Gene-Directed Enzyme Prodrug Therapy with a Mustard Prodrug/ Carboxypeptidase G2 Combination'

[CANCERRESEARCH56, 4735-4742, October 15, 19961 Gene-directed Enzyme Prodrug Therapy with a Mustard Prodrug/ Carboxypeptidase G2 Combination'

Richard Marais, Robert A. Spooner, Yvonne Light, Janet Martin, and Caroline J. Springer@ CRC Centrefor CancerTherapeuticsat the Institute of CancerResearch,15 CotswoldRoad,Sutton,SurreySM2 5NG [R. A. S., J. M., C.J. S.], and CRC Centrefor Cell and Molecular Biology at the Institute of Cancer Research, 237 Fulham Road, London SW3 6JB (R.M., R.A.S., Y.LJ, United Kingdom

ABSTRACT enzyme. Since the foreign enzyme is expressed only in the tumor, the cytotoxic drug will be restricted to the tumor. Theoretically, such an The gene for the bacterial enzyme carboxypeptidase G2 (CPG2) was approach should enhance the therapeutic index of chemotherapeutic expressed internally in mammalian cells. Mammalian-expressed CPG2 agents by minimizing systemic toxicity (3, 5). had kinetic properties Indistinguishablefrom bacterially expressed CPG2. Human tumor cell lines A2780, SK-OV-3 (ovarian adenocarcinomas), A variety of different methods for gene delivery has been consid LS174T, and WLDr(colon carcinomas) were engineered to express con ered. These include retrovirus (6), naked DNA (7), liposomes (8), and stitutively either CPG2 or bacterial 13-galactosidase. These cell lines were adenovirus (9) delivery. Tissue-specific promoters such as those of subjected to a gene-directed enzyme prodrug therapy regime, using the tyrosinase (10, 11), a-fetoprotein (12), and carcinoembryonic antigen prodrug 4-[(2-chloroethyl)(2-mesyloxyethyl)amino]benzoyl-L-glutamic genes (13, 14) have been used to effect selectivity of expression in acid (CMDA). The lines which expressed CPG2 had enhanced sensitivity target tissues. Since expression of the foreign enzyme with these to CMDA. Comparing IC@s, WIDr-CPG2 and SK-OV-3-CPG2 were methods is unlikely to occur in all cells of a targeted tumor in vivo, a 11-16-fold more sensitive, whereas A2780-CPG2 and LS174T-CPG2 were bystander cytotoxic effect is required whereby the prodrug is cleaved â€”95-foldmore sensitive than the corresponding control lines. CPG2- expressing cells and control cells were mixed in differing proportions and to an active drug that kills not only tumor cells expressing the foreign then treated with prodrug. Total kill occurred when only â€”12%ofcells enzyme but also neighboring nonexpressing tumor cells (3). In animal expressed CPG2 with the W1Dr and SK-OV-3 lines and when only 4-5% models, when as few as 2% of the tumor cells express foreign enzyme of cells expressed CPG2 with the LS174T and A2780 lines, indicating a after subsequent treatment with a suitable prodrug, long-term survi substantial bystander effect. These results establish this CPG2 enzyme! vors can be obtained (3). Therefore, an expression efficiency of CMDA prodrug system as an effective combination for the gene-directed 10â€”20%should be enough to achieve 100% cell kill in tumors, and enzyme prodrug therapy approach. efficiencies of 1â€”5%are considered sufficient for a therapeutic re sponse (3). INTRODUCTION A number of different enzyme/prodrug systems has been designed for GDEPT. These include purine nucleoside prodrugs, which are One of the major problems with current cancer therapies is the lack activated by viral thymidine kinase (6, 7, 15â€”24)or thymidine phos of specificity of treatment, which leads to harmful side effects in phorylase (11, 25, 26), 5-fluorocytosine activated by bacterial cyto normal tissues, especially the gut lining and bone marrow (1). Current sine deaminase (3, 27â€”31),cyclophosphamide and isofosphamide research is thus focused on the development of more selective meth activated by rat liver cytochrome P-450 isoenzyme (32), and CB 1954 ods for the delivery of toxic compounds to cancer cells. Gene therapy activated by bacterial nitroreductase (4). may be broadly defined as a genetic technology aimed at modifying In all the prodrug/enzyme examples cited above, the expressed cells for therapeutic gain that has been proposed as one such method enzyme converts the prodrug to an intermediate metabolite, which to achieve greater selectivity (2). requires further catalysis by cellular enzymes within the tumor before In cancer gene therapy, both malignant and nonmalignant cells may the active drug is formed. If the cellular enzymes responsible for this be targeted for therapeutic benefit. The possibility to render cancer second phase of activation become defective or deficient in the tumor cells more sensitive to chemotherapy or toxins, either by suppressing cells, this would lead to resistance of the tumor to the prodrug (5). the expression of resistance genes (e.g., multidrug resistance gene) or Thus, it is preferable that the active moiety be released directly from by introducing â€œsuicidegenesâ€•has been considered (2). The latter the prodrug cleavage by the expressed enzyme. includes two approaches: (a) toxin gene therapy, whereby transfected For maximal benefit, the released drug should be effective against genes are able to generate toxins; and (b) enzyme-activating prodrug both cycling and noncycling cells. Most drugs are active only against therapy whereby transfected genes express foreign enzymes that can cycling cells, whereas mustard alkylating drugs are also cytotoxic to activate prodrugs inside the cancer cells. This latter approach is noncycling cells (33, 34). They have the added advantage that their termed VDEPT (for virally directed enzyme prodrug therapy; Ref. 3), cytotoxicity is dose related, and their use is less prone than other or GDEPT3 (4, 5). classes of drugs to induce resistance (33, 34). We have previously GDEVF is a two-step approach to targeted chemotherapy of human used the bacterial enzyme CPG2 (which has no mammalian homo cancer. In the first step, the gene for a foreign enzyme is delivered to logue) to activate a glutamic acid prodrug derivative of a benzoic acid the tumor in a form that directs tumor-specific expression of the mustard in an ADEPT context (35, 36). We have demonstrated the foreign protein. In the second step, a nontoxic prodrug is administered efficacy of an antibody-CPG2 conjugate that effected CMDA prodrug that is converted to a cytotoxic drug by the action of the expressed activation in tumor xenograft models in nude mice, such as chorio carcinoma (37) and ovarian (38), colorectal (39, 40), and breast Received 5/22/96; accepted 8/15/96. The costs of publication of this article were defrayed in part by the payment of page carcinomas (41). The CPG2 enzyme removes the glutamic acid moi charges. This article must therefore be hereby marked advertisement in accordance with ety from the prodrug releasing the active mustard drug (Fig. 1; Ref. 18U.S.C.Section1734solelyto indicatethisfact. 42). No further enzymatic processing is required to activate the drug I This work was supported by The Cancer Research Campaign, United Kingdom. 2 To whom requests for reprints should be addressed. Phone: 44 1 81 643 8901, ext. (43, 44). 4214. Fax: 44 1 81 770 7899. These properties suggested that the CPG2 enzyme could be a good 3 The abbreviations used are: GDEPT, gene-directed enzyme prodrug therapy; AD EPT, antibody-directed enzyme prodrug therapy; CPG2, carboxypeptidase G2; CMDA, candidate for GDEPT if the corresponding gene could be expressed in 4-[(2-chloroethyl)(2-mesyloxyethyl)aminolbenzoyl-L-glutamic acid; MTX, methotrexate. mammalian cells. The present study was designed to investigate the 4735

