INTRODUCTION

A current major challenge to cancer therapy is to increase antitumor selectivity. One approach to realizing this goal is to use the exquisite selectivity of the antibody:antigen reaction to target therapeutic entities specifically to tumors. While absolutely tumor-specific antibodies are not known, many antibodies are available that can deliver tumor-selective targeting (for review, see Ref. 1).

One investigational therapy that makes use of this principle of antibody targeting is antibody-directed enzyme prodrug therapy (ADEPT).¹ ADEPT is a powerful strategy with the potential for tumor-specific long-term delivery of chemotherapy (2-7). The premise of ADEPT is to target an enzyme of interest specifically to tumor cells by coupling it to a tumor-specific antibody. This conjugate is delivered to the patient systemically and then allowed to bind to the antigen-expressing target cells. Unbound conjugate is allowed to clear from circulation, and when the circulating levels of conjugate are sufficiently low, a prodrug is administered, also systemically, that can be converted to a toxic chemotherapeutic drug by the targeted enzyme-antibody conjugate. The action of the enzyme-antibody conjugate on the prodrug then ideally generates lethal levels of drug specifically at the tumor site. For this therapy to be selective, however, nonspecific activation of the prodrug at sites distant to the tumor must be minimized. This is generally accomplished by using a conjugate enzyme with an activity not endogenous to the host or at least not accessible to the prodrug (2-29).

A number of ADEPT strategies have been reported (2-29). The concept has been shown to be effective both in in vitro and in vivo models, and at least one ADEPT strategy is currently undergoing clinical evaluation (27-29). ADEPT in vitro efficacy has been demonstrated with enzyme-antibody conjugates of (a) carboxypeptidase G2 along with several nitrogen mustards (14-16), (b) alkaline phosphatase with phosphorylated prodrugs of mitomycin, a phenol mustard, and etoposide (3, 10, 11), (c) -lactamase with lactam prodrugs of doxorubicin, vinca alkaloid analogs, and a nitrogen mustard (7, 12, 13, 17, 18), (d) penicillin-G amidase with prodrugs of palytoxin, doxorubicin and melphalan (8, 9), (e) penicillin-V amidase with a prodrug of doxorubicin (2, 7), (f) human or Escherichia coli -glucuronidase with glucuronide prodrugs of epirubicin, doxorubicin and a nitrogen mustard (22-24), (g) cytosine deaminase and 5-fluorocytosine (25, 26), and (h) bovine carboxypeptidase A and -amino acid prodrugs of MTX (19-21). Further, in vivo antitumor efficacy has been shown in a number of systems using the enzymes alkaline phosphatase, carboxypeptidase G2, -lactamase, and -glucuronidase (2, 3, 7, 10-13, 15-18, 24).

An inherent problem with antibody-targeted therapies is the immune response mounted by the host to the foreign proteins and other antigens used in the therapy (1, 28). For example, monoclonal antibodies used in antibody targeting-based therapies are in general rodent in origin and as such recognized by the immune system (1, 28). ADEPT has the additional problem that the enzyme used to generate the site-specific drug synthesis can also be immunogenic, especially when a foreign enzyme is used. The immunogenicity associated with these foreign antibody or enzyme proteins decreases the utility of the antibody-targeting strategies by decreasing the ability of the physician to perform multiple dosing regimens.

Attempts are being made to overcome the immune response to the rodent antibodies through "humanization" of the antibodies (30). In this strategy, much of the sequence of the mouse monoclonal antibody is replaced with corresponding human antibody sequence. Only selected residues at the antigen combining site are left intact, leaving relatively few "rodent residues" remaining in the antibody.

We reasoned that the imunogenicity associated with the use of enzymes of nonhuman origin might be circumvented through a similar strategy. However, we chose not to precisely follow the strategy of antibody humanization, which commences the process with the binding site of a foreign protein. Rather, our approach to generate a composite human/nonhuman enzyme was to start with a fully human enzyme and change the "active site" at one or two residues to produce a >99.5% human enzyme capable of efficiently performing a non-human reaction. This human enzyme with non-human specificity, along with humanized antibodies, should then facilitate the production of enzyme:antibody conjugates having lower immunogenicity and benefit the development of multiple dosing regimen ADEPT strategies.

