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Expression of Endogenously Activated Secreted or Cell Surface Carboxypeptidase A Sensitizes Tumor Cells to Methotrexate--Peptide Prodrugs¹

Departments of Pharmacology [D. A. H., J. M.] and Radiation Oncology [D. A. H., J. M., A. R.], The University of Michigan, Ann Arbor, Michigan 48109-0582, and Department of Biochemistry, The University of Laval, Laval, Quebec, Canada G1K 7P4 [M. P.]

	ABSTRACT

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Methotrexate (MTX) is one of the most commonly used agents in the treatment of solid malignancies; however, the toxicities of MTX to bone marrow and gastrointestinal tract complicate this therapy. We, therefore, propose a gene-dependent enzyme prodrug therapy to limit these toxicities by localizing the production of MTX to the site of the tumor. The combination of MTX--peptide prodrugs, which cannot be internalized by the cellular reduced folate carrier, with carboxypeptidase A (CPA), which can remove the blocking peptide, has been demonstrated previously in vitro using antibody-dependent enzyme prodrug therapy. CPA is normally synthesized as a zymogen that is inactive without proteolytic removal of its propeptide by trypsin. Therefore, to adapt this system to gene-dependent enzyme prodrug therapy, a mutant form of CPA was engineered, CPA_ST3, that does not require trypsin-dependent zymogen cleavage but is instead activated by ubiquitously expressed intracellular propeptidases. Purification, peptide sequencing, and kinetic analysis indicated that mature CPA_ST3 is structurally and functionally similar to the trypsin-activated, wild-type enzyme. In addition, CPA_ST3-expressing tumors cells were sensitized to MTX prodrugs in a dose- and time-dependent manner. To limit diffusion of CPA, a cell surface localized form was generated by constructing a fusion protein between CPA_ST3 and the phosphatidylinositol linkage domain from decay accelerating factor. SDS-PAGE and flow cytometric analysis of infected tumor cells indicated that CPA_DAF was cell surface localized. Finally, after retroviral transduction, this enzyme/prodrug strategy exhibited a potent bystander effect, even when <10% of the cells were transduced, because extracellular production of MTX sensitized both transduced and nontransduced cells.

	INTRODUCTION

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Chemotherapeutics, drugs that are preferentially toxic to tumor cells as compared with host tissues, are a vital part of most current cancer treatments. However, most common chemotherapeutic agents have a small therapeutic index and exhibit profound systemic toxicities, particularly to rapidly dividing tissues such as bone marrow and gastrointestinal tract (1) . These toxicities present a significant morbidity and mortality; in addition, they limit the dose of chemotherapeutic used and thus may also decrease the clinical response. One method proposed to circumvent these toxicities and to increase the therapeutic index of chemotherapy is the development of GDEPT³ (reviewed in Ref. 2 ): (a) tumors are transduced with the gene for an enzyme whose activity is not normally present in the host; and (b) a prodrug is administered systemically, which is nontoxic except when metabolically converted to a toxic form by the enzyme transduced in the first step. The goal of these strategies is to simultaneously increase the local concentration of the toxic agent while also decreasing the associated systemic toxicities.

MTX, a folate analogue antimetabolite, is one of the most commonly used chemotherapeutics for the treatment of solid malignancies (3 , 4) . Prodrugs of MTX have been described where a blocking amino acid is conjugated to the glutamic acid residue in MTX; these prodrugs are unable to be internalized by the cellular reduced folate carrier (5) . CPA, a zinc-metalloprotease, is normally synthesized in the pancreas and released into the lumen of the small intestine, where trypsin-dependent zymogen activation is necessary to remove the inhibitory propeptide and activate the enzyme (6) . CPA has been described previously for use in ADEPT protocols in conjunction with MTX--peptide prodrugs (5 , 7 , 8) . All of these ADEPT strategies relied upon purified CPA that was activated by trypsin in vitro to remove the propeptide. However, ADEPT systems are plagued by a number of problems, including cost and difficulties with development and purification of antibodies, immunogenicity of antibodies, accessibility of tumor to the enzyme/antibody conjugate, stability of the enzyme/antibody conjugate, and background conversion of prodrugs because of localization of antibody conjugates to inappropriate tissues (2) . To adapt this CPA/MTX--peptide-based strategy from an antibody-based therapy to a GDEPT, we endeavored to generate mutant forms of CPA that would be activated in a trypsin-independent manner by endogenous cellular proteases.