GDEFF WiTH A MUSTARD PRODRUG

CH3SO3 @ Fig. 1. Structures of the prodrug CMDA and its CPG2 /=\ ,OH corresponding drug, 4-[(2-chloroethyl)(2-mesy loxyethyl)amino]benzoic acid formed by the action of CPG2 on CMDA are shown. CMDA Prodrug Drug

feasibility of this proposal within COS cells (monkey kidney) and in Oligonucleotides. The following oligonucleotides were used. For oligonu four human tumor epithelial cell lines. Kinetic parameters were meas cleotides nos. 1 and 2, differences from the CPG2 sequence are italicized and ured for all of the modified lines. Cytotoxicity assays in combination contain the engineered restriction sites; sequences are 5'â€”+3':no.1, CGC GGA with prodnig and the bystander effects were studied for GDEPT TCC GTF GCA CAC TFG GGT OCT C; no. 2, CGC GM 7712GAC fTG protocols. GAT GAC AAG GGC; no. 3, C ATG CCG CAT CAC CAC CAT CAT CAC GC; and no. 4, CAT GGC GTG ATG ATG GTG GTG ATG COG. Prodrug Synthesl@ The CMDA prodrug was synthesized and character MATERIALS AND METHODS ized as described previously (53). CPG2 Specific Antisera. Rabbit antisera were raised to H@CPG2* cx Plasmid Construction. All cloning procedures were performed using pressed in Sf9 insect cells by the methods described previously by Marais et a!. standard molecular biology techniques (45). The cpg2 gene was manipulated (54). For purification of H6CPO2*, 1.5 X 108 cells were infected with virus to delete the first 22 amino acids that code for the signal peptide. This was particles, incubated for 48 h, and extracted as reported previously (54), except achieved by PCR, in which the PCR primers were designed to change codons the extraction and dilution buffers contained only 0.5 mMEDTA and 0.06% 21 and 22 to a BamHI site and the stop codon to an EcoRI site. The 5' cpg2 (vlv) 2-mercaptoethanol. The H@CPG2* protein was purified by Ni2@NTA primer (no. 1, see below) was used in conjunction with the 3' CPG2 primer agarose affinity chromatography (Qiagen Ltd.) according to the manufacturer's (no. 2, see below) to amplify the cpg2 gene using the plasmid pNM&3O(46). instructions, eluting the protein with 90 nmi imidazole. The eluted protein was The PCR product was digested with the restriction endonucleases BamHI and judged to be pure by silver-stained SDS polyacrylamide gels (data not shown). EcoRI and cloned into those sites in the polylinker of the mammalian expres Rabbits were inoculated with â€”150 @gof purified protein, and subsequent sion vector pEFplink.2 (47). An initiator ATG is supplied by the vector, which bleeds were examined for production of antibodies by immunoprotcin blotting is in frame with the cpg2 reading frame and is in a highly conserved Kozak using crude insect cell extracts expressing H@CPO2*. motif to ensure efficient translation (48). This plasmid is referred to as Cell Culture and Transfection. The humancarcinomatumorcell lines pEFcpg2* and encodes a protein, the predicted structure of which is MAGS A2780 and SK-OV-3 (ovarian adenocarcinomas), LS174T and WiDr (colon (CPG2 residues 23â€”415)-EFLEID (single letter amino acid code; Ref. 49). carcinomas), and the COS-7 cell lines were obtained from American Type The vector pEFlacZ, which codes for bacterial /3-galactosidase,was con Culture Collection. All cell lines were maintained in DMEM supplemented structed by cloning the NcoIJXbaIfragment from the plasmid pMLVf3lacZ(50) with 10% FCS (DMEMIFCS; 37Â°C,10% C02). Transient transfection of into those sites in the plasmid pEFplink.2. The plasmid pMCEFâ€”wascreated COS-7 cells was performed as described previously for NIH3T3 cells using by cloning the end-repaired Hindffl fragment from the plasmid pEFplink4into LipofectAMlNE (47). For stable line construction, the human tumor cell lines the end-repaired XhoI site of a modified version of pMClNeo Poly(A) (Strat were transfected with either pMCEFcpg2* or pMCEF!acZ using Lipo agene). The pEFplink fragment contains the elongation factor icr promoter fectAMINE. Forty eight h following transfection, the cells were recultured into (51) todirectefficientexpressionoftheforeignproteinandamultiplecloning medium supplemented with 2 