Our initial target to generate a human enzyme with non-human specificity was human pancreatic carboxypeptidase A, recently cloned and expressed in our laboratory (31). Pancreatic carboxypeptidase A is a zinc-containing exopeptidase released into the small intestine from the pancreas as a zymogen (32, 33). The pancreatic CPA has two further subclasses, CPA1 and CPA2, in both humans (31, 34, 35) and rats (36, 37). While the amino acid sequences of CPA1 and CPA2 active sites are similar and both enzymes prefer aromatic C-terminal amino acids, CPA2 enzyme prefers bulkier aromatic C-terminal amino acids (31, 37). This was shown for the rat enzyme with di- and tripeptide substrates and for the human with amino acid prodrugs of MTX (31, 37). High resolution crystal structures for bCPA have been determined (32). Of the nine active site residues within 4.5 Å of the bound substrate, only three vary among bovine CPA (32), rat CPA1 (36), rCPA2 (37), hCPA1 (31, 34), and hCPA2 (31, 35). These changes, at residues 253, 254, and 268, describe a larger binding pocket for CPA2 than CPA1 and provide a rationale for the substrate specificities.

Huennekens and co-workers (19-21) developed an in vitro ADEPT approach using MTX prodrugs in which MTX was modified at the -carbon of the glutamate moiety with one of several natural amino acids. These prodrugs were relatively nontoxic in cell culture but could be activated by bovine CPA or carboxypeptidase B to form MTX. The most successful of these prodrugs was MTX-Phe, which was found to be an excellent substrate for bovine CPA (21). This system was effective under cell culture conditions where the IC₅₀ of MTX-Phe against L1210 cells changed from 2.2 × 10⁶ to 6.3 × 10⁸ M, indistinguishable from MTX itself, in the presence of bovine CPA or a bovine CPA-antibody conjugate (21). An important positive aspect of this system is its use of MTX with its well known efficacy and toxicity profile (38, 39). Thus, MTX maximum tolerated doses are due to well understood gut and bone marrow toxicities. Therefore, specific generation of high concentrations of MTX at tumor sites distal from these sites of known toxicity should not have major side effects. Two potential limitations to the use of the bCPA/MTX-Phe system in humans, however, are the possible background activation of prodrug by endogenous hCPA to liberate MTX systemically and the immune response elicited by the bovine protein.

We sought to improve upon the MTX-Phe/bCPA system in two ways. First, we sought to use the human rather than bovine CPA. Second, we sought to change the catalytic specificity of the human enzyme(s) to accommodate MTX prodrugs that are not substrates for the endogenous wild type carboxypeptidases and would therefore be expected to be stable in vivo. The strategy chosen to accomplish this goal exploited parallel computer-aided design of novel active sites and of new MTX--amino acid prodrugs. Specifically, wild type hCPA and novel hCPA mutant active sites were designed using protein homology model building from the well known high resolution crystal structure of bCPA (40). Then these computer-generated active site mutants and similarly generated modified substrates were evaluated together and compared with wild type enzyme-substrate complexes. Favorable mutants and modified substrates were prepared and tested in vitro and in vivo. We report here MTX prodrugs that are stable in vivo, a one-amino acid mutant of hCPA1 that can efficiently use these in vivo stable prodrugs, and the use of these prodrugs along with a mutant hCPA enzyme-antibody conjugate for antigen-specific cytotoxicity in vitro. The work reported demonstrates proof of principle for the methodology for developing a very efficient mutant human enzyme/prodrug combination for use in ADEPT.

EXPERIMENTAL PROCEDURES

Materials

Human CPAs were obtained as described previously (31). Cell lines were obtained from ATCC (Rockville, MD) and grown in 90% RPMI 1640, 10% fetal calf serum at 37 °C under 5% CO₂. HT-29 cells for in vitro ADEPT experiments in human serum were taken from this medium and grown for 3 weeks in 95% RPMI 1640, 5% human serum at 37 °C under 5% CO₂. Growth rates and MTX cytotoxicity were similar under the two conditions.

