

View larger version (9K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Structures of prodrugs 1, 2, and 3 and the active drugs that are generated by the action of CPG2.

	MATERIALS AND METHODS

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3,5-Difluoro-4-[bis(2-hydroxyethyl)amino]benzonitrile.
A solution of 3,4,5-trifluorobenzonitrile (5 g, 32 mmol) and diethanolamine (8 ml, 85 mmol) in N,N-dimethylacetamide was stirred for 10 days. The solvent was then evaporated, the residue partitioned between CH₂Cl₂ (200 ml) and H₂O (200 ml), and the organic layer dried (MgSO₄) and evaporated to dryness. The residue was chromatographed using CH₂Cl₂-EtOAc as eluant to give, after recrystallization from toluene, 2.7 g (35%) of pure white crystals: mp 63–65°C; ¹H NMR 3.33 (t, 4H, NCH₂, J = 6 Hz), 3.48 (t, 4H, OCH₂, J = 6 Hz), 4.43 (bs, 2H, OH), 7.57 (dd, 2H, H2+6, J_{H2,F3, and H6,F5} = 8.5 Hz, J_{H2,F5 and H6,F3 = 2Hz} = 2 Hz); ¹⁹F NMR -116.0 (d, 2F, F3+5, J_{F3, H2 and F5, 6} = 7.5 Hz); MS m/z 265 (M+Na⁺, 10), 243 (M+H⁺, 100), 211 (M-CH₂OH, 85). Anal. (C₁₁H₁₂N₂O₂F₂) C,H,N.

3,5-Difluoro-4-[bis(2-hydroxyethyl)amino]benzoic Acid.
A solution of the nitrile (3.15 g, 13 mmol) and NaOH (5.2 g, 0.13 mol) in aqueous ethanol (65 ml, 50%) was refluxed for 2.5 h. The solution was then partitioned between EtOAc (800 ml) and HCl (330 ml, 0.4 M), the aqueous layer was washed with EtOAc (3 x 150 ml), and the combined organic layers were dried (MgSO₄) and evaporated to dryness. The solid recrystallized from EtOAc (100 ml) to give 2.94 g (87%) of pure white crystals: mp 146.5–150°C; ¹H NMR 3.3 (t, 4H, CH₂N), 3.46 (q, 4H, CH₂O, J = 5.5 Hz), 4.49 (t, 2H, OH, J = 5 Hz), 7.44 (d, 2H, H2+6, J = 10.5 Hz), 13.1 (bs, 1H, CO₂H); ¹⁹F NMR -117.3 (d, 2F, F3+5, J = 9.5 Hz); MS m/z 284 (M+Na⁺, 15), 262 (M+H⁺, 100), 230 (M-CH₂OH, 40). Anal. (C₁₁H₁₃NO₄F₂) C,H,N.

Di-tert-butyl [3,5-difluoro-4-[bis(2-iodoethyl)amino]benzoyl]-L-glutamate.
(a) To a solution of di-tert-butyl-L-glutamate hydrochloride (2.66 g, 9.0 mmol) in dry dimethylformamide (135 ml) was added Et₃N (2.5 ml, 18 mmol), the acid (2.35 g, 9.0 mmol) followed by diethylcyanophosphonate (1.5 ml, 9.9 ml). After stirring for 3 days, the solvent was evaporated and the residue partitioned between EtOAc (450 ml) and H₂O (375 ml). The organic layer was washed with citric acid (180 ml, 10%), saturated sodium bicarbonate solution (180 ml), and was dried (MgSO₄) and evaporated to dryness, giving 5.6 g of the bis-hydroxy compound.