mgml' neomycin (0418, Geneticin; Life site flanked by the 5' and 3' untranslated regions from the human f3-globin Technologies, Inc.). Individual 0418-resistant colonies were cloned by limit gene, which are provided to give efficient mRNA processing, polyadenylation, ing dilution, and all clones that could degrade MTX were selected for further and translation. The pMCEFâ€”vector,therefore, contains the elongation factor characterization. ha promoter, which directs expression of the foreign gene, and a polyoma Mammalian Cell Extraction and Kinetic Analysis of CPG2. Cells were enhancer which directs expression of the Neo'@gene.These two promoters are grown to confluence, and extracts were prepared by washing twice with 5 ml juxtaposed on opposite strands of the plasmid, giving divergent expression in ofPBSA (137 mMNaCI, 3.4 mMKC1,10 mMNa@HP04,1.8mMKH2PO4@pH opposite directions. The cpg2* coding sequence was cloned from pEFcpg2* as 7.4), followed by the addition of200 @lofextraction buffer (250 mM Tris-HC1, an NcoIJXbaI fragment into the NcoIJSpeI sites of the plasmid pMCEFâ€” to 10% v/v glycerol, and 1% v/v Triton X-100, pH 7.5). The cells were lysed in create the plasmid pMCEFcpg2*. The lacZ gene was cloned into pMCEF situ (5 mm at room temperature), and the extracts were collected and clarified from the plasmid pEFf3lacZ, using the same strategy, to create the vector by centrifugation in a microfuge (5 mm at 14,000 rpm); the supernatant pMCEFIacZ. fractions were stored at â€”70Â°C.Cellextracts were subjected to kinetic anal To express CPG2* in Sf9 insect cells, cpg2* was cloned into the insect cell yses for CPG2 activity by a modification of the method described previously vector pVLH6, a derivative of pVL94l (PharMingen). The EcoRI site in (37). Briefly, cell extract containing â€”200ngof CPG2* was added to 1 ml of pVL941 was destroyed by digesting the plasmid with EcoRI, end-repairing the CPG2 assay buffer (100 mt@iTris-HC1, and 260 @ts@ZnCl2,pH 7.3) containing overhangs and then religating the plasmid. The EcoRV/BamHI fragment from MDCatconcentrationsfrom0.5â€”100pM,andtherateof changeof absorbance pAcYl (52) was then cloned into those sites in pVL941 to create the plasmid at 320 nm was measured. Km5 were calculated from standard regression pVLplink.2. The oligonucleotide adaptors formed by oligonucleotides nos. 3 analysis. and 4 were then cloned into the NcoI site of pVLplink.2 to form the plasmid Cytotoxicity Assays. To determine the sensitivity of the cell lines to the pVLH6. This plasmid contains the multicloning site (4080) CCATG CCG CAT CMDA prodrug in vitro, cells were seeded into 6-well tissue culture plates at CAC CAC CAT CAT CAC 0CC ATG GCG 0CC CGGGTAC CFG CAG 3 X 10@cells/well (SK-OV-3, A2780, and WiDr) or 2 X l0@cells/well ATC TAG AAT TCG GAT CC (4135) and encodes an oligohistidine tag (LS174T) and allowed to grow to confluence. CMDA was prepared in DMSO (numbers refer to base numbering in pVL941; the initiator codon is in italics). immediately prior to use and diluted in DMEM/FCS; then 225 @.dwereused to cpg2* was cloned into pVLH,@ as a NcoI/XbaI fragment, and baculoviruses replace the medium in wells at 6â€”4000 @Mconcentrations.After incubation (1 were prepared using the BaculoGold insect cell virus system (PharMingen). In h), the medium was replaced with 1 ml of medium containing CMDA at the cloning cpg2* into pVLH6, the resultant plasmid contains the cpg2* sequence same concentration and incubated for an additional 18 h. The cells were fused in frame with the histidine tag. Thus, the expressed protein contains a washed and trypsinized; then â€”3%ofthe cells was reseeded into fresh dishes. histidine tag located at its NH2 terminus and has the structure MPHHHHH After 4 days of further growth, cell viability was assessed by incorporation of HAMAGS-(CPG2 residues 23â€”415)-EFLEID;itis referred to as H@CPG2*. [3H]thymidine(0.4 @Ci/mlfor6 h). The cells were washed twice with PBSA, fixed in 5% trichloroacetic acid (4Â°Cfor 20 mm), washed twice with methanol, 4 R. Marais, unpublished data. and air dried. The fixed cells were solubilized (1 ml, 1% SDS/0.2 M NaOH) 4736