Methods

The synthesis of the MTX prodrugs shown in Table I is described elsewhere (40).

Table I. Structures of MTX--R prodrugs Compound  Ra Abbreviation GW1311 Phenylalanine MTX-Phe Negatively charged prodrugs   GW2310 Glutamate MTX-Glu   GW3347 Aspartate MTX-Asp   GW3855 2-Carboxyphenylalanine MTX-2-carboxy-Phe   GW3199 3-Carboxytyrosine MTX-3-carboxy-Tyr   GW4694 3-Carboxyphenylalanine MTX-3-carboxy-Phe Bulky aromatic prodrugs (phenylalanine-based)   GW4160 2-Iodophenylalanine MTX-2-iodo-Phe   GW1667 1-Naphthylalanine MTX-naphthyl-Ala   GW250 2-Cyclopentylphenylalanine MTX-2-cyclopentyl-Phe   GW1442 2-Cyclohexylphenylalanine MTX-2-cyclohexyl-Phe   GW3352 3-Cyclobutylphenylalanine MTX-3-cyclobutyl-Phe   GW1834 3-t-Butylphenylalanine MTX-3-t-butyl-Phe   GW637 3-Cyclopentylphenylalanine MTX-3-cyclopentyl-Phe   GW827 3-(3-n-Pentyl)phenylalanine MTX-3-n-pentyl-Phe Bulky aromatic prodrugs (tyrosine-based)   GW1867 3,5-Diiodotyrosine MTX-3,5-diiodo-Tyr   GW2159 2-Cyclopentyltyrosine MTX-2-cyclopentyl-Tyr   GW5798 3-Cyclobutyltyrosine MTX-3-cyclobutyl-Tyr   GW3335 3-t-Butyltyrosine MTX-3-t-butyl-Tyr   GW5755 3-Cyclopentyltyrosine MTX-3-cyclopentyl-Tyr a All R-groups are L-amino acids attached to the -carboxyl of MTX via a standard peptide bond.

pMP36HCPA1 (31) containing pro-hCPA1-WT cDNA (as a fusion with yeast  factor leader, described below) was restricted with NcoI and SalI to liberate a 481-base pair cDNA fragment. This fragment begins at nucleotide 893, proceeds to the 3-end of the hCPA1 cDNA, and encodes amino acids 186-309 of mature hCPA1.² The CPA1 NcoI-SalI fragment was ligated into the NcoI and SalI cloning sites of pGEM5zf() (Promega) to generate pHCPAINS. pHCPAINS was restricted with SphI and SalI to liberate the hCPA1 NcoI-SalI fragment, and an additional nine base pairs of pGEM5zf() sequence. This SphI-SalI fragment was cloned into M13mp19 (Life Technologies, Inc.) using its SphI and SalI cloning sites to generate M13mp19HCPA1.

Single-stranded M13mp19HCPA1 DNA was used as template for oligonucleotide-directed mutagenesis using the T7-GEN in vitro mutagenesis kit (U.S. Biochemical Corp.). The following mutagenic oligonucleotide primers (Oligos Etc.) were used to mutate residues Ile²⁵⁵ (AAT) and Thr²⁶⁸ (ACC) either separately or in tandem (mutagenic codons underlined): I255A, 5-ggT CCA gTC AgC AgT gCT TCC-3 (Ala = gCT); T268A, 5-gAg CTC gAA ggC gAA ggA gTA-3 (Ala = gCC); T268G, 5-gAg CTC gAA gCC gAA ggA gTA-3 (Gly = ggC). Using these oligonucleotide primers, the following hCPA1 mutants were formed: T268A, T268G, I255A, and I255A/T268G. Each of the mutagenized hCPA1 cassettes was sequenced to verify that only the desired DNA mutations were produced.