b) To a solution of the bis-hydroxy compound in dry CH₂Cl₂ (165 ml) was added 4-dimethylaminopyridine (0.22 g, 1.8 mmol) and Et₃N (6.3 ml, 45 mmol). This solution was cooled in ice; methane sulfonic anhydride (6.3 g, 36 mmol) dissolved in dry CH₂Cl₂ was added over a few minutes, and the reaction was allowed to warm up to room temperature. After 16 h, CH₂Cl₂ (150 ml) was added, and the solution was extracted with 10% aqueous citric acid (375 ml), dried (MgSO₄), and evaporated to dryness to give the bis-mesyl compound as a brown oil.

c) A solution of the bis-mesyl compound and NaI (13.5 g, 0.09 mol) in acetone (150 ml) was refluxed for 5 h. The solvent was removed by evaporation, the residue partitioned between CH₂Cl₂ (375 ml) and H₂O (375 ml), and the organic layer dried (MgSO₄) and evaporated to dryness. The product was purified on silica using CH₂Cl₂ as eluant giving 5.6 g (86%) of pure bis-iodo ester. ¹H NMR 1,39 + 1.41 (2s, 18H, tert-Bu), 2.0 (m, 2H, CH₂CH), 2.33 (t, 2H, CH₂CO), 3.3 (t, 4H, CH₂I), 3.57 (t, 4H, CH₂N, J = 7 Hz), 4.3 (m, 1H, CH), 7.6 (d, 2H, H2+6, J = 10 Hz), 8.60 (d, 1H, NH, J = 7.5 Hz); ¹⁹F NMR -117.2 (d, 2F, F3+5, J = 10 Hz).

[3,5-Difluoro-4-[bis(2-iodoethyl)amino]benzoyl]-L-glutamic Acid.
The bis-iodo ester (5.6 g, 7.8 mmol) was dissolved in trifluoro acetic acid (140 ml). After 1 h, the solvent was removed by evaporation. The acid was crystallized as a pure white solid (4.5 g, 95%): mp 121–123°C; ¹H NMR 1.95 + 2.05 (2m, 2H, CH₂CH), 2.34 (t, 2H, CH₂CO, J = 7.5 Hz), 3.3 (t, 4H, CH₂I), 3.57 (t, 4H, CH₂N, J = 7 Hz), 4.4 (m, 1H, CH), 7.6 (d, 2H, H2+6, J = 10 Hz), 8.6 (d, 1H, NH, J = 7.5 Hz), 12.5 (bs, 1H, CO₂H); ¹⁹F NMR -117.2 (d, 2F, F3+5, J = 10 Hz); MS m/z 633 (M+Na⁺, 23), 611 (M+H⁺, 40), 464 (M-glu, 82). Anal. (C₁₆H₁₈N₂O₅F₂I₂) C,H,N.

Cell Lines and Culture Conditions
The generation of the ß-gal- and stCPG2(Q)3-expressing MDA MB 361 and WiDr stable cell lines has been described previously (26 , 28) . All of the cells were cultured in DMEM supplemented with 10% fetal bovine serum at 37°C in a 5% CO₂ atmosphere.

In Vitro Cytotoxicity Assays
MDA MB 361 (5 x 10⁵) or WiDr (2 x 10⁶) cells were seeded into 6-well tissue culture dishes, yielding similarly confluent monolayers at 48 h. Prodrugs were dissolved in DMSO at 100 times the highest dose immediately before treatment and were administered in a two-stage protocol, as described previously (29) . After exposure to prodrug, the cells were harvested, diluted, reseeded, and grown until the control wells reached confluence and the extent of cell growth was assayed by sulforhodamine-B dye (30) . The results are expressed as the percentage of control growth against log dose, and the IC₅₀ determined by nonlinear regression to a log dose-effect sigmoid, constraining the minimum to be positive (GraphPad Prism, GraphPad Software Inc., San Diego, CA).