A. r4 B. COS C. NQ Fig. 2. Expression of CPG2@ in COS-7 cells. A, immunoprotein blot analysis. COS-7 cells were transfected with either pEFlacZ (Lane 1) or pEF 200â€” cpg2@ (Lane 2), and detergent extracts were pre 100â€¢ pared. For each cell line, 5 @gofsoluble proteins were analyzed on an 8% polyacrylamide gel and probed with the rabbit CPG2-specific polyclonal antiserum. The position of standard molecular mass 96â€” marker proteins is indicated (X 10@),and the CPG25 band is indicated by the arrowhead. B, 68â€” CPG2 enzymatic activity in transfected cells. Deter Fl gentextractsfromcellstransfectedwitheitherpE @5o. FIOCZ (Lane 1) or pEFcpg2@ (Lane 2) were tested forCPO2activity,using5 @gofsolubleproteinand CMDA as a substrate. The degradation of CMDA 45 was monitored at 305 em, and the results are cx pressed in the form of amount CMDA degraded. C, kinetic analysis. Lineweaver-Burk plots, where the X-axis intercept of a linear regression equals â€”1/ @ Km, shown for CPG2@ expressed in mammalian 0@ cells(â€¢)andfor bacteriallyexpressedCPG2(0). 31 12 lIES](M-1) 1 2