PACE/furin is the prototypical member of a family of PCs that include at least seven members (9) . These serine proteases are involved in the maturation of secretory proteins by cleavage after clusters of basic amino acids in proteins such as: growth factors, growth factor receptors, prohormones, bacterial toxins, and viral coat proteins. Some of the PCs exhibit restrictive expression in neuroendocrine tissues; however, at least three members of the family, PACE, PACE4, and PC7, appear to be ubiquitously expressed (9) . Therefore, we felt that a mutant form of CPA, which was engineered to be activated by one or more of these PCs, could prove to be an effective part of a GDEPT strategy. Previously, we have reported just such a mutant, CPA₉₅, where a simple tetra-basic PACE cleavage site (-RQKR-) was introduced into CPA between the propeptide and the mature enzyme (10) . CPA₉₅ was expressed as an active enzyme independent of trypsin treatment and could sensitize cells to MTX-Phe. This activation, however, was dependent upon overexpression of PACE with little or no activation detected by endogenous PCs in absence of PACE cotransfection. To overcome the need for exogenous PACE expression, we report here a mutant form of CPA (CPA_ST3) that is fully activated in a trypsin-independent manner by endogenous propeptidases. The rationale for the construction of CPA_ST3 was that the 10 amino acid sequence (-GLSARNRQKR-) within ST3 sensitizes it to activation by PACE (11) . Because this sequence encompasses the key features of a PACE cleavage site (i.e., basic residues at -1, -2, -4, and -6 relative to cleavage; Ref. 12 ), we hypothesized that the insertion of this decapeptide would also sensitize CPA_ST3 to PC-based activation.

The secretion of active CPA into the extracellular space should allow for a potent bystander effect where a small population of CPA expressing cells could generate sufficient MTX within the tumor milieu to sensitize adjacent, nontransduced cells. This is particularly true for this enzyme/prodrug system because the MTX is generated outside the transduced cell, so both CPA-expressing and bystander cells should be equally sensitized. Unfortunately, if active secreted CPA is able to diffuse out of the tumor, it might result in both a decreased local production and in increased systemic generation of MTX. Therefore, to restrict CPA to the site of transduction, we constructed a cell surface tethered form of the enzyme by fusing CPA_ST3 to the glycophospholipid membrane linkage domain of DAF (13) . This fusion protein is anchored to the surface of the cell by a lipid linkage and thus may afford local production of MTX without systemic release of the protein. In this report, the generation of both the endogenously active soluble and cell surface forms of CPA are described, as is their ability to use MTX--peptide prodrugs and sensitize cells in vitro.

	MATERIALS AND METHODS

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Expression Plasmids.
All common molecular biological techniques were performed according to Sambrook et al. (14) . The expression plasmids for wild-type rat CPA, the single PACE cleavage mutant of CPA at amino acid 95 (CPA₉₅), PACE, or the dominant-negative form of PACE (PACE.SA) have all been described previously (10 , 12) . The endogenously activated mutant of CPA (CPA_ST3) and the cell surface-localized form of this mutant (CPA_DAF) were constructed by overlap PCR (15) using wild-type CPA and CPA_ST3 as template, respectively, and subcloned into the mammalian expression vector pZ (kindly provided by The Genetics Institute, Cambridge, MA). CPA_ST3 introduced the decapeptide sequence (-GLSARNRQKR-; Ref. 11 ) between the prodomain and mature domain of rat CPA (Fig. 1) , and CPA_DAF fused the 37 amino acid region (-PNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTM-GLLT-) from DAF to the COOH terminus of CPA_ST3 (Ref. 13 ; see Fig. 6a ). All plasmids were confirmed by sequencing at the University of Michigan DNA Sequencing Core.

View larger version (31K): [in this window] [in a new window]   Fig. 1. Mutations introduced into the CPA protein to facilitate subtilisin-like propeptidase cleavage. Wild-type CPA, CPA95, and CPAST3 are depicted in diagrammatic form, mutated residues are underlined, and the cleavage sites are indicated with an arrow.

View larger version (37K): [in this window] [in a new window]   Fig. 6. Expression of CPADAF results in cell surface-localized CPA without release into the conditioned medium. CPADAF was constructed by fusing the 37-amino acid glycophospholipid membrane anchoring domain from DAF to the COOH-terminus of CPA (A). 293T cells were mock transfected or transfected with CPAST3 or CPADAF, and cell extracts or conditioned media were then analyzed by immunoprecipitation, SDS-PAGE, and autoradiography after a 30-min pulse with [35S]cysteine/methionine or after a 30-min pulse, followed by a 5-h chase (B). SCCVII cells were infected with LacZ, CPAST3, or CPADAF and then analyzed for cell surface expression of CPA by flow cytometry (C).

Protein Analysis.
For experiments requiring metabolic labeling of proteins, [³⁵S]methionine/cysteine (Pro-mix; Amersham, Arlington Heights, IL) was used according to the protocols described previously (12) . Western blotting was performed as described previously (10) using a rabbit polyclonal anti-bovine CPA antisera (Cemicon, Temecula, CA), followed by enhanced chemiluminescence (Pierce, Rockford, IL).

CPA Purification and Enzymatic Assays.
CPA was purified from conditioned medium using CPA potato inhibitor affinity chromatography (10) or an -CPA immunoaffinity column. The -CPA affinity column was made according to the manufacturer’s instructions (Affi-Gel Hz; Bio-Rad, Hercules, CA) using a rabbit -bovine CPA antibody. Conditioned media were diluted 1:1 with PBS, loaded onto the column, washed (500 mM NaCl in PBS), and eluted (500 mM NaCl, 20 mM Glycine HCl, pH 2.0). Purified CPA was then dialyzed against 500 mM NaCl, 50 mM Tris-HCl (pH 8.0) and stored at 4°C. CPA activity was measured using a spectrophotometric assay for cleavage of a synthetic substrate, N-(3-[2-furyl]acryoyl)-Phe-Phe (Sigma Chemical Co., St. Louis, MO) as described previously (10) . Data were plotted, and kinetic constants were calculated by nonlinear regression using GraphPad Prism (GraphPad Software, San Jose, CA).