In Vivo Analyses
All experiments were conducted in accordance with United Kingdom Home Office regulations and United Kingdom Co-ordinating Committee on Cancer Research guidelines (31) . Xenografts were established in nude (nu/nu) female BALB/C mice (20–22 g) by s.c. inoculation (0.2 ml) in the right flank of a suspension of MDA MB 361 or WiDr cells (10⁷ and 8 x 10⁶ cells, respectively) in PBS. The inocula, (5–10 animals/group), consisted of mixtures of ß-gal- and stCPG2(Q)3-expressing cells, comprising 0, 10, 50, or 100% stCPG2(Q)3-expressing cells. After 4 days, mice were divided randomly into control and treated groups, and those in the treated groups received prodrugs (day 0). Prodrugs were dissolved in DMSO and diluted 20-fold in 1.26% (w/v) sodium bicarbonate just before injection. Each course of prodrug treatment consisted of three i.p. injections over a 2-h (1) or 24-h (2 , 3) period, to a total preestablished maximum tolerated dose of 300, 1200, and 600 mg/kg for prodrugs 1, 2, and 3 respectively. Additional courses of prodrug were administered on days 7, 14, 28, and 36 (±1 day) and, when palpable tumor remained, on days 42, 56, and 63. When eventual tumor regrowth followed a prolonged tumor-free period, an additional course was administered. Animals were culled if the tumor exceeded 1.5 cm in any dimension. At the time of cull, the tumors were excised and analyzed for specific CPG2 activity by enzyme assay as described previously (32) . In a separate experiment, WiDr xenografts consisting of 0, 10% and 50% stCPG2(Q)3-expressing cells were established as above and were treated with similar courses of the prodrugs. Courses were repeated on days 7 and 14. Twenty-four h after the end of the last course, the tumors were excised and fixed in formol-saline. After paraffin mounting, sections (5 µm) were cut, stained with H&E, and examined microscopically (x40). Representative fields of view were photographed.

	RESULTS

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In Vitro Cytotoxicity of Prodrugs in MDA MB 361 and WiDr Cells Expressing ß-gal or stCPG2(Q)3.
We compare the cytotoxicity (Table 1) of the prodrugs 1, 2, and 3 (Fig. 1) for efficacy in GDEPT toward MDA MB 361 breast carcinoma and WiDr colon carcinoma cell lines engineered for stable expression of stCPG2(Q)3, with parental cells expressing ß-gal as controls. Relative dye binding in control wells is similar for ß-gal- and stCPG2(Q)3-expressing cells, implying equal growth rates. Cell monolayers are exposed to a range of concentrations of the prodrugs, and the resultant IC₅₀ values are shown in Table 1 . When the survival of the prodrug-treated cells did not fall below 50% of the control untreated cells, a value of greater than the highest dose used is quoted. In both the MDA MB 361 and WiDr cell lines, prodrug 1 is the most potent, toward both the ß-gal- and stCPG2(Q)3-expressing variants (Table 1) . Prodrug 3 is more potent than 2 in ß-gal-expressing MDA MB 361 and WiDr cells, but very similar in both MDA MB 361 and WiDr stCPG2(Q)3-expressing cells. In MDA MB 361 and WiDr, the magnitude of the dose modification factor ranks prodrug 1 > prodrug 2 > prodrug 3, and is as high as 450-fold in the case of WiDr stCPG2(Q)3 treated with prodrug 1 (Table 1) .

View larger version (22K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. The CPG2 activity of MDA MB 361 (A), or WiDr (B) xenografts arising from inocula of 100, 50, 10, or 0% stCPG2(Q)3-expressing cells, excised at the time of cull after treatment with nothing (, ), prodrug 1 (, ), prodrug 2 (, ····), or prodrug 3 (, · ). The xenografts were homogenized in CPG2 assay buffer/10% glycerol. After 30 min incubation of the homogenate with a fixed concentration of the model substrate methotrexate, the reaction product was quantified by high-performance liquid chromatography, and the activity was calculated from an external calibration curve that was derived using authentic enzyme. The activity is expressed in units of enzyme per gram wet weight of tumor.