and the soluble fraction was added to 6 ml of scintillation fluid to determine the pEFcpg2* or pEF!acZ (which codes for the bacterial (3-galactosidase thymidine incorporation; the results are expressed as percentage growth of enzyme), and detergent extracts were prepared. These extracts were control cells that were treated with vehicle alone. For bystander cytotoxicity examined using a CPG2-specific rabbitpolyclonal antiserum.The results assays, mixtures of CPG2*@expressingcelllines and their appropriate lacZ cell show that an immunoreactive band with an apparent molecular weight of line were seeded as above and treated with either 1 mM(A2780 and LS174T) â€”42,000was detected in cells transfected with pEFcpg2*, which was or 2 sims(SK-OV-3 and WiDr) CMDA. absent in the cells transfected with pEFlacZ (Fig. 2A). We also found that Miscellaneous. Immunoproteinblottingwas performedby standardtech niques using â€˜25I-labeledproteinA to detect specific antibody interactions when CPG2* was analyzed in nondenaturing gels, it migrated with a (55). The primary antiserum was used at a concentration of 1:2000. SDS mobility that is consistent with the protein being a dimer (data not PAGEwasperformedunderstandardconditions(56). shown). The detergent extracts were then analyzed for CPG2 activity, using CMDA as a substrate. The extract from the cells transfected with RESULTS pEFcpg2* was able to degrade CMDA, whereas there was no such activity in the control f3-galactosidase extract (Fig. 2B). Conversely, using Expression of CPG2 in COS Cells. Our initial experiments were O-nitrophenyl f3-o-galactopyranoside as a substrate, we could detect designed to address whether the bacterial enzyme (CPG2) could be j3-galactosidase activity in the extract from the cells transfected with expressed in mammalian cells in an active form. Wild-type CPG2 pEF!acZ but not in the extract from the cells transfected with pEFcpg2* is a secreted enzyme found in the bacterial periplasm (46). The (data not shown). mature protein is a homodimer consisting of subunits, the molec Using MTX as a substrate, the mammalian-expressed CPG2* was ular weight of which is â€”42,000, and the monomers are inactive. found to have a Km of 7 @tM,whichis in close agreement with our own We were concerned that if CPG2 were secreted from tumor cells, fmdings for the bacterially expressed wild-type protein (Fig. 2C) and it could escape from the tumor, giving rise to nonspecific toxicity with those published previously for CPG2 purified from the periplasm owing to production of active drug at distant sites. Therefore, we of Escherichia co!i and Pseudonwnas sp. strain RS16 (46). To deter expressed CPG2 internally in a form that could not be secreted mine whether CPG2* expressed in COS-7 cells was secreted, we from mammalian cells. We also wished to establish whether the examined the tissue culture supematant from the pEFcpg2*@trans@ CPG2 in mammalian cells was active, since problems due to fected cells for CPG2 activity, and none was detectable (data not incorrect folding of the protein, lack of formation of dimers, or to shown). These data, taken together with the lack of the signal peptide sequestration into a hostile cell subcompartment might render this and the fact that CPG2* can be released from the transfected cells enzyme inactive. To this end, we chose to use a transient trans with the detergent Triton X-lOO, suggest that CPG2* is not secreted fection system based on COS-7 cells to examine whether CPG2 by mammalian cells. could be expressed in mammalian cells. Establishment of Cell Lines Constitutively Expressing CPG2*. Our first priority was to create a protein that would not be secreted The results presented above show that when CPG2* is expressed from mammalian cells. The first 22 codons of the cpg2 gene encode internally in mammalian cells, it is soluble and fully active, indicating a signal peptide that is responsible for targeting the protein to the that neither the alterations that have been introduced into the coding periplasm of bacteria and which is removed by proteolysis following sequence nor the intracellular location affect the enzymic properties of membrane translocation (57). We used PCR-directed mutagenesis to CPG2. Because we wished to create a model system to examine the remove the sequences encoding the signal peptide and cloned the potential 0fCPG2* in a GDEPT approach, we established mammalian altered gene into the mammalian expression vector pEFplink.2, which tumor cell lines that constitutively expressed CPG2*. This was uses the promoter from the elongation factor liz gene to direct achieved by cloning the gene encoding CPG2* into the mammalian expression of foreign proteins in mammalian cells (47). This promoter expression vector pMCEFâ€” to create the plasmid pMCEFcpg2*. The was chosen because it is active in a wide variety of cell types (51); the vector pMCEFâ€” uses the elongation factor lea promoter to direct expression construct is referred to as pEFcpg2*. foreign gene expression, but it also contains the Neo't gene; therefore, We used immunoproteinblotting to determinewhetherCPG2* protein the vector can be used to select cells with the cytotoxic drug G418. was expressed in COS-7 cells. COS-7 cells were transfected with either We chose the human colon carcinoma cell lines LS174T (58) and 4737

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1996 American Association for Cancer Research. GDEPT WITH A MUSTARDPRODRUG WiDr (59) and the human ovarian adenocarcinoma cell lines SK A. A2780LS174TSK-OV-3WiDr OV-3 (60) and A2780 (61) for this analysis. * * * * C'4 Cl @4 For each tumor line, cells were transfected with either pMCEF NQ NQ NQ NQ @ cpg2* or the plasmid pMCEF1acZ (which directs expression of bac 1.5 @b @b tenal j3-galactosidase), and G4l8-resistant colonies were selected. G418-resistant colonies were examined for CPG2* expression by enzymic assay, using MTX as a substrate, and for j3-galactosidase activity using O-nitrophenyl @-D-galactopyran05ide as a substrate (data not shown). All the cell lines were found to express >0.4 Hi @1.o CPG2*/mg soluble protein (data not shown). We then examined the susceptibility of the cell lines to prodrug treatment in tissue culture by incubating each line with increasing concentrations of the prodrug