Retroviral Production and Infection.
The cDNAs for CPA_ST3 or CPA_DAF were subcloned into Lzrs.pBMN (kindly provided by Gary Nolan, Stanford, CA), yielding Lz.CPA_ST3 and Lz.CPA_DAF. To generate retroviruses coding for both CPA_ST3 or CPA_DAF and the neomycin resistance gene (neo^R) from one bicistronic retrovirus using an IRES, the entire CPA_ST3/IRES/neo^R or CPA_DAF/IRES/neo^R expression cassettes were amplified by PCR and subcloned into Lzrs.pBMN, yielding Lz.Neo.CPA_ST3 and Lz.Neo.CPA_DAF. Retroviruses were produced by transfecting the nX-ampho packaging cell line (kindly provided by Gary Nolan, Stanford, CA) using calcium phosphate precipitation, and 48 h after transfection, the producer cells were selected in 0.5 µg/ml puromycin (Sigma). Retroviral supernatants were generated by plating puromycin-selected producer cells at a density of 40,000 cells/cm² in 100-mm plates and culturing at 32°C for 4 days, with daily harvests. At this time, the supernatants were pooled, filtered through a 0.4 µm filter, aliquoted, and frozen at -70°C. Cells were infected using retroviral supernatants in the presence of Polybrene (16 µg/ml; Sigma). The titer of each reach retroviral batch was determined using SCCVII cells and G418 selection for CPA constructs and 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside staining for ß-galactosidase constructs. The titers achieved were 2.4 x 10⁶ ± 0.7 x 10⁶, 2.5 x 10⁶ ± 0.6 x 10⁶, and 1.1 x 10⁶ ± 0.3 x 10⁶ colony-forming units/ml for LacZ, CPA_ST3, and CPA_DAF respectively.

HPLC Analysis.
Tissue culture media were acidified with 1/10th volume 1 M HCl and extracted with -20°C CH₃CN (5:2 CH₃CN:media). Extracts were then analyzed on a C₁₈ reverse phase column (Waters, Milford, MA) by HPLC on a gradient from 10:90:0.1 to 50:90:0.1 CH₃CN:H₂O:trifluoroacetic acid at a flow rate of 1 ml/min over 15 min. HPLC was performed on a Waters gradient system composed of two model 501 pumps, a U6K injector module, and a model 996 photodiode array detector; the system was controlled by Millennium 2010 software. Absorbance was monitored at 315 nm, and under these conditions, MTX had a retention time of 6.4–6.6 min, and MTX-Phe had a retention time of 8.4–8.6 min.

Flow Cytometry.
To evaluate cell surface expression of CPA, SCCVII cells were infected with LacZ, CPA_ST3, or CPA_DAF retrovirus; 48 h later, they were detached from dishes using trypsin, and the trypsin was inactivated by the addition of serum. Cells were then incubated on ice for 30 min in medium supplemented with a 1:200 dilution of -CPA antibody. Subsequently, they were centrifuged through a 1/2 ml of FBS to isolate them from unbound antibody and then resuspended in medium supplemented with a 1:200 dilution of R-phycoerythrin conjugated goat -rabbit IgG secondary antibody (Fischer, Pittsburgh, PA). After 30 min, the unbound antibody was again removed, and the cells were resuspended in PBS for analysis at the University of Michigan Flow Cytometry Core.

Cytotoxicity Assays.
For some experiments, growth inhibition was assayed using the sulforhodamine B assay (Sigma; Ref. 16 ). Cells were plated at a density of 3000 cells/cm² in a 96-well plate; 12–18 h after plating, cells were infected with retroviral supernatants. Twenty-four h after infection, the medium was changed to that supplemented with vehicle (PBS), MTX, or MTX--peptides (17) . The cells were left cultured with the drug for 72 h, at which point they were fixed and stained according to Skehan et al. (16) . Data represent the mean ± SE of at least eight replicate wells. For other experiments, cells were assayed using a CFA (18) . Cells were plated in 60-mm dishes at a density of 2000 cells/cm²; 18–24 h later, they were infected with retroviral supernatants for 24 h, at which time the medium was changed to that supplemented with vehicle (PBS), MTX, or MTX-Phe. Cells were cultured with the drug for the indicated time and then plated for colony formation; and after 7–10 days, the dishes were fixed and stained with crystal violet before counting. Data plotted represent the mean and SE of at least three experiments.