View larger version (155K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Histology of WiDr xenografts arising from inocula composed of 0% (top row), 10% (middle row), or 50% (bottom row) stCPG2(Q)3-expressing cells, after treatment with prodrug 1 (left column), prodrug 2 (middle column), or prodrug 3 (right column). Three courses of prodrug were administered to nude mice bearing xenografts as above. The tumors were excised and fixed in formol-saline 24 h later. Sections (5 µm) were cut from paraffin-embedded blocks and stained with H&E. Photomicrographs were taken of representative fields of view at x40.

View larger version (23K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. The antitumor effect of prodrug 1 (), prodrug 2 (), and prodrug 3 () in MDA MB 361 (A), and WiDr (B) xenografts arising from inocula consisting of 0, 10, 50, or 100% stCPG2(Q)3-expressing cells. Horizontal line, the median survival of the untreated control groups for all of the inocula.

	DISCUSSION

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We test the three new prodrugs for therapeutic efficacy in the previously described stCPG2(Q)3- and control ß-gal-expressing MDA MB 361 xenograft models and in the newly described stCPG2(Q)3- and control ß-gal-expressing WiDr xenograft model. To assess the ability of the prodrugs to mount a bystander effect when activated by CPG2, we assess their therapeutic effect in xenografts arising from inocula composed of mixtures of stCPG2(Q)3- and control ß-gal-expressing cells. At the termination of each experiment, the tumors were excised, and the specific activities of CPG2 assayed and expressed per gram of wet weight of tumor. The concentrations of CPG2 observed in the tumors at the time of cull suggest that the prodrugs can act as selective agents against stCPG2(Q)3-expressing cells in a mixed population with differing efficiency. Prodrug 1 greatly reduces the specific activity of CPG2 in tumors that arose from inocula in which only 10 or 50% of the cells expressed CPG2. This suggests that prodrug 1, although the most potent and generating the greatest cytotoxic differential between stCPG2(Q)3- and control ß-gal-expressing cells, is mounting only a limited bystander effect. This is because a drug with a poorer bystander effect will kill only the stCPG2(Q)3-expressing cells in which it is generated, leaving the non-CPG2-expressing bystanders to regrow, giving rise to a tumor with little remaining CPG2. In this situation, it would be expected that tumors that regrew from inocula of 10 and 50% mixtures of stCPG2(Q)3-expressing cells display reduced CPG2-specific activity, because the drug derived from prodrug 1 has acted as a selective agent. That the same reduced concentration of CPG2 is seen in tumors that regrew from an inoculum of 100% stCPG2(Q)3-expressing WiDr cells suggests that, at the time of prodrug treatment, the actual percentage of stCPG2(Q)3-expressing cells was less than the inoculated 100%. This may be because, although the cell lines were cloned in and periodically reselected in G418, they are selection-free once they are in vivo, and the expression of CPG2 may become extinguished in a proportion of the cells. Tumors that regrew after treatment with prodrugs 2 and 3 possess concentrations of CPG2 that were essentially unchanged, which suggests that these prodrugs mount a considerable bystander effect, to the extent that even when only 10% of the cells of the tumor express CPG2, all of the cells experience similar cytotoxicity. Indeed, the results with prodrug 1 suggest that the actual percentage may be less than 10%, indicating an even more powerful bystander effect.

The histological appearance of sections cut from WiDr xenografts arising from inocula in which only 10% of the cells expressed CPG2 is supportive of the proposal that, although prodrug 1 is more potent than prodrugs 2 and 3, it exerts a poorer bystander effect. We see that although prodrug 1 produces some visible damage in tumors composed solely of control ß-gal-expressing cells, it produces no additional effect in tumors arising from inocula of 10% CPG2-expressing cells. By contrast, prodrugs 2 and 3 produce a visible effect in tumors arising from inocula of 10% CPG2-expressing cells, which indicates the presence of a bystander effect when compared with no effect produced in tumors composed entirely of control ß-gal-expressing cells. When 50% of the cells of the inoculum expressed CPG2, a marked bystander effect is seen with all three prodrugs.