CMDA. For these experiments, the cells were treated with CMDA 4@ 0.5 c'4 twice, and then the medium was replaced with fresh prodrug-free Q medium. Thus, the cells were incubated with the prodrug for only 19 h. The rate of survival of the cells was determined by [3H]thymi dine incorporation. 0 The results show that the cell lines fall into two different categories (Table 1): those that are highly susceptible to CMDA, with IC50s in 12345678 the 5â€”50/.LMrange (LS174T and A2780); and those that are less B. sensitive to CMDA, with IC50s in the range 250â€”600p.M (WiDr and A2780 LS174T SK-OV-3 WiDr * * * * SK-OV-3). For each tumor cell line, one clone was selected to express C'4 C@4 e'1 N N Q N C., N Q N Q @ either CPG2* or @3-galactosidase for further study. The CPG2*@ex@ .@V U ,@V LI ,@V LI ,@U U pressing lines were selected to express similar amounts of CPG2 activity (â€”1unit/mg of detergent-soluble proteins; Fig. 3A and Table 200 2). Each selected line was then subjected to immunoprotein blotting analysis. An immunoreactive band with Mr @42,OOOwasdetected in the cell lines that contained CPG2 activity but not in those that contained @-galactosidase activity, confirming the presence of the 96 â€” cpg2* gene (Fig. 3B). Following G4l8 selection, the selected cell lines were transferred into medium lacking G418 to determine the stability ofexpression and 68â€” to assess whether CPG2* was toxic to mammalian cells. No differ ences in the levels of CPG2* enzyme activity were detected, even when the cells were maintained in the absence of G418 selection for 4 months, and no significant differences were detected in the rate of 45 â€” cell growth of the CPG2*@expressing cells compared to control (3-ga .4 lactosidase-expressing cells of the same lineage (data not shown). Because we did not observe any reduction in the expression of the protein with time or major differences in the rate of growth rate of cells, we conclude that the expression of CPG2* in these cell lines is stable, and CPG2* is not toxic to mammalian cells. 31â€” The IC50s for the selected CPG2*@expressing cell lines were com pared to their corresponding (3-galactosidase lines. The IC50 for the 1 2 3 4 5 6 7 8 selected WiDr@CPG2* line was found to be >11 fold lower than the Fig. 3. Constitutive expression of CPG2* in human tumor cell lines. A, CPG2* enzyme activity in human tumor cell lines. The levels of CPG2 enzyme activity in the pEFlacZ transfected cell lines (Lanes 1, 3, 5, and 7) or the pEFcpg2@-transfectedcell lines (Lanes Table 1 Expression of CPG2@in human tumor cell lines 2. 4, 6. and 8), for A2780 cells (Lanes I and 2); LS174T cells (Lanes 3 and 4); SKOV-3 cells (Lanes 5 and 6); and WiDr cells (Lanes 7 and 8) were determined by standard kinetic The sensitivity of the cell lines to CMDA was determined by treating the cells with analysis and are expressed as a function of activity with respect to the soluble protein in varying concentrations of prodrug. The IC@s are shown and are defined as the concen the cell extracts. B, immunoprotein blot analysis. Immunoblot analysis was performed tration of prodrug required to kill 50% of the cells. with the CPG2-specific rabbit antiserum on 5 @gofdetergent-extracted protein for each CellCMDA)A2780 lineCloneIC50 (@tM of the cell lines stably transfected with either pMCEFIacZ (Lanes I, 3, 5, and 7) or pMCEFcpg2* (Lanes 2, 4, 6, and 8), for A2780 cells (Lanes I and 2); LS174T cells l'@ Â±2.9 (Lanes 3 and 4); SKOV-3 cells (Lanes 5 and 6); and WiDr cells (Lanes 7 and 8). The A2 44.4Â±8.1 position of migration of standard molecular mass markers (X l0@) is indicated, and the LS174T Ll 7.6Â±0.36 CPG2* band is indicated by the arrowhead. L2 25.8Â±1.6 L3 27.1Â±4.4 L4a 29.1 Â±8.9 IC50for the WiDr-lacZ line (Fig. 4; Table 1); the IC50of the selected L5 39.7Â±0.8 SK@OV@3@CPG2*line was >16 fold lower than the IC50 for the SK-OV S 1Â° 258 Â± 14 3 SK-OV-3-lacZ line; the IC50 for the selected A278O@CPG2* line was S2 459Â±36 >92-fold lower than the IC50 for the A2780-lacZ line, and the IC50 of S3 535Â±12 the selected LSl74T@CPG2* line was â€”95-fold lower than the IC50 S4 546Â±17 WiDrA wlâ€• 257Â±15 for the LS174T-lacZ line. These data show that for each each tumor 277Â±38a W223.2 cell line, expression of CPG2* significantly increases the sensitivity The clone from each celi line type picked forfurther study. of the cells to the CMDA prodrug. 4738