Expression of hCPA enzymes in Saccharomyces cerevisiae was performed according to the strategy of Gardell et al. (43) as described by Laethem et al. (31). The cDNAs for pro-hCPA1 or pro-hCPA2 were cloned into the pMP36 vector and fused in frame to the yeast  factor leader of this vector using a polymerase chain reaction approach (44).

pMP36HCPA1 was restricted with NcoI to liberate a 1.2-kilobase pair fragment that was cloned into the NcoI site of the M13mp19HCPA1 mutants. The correct orientation of the NcoI fragment within M13mp19pro-hCPA1 mutants was verified, and the DNAs were restricted with HindIII and SalI liberating a 1.2-kilobase pair cDNA encoding the entire pro-hCPA1 mutant enzyme. This fragment was ligated into the HindIII and SalI sites of pMP36 yielding pMPHCPA1 mutants. These DNAs were restricted sequentially with BamHI, SalI, and SspI with intervening purifications by either phenol extractions or use of Promega Magic Mini Columns with manufacturer supplied procedures. Following SspI restriction, the 2.8-kilobase pair band, containing hCPA1 mutant with the yeast  factor leader in frame, was gel-purified from a 1% low melting agarose gel. The BamHI-SalI fragment was ligated into the pBS24.1 shuttle vector overnight at 16 °C. This vector (pBSHCPA1-mutant) was electroporated into DH5, and plasmid DNA was isolated using the Wizard Miniprep Kit (Promega).

Approximately 500-2000 ng of pBSHCPA1 mutant DNA was electroporated into 40 µl of electrocompetent DLM101 S. cerevisiae. One ml of 1 M sorbitol was added immediately after electroporation to rescue the cells. 100-µl samples were plated out on dishes of yeast nitrogen broth-uracil selection medium and were incubated at 30 °C for 2-3 days (31). Positive colonies were picked and grown in yeast nitrogen broth-leucine selection medium containing 8% glucose at 30 °C for 48 h. This culture was used to seed 350 liters of YP, 1% glucose in a 500-liter New Brunswick fermenter.

Mutagenesis of hCPA2 was analogous to CPA1 described above. The following mutagenic oligonucleotide primer (Oligos Etc.) was used to mutate residue Ala²⁶⁸ (gCC): A268G, 5-CAg TTC AAA gCC AAA TgA gTA-3 (Gly = ggC). Using this oligonucleotide primer, the following hCPA2 mutant A268G was formed. The mutagenized hCPA2 cassette was sequenced to verify that only the desired DNA mutations were produced.

The hCPA2-A268G mutant was subcloned into pMP36, then into pBS, and expressed in yeast (described above).

The wild type and mutant hCPA1 and hCPA2 enzymes were purified to electrophoretic homogeneity using a combination of hydrophobic and ion exchange chromatography as described previously (31).

Enzymatic activity was determined in one of two ways. Hippuryl-L-phenylalanine and hippuryl-DL-phenyllactate were determined spectrophotometrically at 255 nm as described (45). Reactions contained either 0.5 mM hippuryl-L-phenylalanine or 1.0 mM hippuryl-DL-phenyllactate in 25 mM Tris-HCl (pH 7.4), 100 mM NaCl. Reactions were initiated by the addition of enzyme and were monitored by the change in absorbance at 255 nm at 25 °C. Enzyme kinetic rates were determined from initial velocities using  = 390 M¹.

Hydrolysis of MTX prodrugs was measured using a modification of the coupled assay described by Kuefner et al. (19). Reactions were carried out in 1 ml of 25 mM Tris-HCl (pH 7.4), 100 mM NaCl at 25 °C. Buffer was added to the cuvette along with 0.026 units of carboxypeptidase G; then prodrug was added, and the absorbance was determined to calculate the concentration. Reactions were initiated by adding a known amount of CPA enzyme, and the decrease in absorbance at 315 nm was monitored. Enzyme kinetic rates were determined from initial velocities using  = 9.57 mM¹ for MTX. One unit of enzyme activity is defined as the hydrolysis of 1 µmol of substrate/min at 25 °C.