CPA Diffusional Assay.
To measure the ability of secreted, soluble CPA to sensitize nontransduced cells, a two-chamber tissue culture plate was used. A 50/50 mixture of CPA_ST3- or CPA_DAF-expressing SCCVII cells and parental SCCVII cells (4000 total) were plated into the top wells of a six-well transwell plate (Costar Transwell-clear; Fischer, Pittsburgh, PA), and at the same time, an equal number (4000 cells) of parental SCCVII cells were plated into the bottom chambers. The top and bottom chambers were separated by a permeable membrane with 0.4 µM pores such that CPA or small molecules like MTX or MTX-Phe could freely move between the chambers, but whole cells were prohibited from crossing the barrier. The cells were left seeded in the chamber for 48 h, at which time MTX-Phe was added directly to the top and bottom chambers at a uniform concentration of 1 µM. Both the top and bottom chambers from parallel wells were subsequently trypsinized and plated for CFA at 12-h intervals.

	RESULTS

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CPA_ST3 Is Activated by Endogenous Prohormone Convertases.
To use CPA in a GDEPT strategy for cancer therapy, we constructed a mutant that is expressed as an active enzyme in the absence of trypsin-dependent propeptide cleavage. Previously, we reported that CPA₉₅, a mutant into which a PC cleavage site was introduced by two amino acid substitutions (FQAR RQKR), is activated but only in the presence of PACE overexpression (Fig. 1 ; Ref. 10 ). We, therefore, constructed CPA_ST3, which includes a 10-amino acid linker region (-GLSARNRQKR-) between the propeptide and mature domain of CPA (Fig. 1) , where the underlined amino acids represent a prototypical PACE/furin cleavage site. This linker region is derived from the matrix-metalloprotease ST3, where it has been demonstrated to sensitize ST3 to PC-dependent activation (11) .

To examine the expression and processing of CPA_ST3, 293T cells were transiently transfected with the expression plasmids for wild-type CPA, CPA₉₅, or CPA_ST3 in the absence or presence of a PACE expression plasmid. Conditioned media were collected from transfected cells and analyzed by SDS-PAGE and Western blot. Expression of wild-type CPA alone as well as with cotransfected PACE resulted in a M_r 43,000 protein, which is characteristic of pro-CPA (Fig. 2 , Lanes 2 and 3). When CPA₉₅ was expressed in 293T cells, there was a small amount of mature CPA generated, as evidenced by the band at M_r 34,000, yet the majority of the protein was still in the pro- form (Fig. 2 , Lane 4). However, in the presence of PACE cotransfection, >50% of CPA₉₅ was processed to the mature form, while the rest remained in the larger pro- form (Fig. 2 , Lane 5). These results are consistent with previous observations for CPA₉₅ when expressed in COS-1 cells and in squamous cell carcinoma lines (10) .

View larger version (48K): [in this window] [in a new window]   Fig. 2. Expression of CPAST3 results in mature CPA in the absence of PACE coexpression. Expression plasmids for wild-type, CPA95, and CPAST3 constructs were transfected into 293T cells either alone or in cotransfection with PACE or dominant-negative PACE (PACE. SA). A total of 5 µg of the different CPA expression plasmids were used in each condition supplement with 5 µg of the PACE constructs or empty vector. Forty-eight h after transfection, conditioned media were harvested and analyzed by SDS-PAGE and Western blot analysis using a CPA-specific polyclonal antibody. Arrows, pro and mature forms of CPA. As a control, 293T cells were transfected in the absence of DNA and analyzed as above (Mock).

Endogenously Activated CPA_ST3 Is Indistinguishable from Trypsin-activated Wild-Type CPA.
293T cells were transiently transfected with the CPA_ST3 expression plasmid, and the protein was purified from the conditioned medium using an anti-CPA immunoaffinity column. Enzymatic analysis using a synthetic substrate, N-(3-[2-furyl]acryoyl)-Phe-Phe (1 x 10^-5 M to 5 x 10^-4 M), demonstrated that endogenously activated CPA_ST3 had a similar kinetic profile to trypsin-activated wild-type CPA over the range of substrate concentrations studied with K_m and kcat values, which were virtually identical (Table 1) . In addition, conditioned medium from 293T cells transiently transfected with CPA_ST3 was submitted to the University of Michigan Protein Structure Core for electrophoresis and NH₂-terminal sequencing. Ten consecutive amino acids were identified (-ALSTDSFNYA-), which correspond to the first 10 amino acids of mature rat CPA that were immediately COOH-terminal of the PC cleavage site introduced via the ST3 linker region (see Fig. 1 ; Ref. 19 ).

View larger version (28K): [in this window] [in a new window]   Fig. 4. Retroviral infection with CPAST3 results in secretion of mature CPA and sensitization to MTX-Phe. The supernatants from Lzrs.LacZ or Lzrs.CPAST3 retroviral producer cells were analyzed by SDS-PAGE and Western blotting for CPA expression (A, leftmost panel). Three tumor cell lines (SCCVII, MCF7, and UMSCC6) were infected with LacZ or CPAST3 retrovirus, metabolically labeled with [35S]methionine/cysteine, and conditioned media were analyzed by immunoprecipitation, SDS-PAGE, and autoradiography (A, right three panels). Arrows, pro and mature forms of CPA. In parallel cultures, LacZ or CPAST3-infected cells were treated with MTX-Phe for 48 h and plated for surviving fraction (B). Bars, SE.