GDEPT WITH A MUSTARD PRODRUG

Table2 ExpressionofCPG2*in selectedhumantumorcelllines results with the SK-OV-3 and the WiDr clones were similar, 50% of For each cell line, the levels ofCPO2 activity have been determined by standard kinetic the cells in the culture mixture could be killed when only 1â€”2%ofthe analysisandareexpressedasa functionofunitsof activity/mgofcellproteininthecell extracts (units/mg). The sensitivity ofthe cell lines to CMDA was determined by treating cells were expressing CPG2*; 90% of the cells were killed when â€”8% the cells with varying concentrations of prodrug. The IC5@sare shown and are defmed as of the cells expressing CPG2*, and 100% cell kill was achieved when theconcentrationofprodrugrequiredtokill50%ofthe cells,comparedtovehicle-treated about 12% of the cells expressed CPG2* (Fig. 5; Table 3). The A2780 controls. and LS174T clones were able to direct an even greater bystander activity effect; 50% cell kill occurred when only 0.1 and 1.6%, respectively, CellCMDA)A2780lacZ lineCloneCPG2 (units/mg)ICso (p.M of the cells were CPG2* expressors; 90% of the cells were killed Â±180 2.9LS174TlacZ CPG2@0 0.9642150.0 23.2 Â± when 2.0 and 3. 1%, respectively, of the cells were CPG2* expressors. Â±260 Total cell kill occurred when only an estimated 5 and 3.7%, respec 8.9SK-OV-3lacZ CPG2@0 1.2562770.0 29.1 Â± tively, of the cells were CPG2* expressors (Fig. 5 and Table 3). Â±452 14WiDrlacZ CPG2*0 1.0134180.0 258.0 Â± Â±120 CPG2@0 0.7873230.0 277.0 Â±38 DISCUSSION We have analyzed the potential use of the enzyme CPG2 together with the prodrug CMDA as a mammalian GDEVF model. To prevent @150 CPG2 secretion, we removed the signal peptide from the cpg2 gene to a) make CPG2* and cloned the altered gene into mammalian expression vectors. The altered protein had the same kinetic properties as wild type CPG2 purified from bacterial cells but was located intracellu larly. The altered gene was cloned into a variety of mammalian tumor 11: cells for constitutive expression. The stable cell lines expressing CPG2* were subjected to GDEPT protocols, using the CMDA pro drug. The sensitivity of the CPG2*@expressing lines fell into two categories: the more susceptible A2780 and LS174T lines; and the @150 less sensitive WiDr and SK-OV-3 lines. Clearly, this is not lineage dependent because A2780 and SK-OV-3 are derived from ovarian adenocarcinomas, whereas WiDr and SK-OV-3 are derived from colon carcinomas. We found that in each case, the selected cell lines expressing CPG2* were significantly more sensitive to the prodrug than the 11: (3-galactosidase controls, with IC50 differentials between 11- and 95-fold. Bystander assays of mixtures of the CPG2* and control lines

150 â€˜- â€” 1-4 V [CMDAI(jiM) 100 Fig. 4. Susceptibility to CMDA of human tumor cell lines expressing [email protected] of the cell lines was treated with increasing concentrations of CMDA. and survival was determined by [tmHlthymidineincorporation.The results are expressed as the proportion of 50 surviving cells relative to the vehicle-treated controls. For each cell line, the control cell lines expressing @-galactosidase(0)are shown with their corresponding CPG2@-express ing line(I). Bars,SD. 0 0 a) 150 1.4 The Bystander Effect. The modified cell lines were examined for their ability to mount a bystander effect in vitro. The bystander effect E is defined as the ability of CPG2*@expressing cells to kill neighboring 0 100 V cells that do not express CPG2* in the presence of the prodrug. We determined this effect by growing mixtures of each of the clones expressing CPG2* with their corresponding (3-galactosidase control, 0 followed by treatment of the cells with the CMDA prodrug. The 1.4 concentration of the prodrug used was determined by the sensitivity of

the (3-galactosidase-expressing lines to CMDA and was chosen to be 0 25 50 75 1000 25 50 75 100 approximately one-half the IC50 concentration of those lines. Thus, for the A2780 and LS174T clones, the concentration used was 1 nmi CMDA, and for the SK-OV-3 and the WiDr clones, the concentration Proportion of CPG2*@ expressing cells (%) was 2 mM CMDA (Table 3). At these concentrations, between 80 and Fig. 5. Bystander effect of cells expressing CPG2*. For each of the tumor models, the cell line expressing @-galactosidase and the cell line expressing CPG2* were mixed in 100% of the lacZ-expressing cells survive. various proportions in tissue culture and treated with the appropriate concentration of Mixtures of cells were treated with the prodnig, and cell survival CMDA (1 mMfor A2780 and LS174T; 2 mat for SK-OV-3 and WiDr). The proportion of was determined by [3H]thymidine incorporation. The results are cells surviving was determined by [3H]thymidine incorporation and is expressed as the proportion of surviving cells relative to the cells expressing 100% (3-galactosidasetreated shown in Fig. 5 and expressed in Table 3. They indicate that all of the with the prodrug. The dashed line predicts cell survival if there were no bystander effect. clones expressing CPG2* were able to direct a bystander effect. The Bars, SD. 4739