When the therapeutic effect is examined, in MDA MB 361 xenografts arising from 100% stCPG2(Q)3-expressing cells, the most therapeutically effective prodrug was 1, consistent with its being the most potent prodrug, and having the greatest cytotoxic differential. However, in tumors arising from inocula of 50% and 10% stCPG2(Q)3-expressing cells, prodrug 1 becomes progressively less effective. This is consistent with the histological and CPG2 activity findings that suggest that this prodrug exerts a relatively small bystander effect. Prodrugs 2 and 3, although not as effective as prodrug 1 in tumors arising from inocula of 100% stCPG2(Q)3-expressing cells, retain their efficacy in tumors arising from inocula of 50% and 10% stCPG2(Q)3-expressing cells, consistent with their correspondingly larger bystander effect, as evidenced in the histological and CPG2 activity sections of the study. In WiDr xenografts, the pattern is less clear, with a much smaller therapeutic effect being seen in tumors arising from inocula of 50% stCPG2(Q)3-expressing cells, and none at all in tumors arising from inocula of 10% stCPG2(Q)3-expressing cells.

The extent of the bystander efficacy as compared with the cytotoxic potency may reflect the stability of the active drugs. The phenol mustard drug deriving from prodrug 1 will be the most reactive of the drugs, and will, thus, tend to react primarily at its site of production, becoming inactivated before it can diffuse to bystander cells. The less reactive, and less potent, drugs will be able to diffuse to a greater extent before inactivation. Clearly, there is a balance between a drug being too reactive with a poor bystander effect, or being insufficiently reactive and leaking from the tumor to cause systemic toxicity. The high cytotoxic differential of prodrug 1 may reflect a larger difference in lipophilicity between prodrug and drug than for prodrugs 2 and 3, which could also influence bystander efficacy. The optimum lipophilicity will enable a drug to permeate nearby cells efficiently, but not to be so completely sequestered in their lipid membranes that it cannot reach bystander cells. Future experiments will endeavor to establish the quantitative relationship between the half-life, lipophilicity, cytotoxic potency, cytotoxic differential, and bystander efficacy of these and other prodrugs and drugs. This will enable the design of prodrugs with the optimum combination of characteristics for use in GDEPT.

In summary, we have synthesized and studied three new prodrugs for use with CPG2 in GDEPT. In one tumor model, the prodrugs with the poorer cytotoxic differential but greater bystander effect produce a relatively larger extension of life span in tumors composed of a low percentage of stCPG2(Q)3-expressing cells. This model is, thus, more relevant to GDEPT, in which expression of the transgene is more likely in only a small proportion of the tumor cells, than in the commonly used systems, in which 100% of the cells of the tumor express the transgene. These results show the need to optimize several properties of prodrugs for GDEPT for performance in different tumor types, and for tumors composed of different percentages of cells expressing the therapeutic enzyme. These results also highlight the versatility of prodrug design available in CPG2-based GDEPT that is precluded in many other commonly used GDEPT systems.

	ACKNOWLEDGMENTS

 
We thank Geoffrey Boxer and Dr. R. Barbara Pedley for helpful advice on histology.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom requests for reprints should be addressed, at CRC Centre for Cancer Therapeutics at the Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG United Kingdom. Phone: 44-(0)-20–8722-4214; Fax: 44-(0)-20–8643-6902; E-mail: caroline{at}icr.ac.uk

² The abbreviations used are: GDEPT, gene-directed enzyme prodrug therapy; CPG2, carboxypeptidase G2; CMDA, 4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl-L-glutamic acid; stCPG2(Q)3, surface-tethered CPG2; ß-gal, ß-galactosidase.

Received 8/ 3/01. Accepted 1/16/02.

	REFERENCES

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