View larger version (18K): [in this window] [in a new window]   Fig. 3. CPAST3-expressing cells convert MTX-Phe to MTX and are sensitized to the prodrug in a time-dependent manner. SCCVII cells expressing LacZ or CPAST3 were exposed to MTX or MTX-Phe for 0–72 h and then evaluated by CFA (A), and HPLC analysis was performed on the condition media at the time of plating (B). LacZ-expressing cells treated with MTX () or MTX-Phe () and CPA ST3-expressing cells exposed to MTX-Phe (•) are shown.

Retroviral Transduction of Tumor Cell Lines Leads to Production of Mature CPA and Sensitivity to MTX-Phe.
Because the use of stable cell lines may not accurately represent a gene therapy strategy, a CPA_ST3 retrovirus was produced by subcloning the CPA_ST3 cDNA into the Laz.pBMN expression plasmid and transfecting the Nxa retroviral producer line. A Western blot of the conditioned medium from this retroviral producer line indicated that these cells secrete mature CPA_ST3 (Fig. 4a) . Three tumor cell lines, SCCVII murine squamous cell carcinoma, UMSCC6 human squamous cell carcinoma, and MCF7 human breast carcinoma, were infected with either CPA_ST3- or LacZ-expressing retrovirus. LacZ infection, followed by ß-galactosidase staining, revealed that 50% of SCCVII cells were infected, whereas for the other two cell lines, the infection rate was 25–30% (data not shown). Forty-eight h after infection, the cells were labeled with [³⁵S]methionine/cysteine for 30 min, followed by a 4-h chase, and the conditioned media were immunoprecipitated using an -CPA antibody and then analyzed by SDS-PAGE and autoradiography. Cells infected with LacZ virus did not produce any detectable CPA; however, cells infected with the CPA_ST3 retrovirus produced CPA that was predominantly in the mature form, as evidenced by the band at M_r 34,000 (Fig. 4a) . In addition, in a parallel series of plates, infected cells were exposed to 1 µM MTX-Phe for 72 h before plating to analyze their SF. For all three lines tested, LacZ-infected cells were resistant to MTX-Phe (SF >0.75), whereas CPA_ST3-infected cells were potently sensitized to the prodrug (SF of 0.01 to 0.00 liter).

CPA_ST3-expressing Cells Are Sensitized to MTX-Phe in a Dose-dependent Manner.
More detailed studies on prodrug activation by CPA_ST3 were performed using SCCVII cells at a range of MTX and MTX-Phe concentrations from 1 to 1000 nM. Both LacZ- and CPA_ST3-expressing cells were sensitive to MTX exhibiting a SF of 0.001 at 1000 nM MTX and IC₅₀ and IC₉₅ values of 1 and 25 nM, respectively (Fig. 5 and Table 2 ). There was no toxicity to LacZ-infected cultures, even when exposed to 1000 nM MTX-Phe (Fig. 5 and Table 2 ). However, infection of SCCVII cells with CPA_ST3 retrovirus, followed by exposure to MTX-Phe, resulted in cytotoxicity in a dose-dependent manner that paralleled that of MTX exhibiting a SF of about 0.001 at 1000 nM MTX-Phe and IC₅₀ and IC₉₅ values of 2.5 and 35 nM, respectively (Fig. 5 and Table 2 ).

View larger version (20K): [in this window] [in a new window]   Fig. 5. Retroviral infection with CPAST3 sensitizes cells to MTX-Phe in a dose-dependent manner. SCCVII cells were infected with LacZ or CPAST3 retrovirus and then treated with increasing doses of MTX or MTX-Phe for 48 h prior to plating for SF. CPAST3-infected cells treated with MTX (), LacZ-infected cells treated with MTX-Phe (), and CPA ST3-infected cells treated with MTX-Phe (•) are shown. Bars, SE.

Both the soluble (CPA_ST3) and the cell-surface tethered form (CPA_DAF) of CPA were transiently expressed in 293T cells; 48 h after transfection, the cells were labeled with [³⁵S]methionine/cysteine for 10 min, and cell extracts were collected. In parallel plates, labeled cells were chased in serum-free medium for 5 h prior to the collection of both cell extracts and conditioned medium. Cell lysates from both the early and late time points were then lysed in a Dounce homogenizer and submitted to a 100,000 x g spin to precipitate cellular membranes (S100). The S100 fractions as well as the conditioned medium were analyzed by immunoprecipitation, followed by SDS-PAGE and autoradiography. CPA_ST3 was initially synthesized as a M_r 43,000 pro-form and subsequently converted to the M_r 34,000 mature form and secreted from the cell such that after a 5-h chase, it accumulated in the conditioned medium and was no longer detectable in the cell extract (Fig. 6b) . In contrast, CPA_DAF was initially synthesized as a M_r 48,000 form and then converted to a M_r 38,000 form, which remained cell associated and was undetectable in the conditioned medium (Fig. 6b) , thus indicating that CPA_DAF is cell associated whereas CPA_ST3 is secreted.