Table 3 The bystander effect in vitro For each tumor model, the line expressing CPG2* was mixed with the line expressing @-galactosidaseandtreatedofProportion with the indicated concentration CMDA. The proportion cells required to kill 50, 90. and 100% of the cells was determined by logarithmic regression of the data presented in Fig. 5.of

of CPG2*@expressing cells required for: CMDA conc.'@ deathA2780Cell line (mM) 50% cell death 90% cell death100% cell 2.0%5.0%LS174T 1 0.1% 3.1%3.7%SK-OV-3 1 1.6% 7.7%12.0%WiDr 2 1.3% 8.3%11.5%a 2 2.2%

cone., concentration. demonstrated that total cell kill could be obtained when only 3.7 to enzyme-modified cells leads to killing of adjacent unmodified cells. 12% of the cells were expressing CPG2*. Herein, the bystander effect was examined by mixing cells expressing To obtain selective toxicity in GDEPT, there should be no endog CPG2* with those of the same lineage that did not express CPG2, enous enzyme capable of catalyzing the prodrug to the cytotoxic followed by treatment with CMDA. We have demonstrated that moiety. In previous ADEPT clinical trials, when the CMDA prodrug effective bystander effects occurred when we mixed CPG2*@express@ was administered to patients without prior injection of an antibody ing and nonexpressing cells in combination with CMDA. Total cell CPG2 conjugate, there was no CMDA related toxicity (62), nor was kill was obtained when as few as 3.7% of the cells were expressing there any conversion of the prodrug to the active drug, as monitored CPG2*. This compares very favorably with the data on in vitro in plasmaby high-performanceliquidchromatographyandthe more bystander cytotoxicity of other enzyme/prodrug systems: (a) total cell sensitive technique of liquid chromatography-mass spectrometry (63). death in combination with ganciclovir required expression of thymi Taken together, these data demonstrate that there is no human enzyme dine kinase in 60â€”90%of the cell population (7, 68); (b) expression capable of converting CMDA to the active drug when it is adminis of cytosine deaminase in 33% of cells in vitro resulted in 100% cell tered systemically. Thus, the required selectivity of GDEPT can be kill with 5-fluorocytosine (3); (c) in a cytochrome P-450-activating attained by the CPG2 enzyme. enzyme GDEPT system, >50% cells were required to express the In clinical trials with CMDA in ADEPT, doses of 2.2â€”5.5mmols/ enzyme to obtain total cell kill with cyclophosphamide (69); and (d) m2/days of prodrug were administered to patients (62), and concen only 1% (25) to 2% (11) of cells expressing purine nucleoside phos trations in excess of 3 mt@ihave been measured in the plasma of phorylase were required to effect almost total cell kill with purine patients.5 The concentrations of 1â€”2mr@iCMDA used in the in vitro nucleoside prodrugs. However, in each case with the purine nucleo bystander cytotoxicity studies described herein, therefore, fall within side phosphorylase, there was an extended 6-day incubation with the range of concentrations that could be achieved in patients. At these prodrug required to obtain such an effect; in order to obtain greater concentrations, the CMDA was not cytotoxic to the cells in vitro that than 90% cell kill with the prodrug CB1954, 30â€”50%of cells were were not expressing CPG2. However, in GDEPT this level of prodrug required to express nitroreductase (4). concentration would be effective to kill those cells that express the In summary, CPG2 represents an effective enzyme for use in enzyme and would be sufficient to direct a substantial bystander GDEPT, in combination with the CMDA prodrug, providing a novel response. GDEPT system with advantages over the other enzyme/prodrug com Activated CMDA exerts its cytotoxicity by cross-linking DNA; binations. We are currently investigating the utility of this combina thus, it is able to kill both cycling and noncycling cells. This contrasts tion in vivo in CPG2*@modified tumor xenografts of the ovarian with the most commonly used GDEPT enzyme/prodrug system, thy adenocarcinoma and the colon carcinoma cell lines described here. midine kinase/ganciclovir, which is cytotoxic only during the S phase of the cell cycle (64). It has been proposed that resistance to ganci ACKNOWLEDGMENTS clovir in GDEPT is due to the proportion of cells in G0 at the time of ganciclovir administration (65). Resistant tumor outgrowth occurred, We thank Professors K. R. Harrap and C. J. Marshall for support and despite up to 30 days of continuous ganciclovir administration, mdi Professor I. Niculescu-Duvaz for helpful discussions. We are grateful to Dr. R. cating that some tumor cells can remain in G0 for long periods. Treisman for the pMLV(3lacZ plasmid, Dr. N. Minton for the pNM83O Tumors that grew out remained sensitive to ganciclovir on additional plasmid, Dr. R. Sherwood for bacterially expressed CPG2, and 0. 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Richard Marais, Robert A. Spooner, Yvonne Light, et al.

Cancer Res 1996;56:4735-4742.

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