Thermal inactivation of the enzymes was determined spectrophotometrically using the CPA assay described above. Inactivation reactions were carried out by incubating 0.1 mg/ml enzyme in Dulbecco's phosphate-buffered saline at the desired temperature. Aliquots were sampled at various times and assayed with hippuryl-DL-phenyllactate.

This parameter was determined by incubating the prodrugs in trypsin activated human pancreatic juice. The percentage of conversion to MTX was determined as a function of time from linear conversion versus time plots. Pancreatic juice was a generous gift of Dr. T. Pappas of Duke University Medical School (Durham, NC). As obtained, the material had little or no CPA activity and did not metabolize MTX prodrugs. The pancreatic juice was activated fresh for each experiment by trypsinization with 1 mg/ml trypsin for 10 min at 37 °C. (This high concentration of trypsin was required to overcome endogenous trypsin inhibitors.) This activated solution was used directly for stability tests of the prodrugs. Activated pancreatic juice was diluted 1:40 to 1:2000 into 25 mM Tris-HCl, 100 mM NaCl, pH 7.5. Prodrug was then added to a final concentration of 50 µM. The solution was then incubated at 25 °C for up to 24 h. During this incubation period, aliquots were removed and analyzed by HPLC for prodrug and MTX. HPLC conditions were as follows. Chromatography was performed on a Waters C-18 Nova Pak column with a flow rate of 1 ml/min. Mobile phase conditions were a step gradient system composed of 0.1% trifluoroacetic acid in water (component A) or in acetonitrile (component B). For chromatography, the column was equilibrated with 82% A, 18% B. At 3 min, conditions were switched to 50:50 A:B. Then the column was reequilibrated starting at 7 min for another 10 min with 82:18 A:B. Under these conditions, MTX eluted at 4 min, and the prodrugs eluted at 6-8 min, depending upon hydrophobicity. Elution was monitored at 310 nm. No significant conversion of prodrug to MTX occurred when a suitable dilution of trypsin replaced activated pancreatic juice, indicating that conversion was due solely to materials contained in the pancreatic juice.

This parameter was measured as described elsewhere (42). Briefly, animals were dosed with prodrug intravenously. At specified times, plasma and tissues were collected and snap-frozen. The samples were then homogenized in 0.1 M HCl and extracted with a mix of 2 volumes of the HCl homogenate and 5 volumes of 20 °C acetonitrile. Then prodrug and MTX were measured by HPLC as described (42).

The enzyme was modified at free amines with Sulfo-SMCC. This bifunctional reagent has both an amine-reactive N-hydroxysulfosuccinimide group and a thiol-reactive maleimide group. Since carboxypeptidase has no free thiols, the compound reacts with enzyme amines to place a maleimide on the enzyme for subsequent reaction/coupling with free thiols on the antibody.

Four mg of mutant or wild type carboxypeptidase A were combined with 0.15 mg of Sulfo-SMCC (Pierce) in 400 µl of Dulbecco's phosphate-buffered saline. The resulting solution was stirred for 45 min at 25 °C. The modified enzyme then resolved from reagent through a 1 × 13-cm G-25 medium column equilibrated with Dulbecco's phosphate-buffered saline.

Maleimide content was determined as follows. A 0.5-ml aliquot of a 6.3 µM solution of modified enzyme was mixed with 6 µl of 1 mM mercaptoethanolamine. The mixture was allowed to sit for 30 min to permit the enzyme-bound maleimide to react with the mercaptoethanolamine. Then 20 µl of 4 mg/ml Ellman's reagent was added to react with the remaining free mercaptoethanolamine. After another 20 min, absorbance at 412 nm was determined. The amount of enzyme-bound maleimide was inferred from the amount of mercaptoethanolamine that was consumed during reaction with the enzyme. Based upon a molar absorptivity of 13.6 mM¹, the purified product was typically found to contain 1 maleimide/enzyme molecule. The derivatization had no effect upon enzyme activity measured with hippurylphenylalanine.