In a separate experiment, 293T cells were mock-transfected or transfected with either CPA_ST3 or CPA_DAF. Conditioned medium and S100 fractions were collected from the transfected plates and assayed for CPA activity. The S100 cell pellet from mock or CPA_ST3-transfected cells had little or no catalytic activity, whereas the S100 fraction from CPA_DAF transfected cells rapidly cleaved the synthetic substrate (data not shown). Finally, unlike CPA_ST3 which contained significant catalytic activity in the conditioned medium, such activity was undetectable in the conditioned medium derived from mock or CPA_DAF-transfected cells (data not shown).

To further verify that CPA_DAF was expressed on the cell surface, a CPA_DAF retrovirus was generated by subcloning the cDNA for CPA_DAF into Laz.pBMN. SCCVII cells were infected with LacZ, CPA_ST3, or CPA_DAF virus, and 48 h after infection, they were analyzed for cell surface expression of CPA by flow cytometry. Cells infected with CPA_DAF had a >100-fold increase in staining using an anti-CPA antibody when compared with LacZ- or CPA_ST3-infected cells, thus demonstrating that not only is CPA_DAF cell associated, but it is also cell-surface exposed (Fig. 6c) .

To evaluate whether the CPA_DAF molecule retained a substrate specificity similar to the native enzyme, we performed sulforhodamine B growth inhibition assays using five different MTX--peptides (17) to compare the substrate specificity of CPA_ST3 and CPA_DAF, as measured by their ability to sensitize cells to these prodrugs using the sulforhodamine B growth inhibition assay (Table 3) . Both CPA_ST3 and CPA_DAF showed a preference for the large aromatic side chain prodrugs, with MTX-Phe being the best used substrate, followed by MTX-Tyr. Both forms of CPA had slight activity against MTX-Met with little or no activity versus MTX-Gln and MTX-Trp. Although the trend of substrate specificity was consistent between CPA_ST3 and CPA_DAF, the absolute level of activity varied with CPA_ST3 consistently having greater activity than CPA_DAF; however, this difference may not reflect actual differences in activity and instead most likely is an indication of different titers of retroviruses (see below).

View this table: [in this window] [in a new window]   Table 3 Retroviral transduction with CPAST3 or CPADAF sensitizes cells to MTX--peptide prodrugs The IC50s of five MTX--peptide prodrugs were evaluated using a 96-well plate growth inhibition assay after retroviral transduction of SCCVII cells with LacZ, CPAST3, or CPADAF retrovirus. Data represent the average of at least eight replicate wells.

View larger version (20K): [in this window] [in a new window]   Fig. 7. CPAST3- and CPADAF-expressing cells both exhibit a potent bystander effect. SCCVII cells were infected with increasing dilutions of CPAST3 or CPADAF retrovirus and plated for colony formation in the absence or presence of G418 to determine percentage of infected cells. In parallel plates, CPAST3 () or CPADAF () infected cells were treated with MTX-Phe for 48 h before plating for SF. Bars, SE.

CPA_DAF Partially Protects from Collateral Cytotoxicity.
Finally, to determine whether the release of secreted CPA_ST3 would sensitize cells distant from the site of production, a unique coculture assay was developed to measure the impact of the diffusion of CPA_ST3 on the cytotoxicity of nontransduced cells located some distance from the CPA-expressing cells. SCCVII cells that were 50% CPA_ST3 or 50% CPA_DAF expressing were plated in the top chambers of a two-chamber, six-well tissue culture plate, and an equal number of nontransduced SCCVII cells were plated in the bottom chamber. The membrane dividing the two chambers had a 0.4 µm pore size, which was small enough to prohibit the passage of whole cells between the chambers; however, released CPA_ST3 and both MTX-Phe and MTX would readily diffuse between the chambers. The seeded cells were left in culture for 48 h, at which point MTX-Phe was added to the medium in both the top and bottom chambers to a final concentration of 1 µM, and the cells were plated for CFA at 12-h intervals.

Consistent with previous results, nontransduced parental cells exhibited no toxicity when exposed to the prodrug (Fig. 8) . However, for CPA_ST3- and CPA_DAF-expressing cultures, cytotoxicity was observed for both the transduced wells (the top chamber) and the bystander cells (the bottom chamber). The toxicity appeared first in the top chambers and increased with the time of exposure to the prodrug. The level and rate of cytotoxicity in the top chambers was similar between CPA_ST3 and CPA_DAF cultures throughout the course of the experiment (Fig. 8) . However, there was a difference in collateral cytotoxicity to cells in the lower chambers, with cytotoxicity apparent earlier and to a greater extent in CPA_ST3 cultures than in CPA_DAF cultures (Fig. 8) . Therefore, the anchoring of CPA_DAF to the surface of the cell conferred potent sensitization to both CPA_DAF-expressing and bystander cells in close proximity to each other (those in the top chamber) in a manner similar to CPA_ST3-expressing cells. Yet, although there was collateral cytotoxicity to cells some distance from the CPA_DAF-expressing cells (those in the bottom chamber), this cytotoxicity appeared later and to a lesser extent than that seen for CPA_ST3-expressing cells, where the enzyme could diffuse into the lower chamber and generate MTX in situ.

View larger version (18K): [in this window] [in a new window]   Fig. 8. CPADAF expression sensitizes infected and "bystander cells" to MTX-Phe while exhibiting reduced collateral toxicity. CPAST3- or CPADAF-expressing SCCVII cells were mixed at a 50:50 ratio with parental SCCVII cells and plated in the top of a dual chamber tissue culture plate. In the bottom chamber, an equal number of parental SCCVII cells were plated. Both chambers were then treated with 1 µM MTX-Phe, and parallel wells were plated for SF at 12-h intervals. Parental cells alone exposed to MTX-Phe (), cells from the top culture (, •), or cells from the bottom culture (, ) are shown. A, CPAST3 cocultures (, ). Bars, SE. B, CPADAF cocultures (, •). Bars, SE.

	DISCUSSION

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In addition, for both the HSV-TK/ganciclovir and the CD/5-FC systems, it has been demonstrated that the "factory" or transduced cells are killed earlier and at lower doses of prodrug than nontransduced cells because of the intracellular accumulation of the toxic metabolites (22, 23, 24, 25, 26) . In the case of HSV-TK/ganciclovir, this results in very little cytotoxicity when <50% of the cells in culture are expressing HSV-TK. Because of the fact that 5-fluorouracil is membrane permeable, there is a greater bystander effect for the CD/5-FC system. Unfortunately, we still have witnessed limited cytotoxicity with a low percentage of CD expressing cells both in culture (25) and in animal models (26) . In the GDEPT reported here, extracellular production of MTX by soluble or cell surface CPA should equally sensitize both transduced and bystander cells with minimal preferential cytotoxicity.

One additional advantage of this system is that because MTX is one of the most widely used agents in the treatment of solid tumors, its pharmacokinetics, dose-limiting toxicities, and mechanisms of resistance are well understood (3 , 4) . HD-MTX therapy has been suggested as a means to circumvent tumor-derived resistance to MTX and is used quite commonly in current oncological practice. Recently, other strategies have been developed to genetically modify bone marrow stem cells to make them resistant to MTX so that HD-MTX treatment could be undertaken while biochemically protecting the bone marrow. These protective strategies have been demonstrated both for human bone marrow in culture (27) and for the protection of mice from MTX toxicity after transplantation of MTX-resistant bone marrow (28) . Although these strategies have provided promising results, they do not offer any protection to the gastrointestinal tract, which is also highly sensitive to MTX-induced toxicity. The GDEPT proposed here by localizing HD-MTX to the tumor site may be able to increase the cytotoxic dose delivered to the tumor while protecting both the gastrointestinal tract and the bone marrow. An additional advantage for the use of CPA in an enzyme/prodrug strategy is that other antifolates, which have proven cytotoxic even in MTX-resistant cell lines (29) , also rely upon transport through the reduced folate carrier. Therefore, these drugs could be converted to -peptide blocked prodrugs to be used in this enzyme/prodrug strategy (30 , 31) .

To render CPA active in the absence of trypsin-dependent zymogen cleavage, the 10-amino acid linker region from ST3 was incorporated into CPA between the pro- and mature domains such that if cleavage occurred at the expected site, the mature peptide released would be identical to trypsin-activated CPA. Enzymatic analysis of purified CPA_ST3 confirmed the correct activation of the zymogen to the catalytic form for its kinetic profile was indistinguishable from the trypsin-activated CPA. NH₂-terminal sequencing of endogenously activated CPA _ST3 also revealed that cleavage occurred at the expected location, liberating a mature peptide that is identical to the native mature protein. This is the first direct biochemical demonstration of the cleavage site for the ST3 linker region; the work that identified this cleavage domain was based upon site-directed mutagenesis to indirectly ascertain which site was cleaved (11) .

CPA_ST3-transduced squamous cell carcinoma cells were able to generate MTX from MTX-Phe. Further evidence for the specificity of this activation was demonstrated by the inhibition of the conversion by the carboxypeptidase inhibitor derived from potatoes (data not shown). MTX was first detected 12 h after exposure to the prodrug, and by 72 h, >200 nM MTX was generated. Although this only amounted to a 20% conversion of MTX-Phe to MTX, the amount of MTX generated was still almost 10 times higher than the IC₉₅ of MTX in this cell line and thus was more than sufficient to cause potent cytotoxicity (see Table 2 ). These data are consistent with the notion that the MTX generated within the first 24 h inhibited cellular growth and the further production of CPA and, therefore, limited the final conversion of MTX-Phe to MTX. However, this MTX-mediated inhibition of further CPA production has not been proven.

Having ascertained that secreted active CPA_ST3 could indeed sensitize a number of different tumor cell lines to MTX-Phe after retroviral infection, we next developed a cell surface-associated form of endogenously active CPA. Unlike previous reports where carboxypeptidase G2 was still highly active both in vitro and in vivo when fused to the transmembrane domain of a cell surface receptor (32) , we were unable to detect any functional CPA after construction of a similar fusion protein with CPA (data not shown). However, the use of the phospholipid membrane anchor from DAF resulted in a CPA molecule that not only remained cell tethered but was also functional, perhaps because of the increased conformational flexibility allowed by the lipid linkage as compared with a more rigid peptide transmembrane domain. This molecule, CPA_DAF, was able to sensitize SCCVII cells to MTX-Phe in a manner similar to the secreted form, and it appears to have retained the substrate specificity of the native molecule.

CPA_DAF also sensitized SCCVII cells to MTX-Phe when only a very small fraction of cells (5%) were expressing the protein. This may be attributable to the fact that time is not necessary for the molecule to build-up in the culture medium, because CPA_DAF is anchored to the surface of the cell and does not diffuse away, and thus it is not removed when the culture medium is changed. The heightened cytotoxicity of CPA_DAF when compared with CPA_ST3 also may be attributable to the local production of MTX at the cell surface, thus requiring lower total conversion levels of MTX-Phe to MTX to sensitize cells. This theory has been suggested for ADEPT protocols using CPA. Kuefner et al. (5) determined that when using antibody-conjugated CPA localized to the surface of the cell, 100-fold less enzyme was required to achieve equal sensitization as that found when purified CPA was simply added to the culture medium. They attributed this enhanced cytotoxicity to the production of MTX within the microenvironment around cell surface-localized CPA-antibody conjugates. In addition, by prohibiting the diffusion of the catalytically active enzyme away from transduced cells, CPA_DAF also partially inhibited collateral toxicity to cells more distant from the site of enzyme production than that seen for CPA_ST3, a fact that may be more readily apparent and more critical when this strategy is evaluated in vivo.

The studies reported here have focused on MTX-Phe, because it was identified as the best substrate for wild-type CPA (17) . However, it has recently been demonstrated that this compound, unlike predictions, is not stable in vivo for it is rapidly converted to MTX after injection into mice, which prohibits direct evaluation of this GDEPT in an animal model (8) . To overcome the unsuitability of MTX-Phe, modified MTX--peptide prodrugs with nonnatural amino acid blocking groups have been described that are poor substrates for endogenous systemic CPA-like activities and are thus highly stable in vivo. For example, the MTX-3-cyclopentyl-Tyr prodrug is 50,000-fold more stable in the presence of wild-type CPA than MTX-Phe (8) . In addition, although these compounds are poor substrates for wild-type CPA, they are efficiently cleaved by the T268G mutant of CPA, which has an alteration in the substrate binding pocket. The efficacy of the endogenously active soluble (CPA_ST3) and cell surface forms of CPA (CPA_DAF) when combined with the T268G altered specificity form of the enzyme are now being evaluated. Preliminary evidence, which is in accordance with Smith et al. (8) , suggests that expression of this T268G mutant in culture confers sensitization to MTX-3-cyclopentyl-Tyr, whereas expression of the wild-type enzyme has no such capacity.

Recently, studies using the T268G mutant of human CPA in an ADEPT protocol were unable to demonstrate a clinical response because of the rapid inactivation of CPA in vivo (31) . To allow time for distribution, binding, and subsequent clearance of unbound CPA/antibody conjugates, the authors waited 24 h after injection before they initiated prodrug treatment. However, the half-life of the enzyme/antibody conjugate in vivo was found to be significantly less than this, and as a result, there was little conversion of MTX-prodrugs. The CPA GDEPT strategy described here may circumvent this limitation through the continuous production of CPA by virally transduced cells, thus limiting the impact of protein inactivation. Because of the clinical efficacy of MTX in the treatment of squamous cell cancer of the head and neck and to the difficulty in achieving local control of head and neck cancer (33) , future studies will be focused on the use of this GDEPT in the treatment of head and neck cancer by direct injection of CPA_ST3- or CPA_DAF-expressing adenoviruses into submental tumors in an animal model of head and neck cancer (26 , 34 , 35) .

	ACKNOWLEDGMENTS

 
We thank Leo Ostruszka and Donna Shewach for assistance with HPLC analysis of MTX and MTX-Phe. We also thank Amy Pace for excellent help with graphic arts.

	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 Supported in part by NIH Award 1R29CA7390401 (to A. R.), USAMRC Breast Cancer Research Pre-Doctoral Fellowship DAMD17-97-1-7127 (to D. H.), as well as a Developmental Student Award (to D. H.) as part of the UM Specialized Programs of Research Excellence in Prostate Cancer P50 CA69568. D. H. is a fellow in the Medical Scientist Training Program.

² To whom requests for reprints should be addressed, at The University of Michigan Medical School, 1331 East Ann Street, Ann Arbor, MI 48105-0582. Phone: (734) 764-4209; Fax: (734) 763-1581; E-mail: alnawaz{at}umich.edu

³ The abbreviations used are: GDEPT, gene-dependent enzyme/prodrug therapy; MTX, methotrexate; MTX-Phe, methotrexate--phenylalanine; CPA, carboxypeptidase A1; ADEPT, antibody-dependent enzyme/prodrug therapy; PACE, paired basic amino acid cleaving enzyme; PC, prohormone convertase; HPLC, high-performance liquid chromatography; HSV-TK, herpes simplex virus thymidine kinase; CFA, colony formation assay; SF, surviving fraction; IRES, internal ribosomal entry site; ST3, stromelysin 3; DAF, decay accelerating factor; 5-FC, 5-flucytosine; HD, high dose.

Received 3/31/99. Accepted 12/ 2/99.

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Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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