The antibody was modified with the amine-reactive reagent 2-iminothiolane (Traut's Reagent; Pierce), which replaces amines with free thiols. The thiols generated on the antibody by this procedure were then used for subsequent coupling to carboxypeptidase through the enzyme-bound, thiol-reactive, maleimide.

1.8 mg of ING-1 (46) or Campath-1H (47) were combined with 0.22 mg of 2-iminothiolane in 1.3 ml of a 50:50 mixture of Dulbecco's phosphate-buffered saline and 0.1 M triethanolamine-HCl, 2 mM EDTA, pH 8.0, under anaerobic conditions. This was allowed to react with stirring for 1 h and 45 min. The modified antibody was purified through a 1 × 13-cm G-25 medium column equilibrated with 0.02 M sodium acetate, 0.1 M NaCl, pH 5.8, bubbled with and maintained under a helium atmosphere.

The modified antibody was collected directly from the G-25 column into the solution of the modified carboxypeptidase A. The resulting mixture was then carefully adjusted to pH 7.4 with NaOH, made anaerobic by repeated N₂ gassing and evacuation, and allowed to react with stirring at 4 °C for 18 h. Excess free maleimide groups were removed by reacting the solution with 0.3 mM mercaptoethanolamine at room temperature for 1 h, and the solution was then concentrated to 1 ml. The resulting concentrated conjugate was purified from aggregates, unreacted enzyme, and small molecules by chromatography on Superose 12 HR 10/30.

IC₅₀ values for drugs and prodrugs alone were determined as described previously (48). For ADEPT experiments, HT-29 cells were seeded at 7500 cells/well in 96-well plates containing 200 µl of 95% RPMI 1640/5% human serum (human growth medium). The cells were allowed to grow for 24 h at 37 °C. Then medium was removed, and 50 µl of conjugate composed of either ING-1·hCPA1-T268G or Campath-1H·hCPA1-T268G in fresh human growth medium was added to triplicate wells. Conjugate concentrations of 0, 2, 10, and 50 µg/ml were used. After 1 h at 37 °C in a 5% CO₂ incubator, conjugate was removed, and the plates were washed three times with fresh human growth medium without conjugate. Finally, prodrugs at 0, 0.01, 0.03, 0.1, 0.3, 1, 3, and 30 µM were added in 200 µl of fresh human growth medium, and cells were allowed to grow for 72 h. The plates were then stained with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium to assay for proliferation as described (48). MTX was included in separate wells in all experiments as an internal control for IC₅₀ reproducibility.

RESULTS

Vitols et al. (21) reported that MTX-Phe is a good substrate for bovine CPA. Only one pancreatic CPA isozyme has been found in this species; however, in rodents and humans two isozymes exist. We reported previously that the compound is a good substrate for both human isozymes, hCPA1 and hCPA2 (31). Thus, an hCPA-antibody conjugate should effectively hydrolyze MTX-Phe for human enzyme-based ADEPT as had been shown previously for a bovine CPA-antibody conjugate (21).

For ADEPT, it is desirable that the active agent (MTX) is generated selectively at the site of the tumor by the action of a targeted enzyme (CPA). Therefore, the prodrugs used must not be converted to the active agent systemically by the host. The in vivo conversion of MTX-Phe to MTX was tested in mice after intravenous administration. After administration of 50 mg/kg prodrug, animals were killed at 0.5 and 2 h, and tissues and blood plasma were collected and analyzed for both MTX-Phe and MTX. Table II shows the results of this experiment, where the data are presented as the percentage of sample found as MTX-Phe and as absolute levels of both MTX and MTX-Phe. Unfortunately, even at the 30-min time point, 69% of the sample in circulation was found as MTX (31% as prodrug), and this increased to 97% by 2 h (3% as prodrug). The liver was the site of the largest accumulation of prodrug at 30 min (335 nmol/g, and 95% of the sample found in the liver was prodrug); however, by 2 h, MTX predominated in this tissue as well (0.6 nmol/g and 8% prodrug). The site of most rapid accumulation of MTX was the small intestine and pancreas, which was found to to have 84.5 nmol/g MTX and 17.3 nmol/g MTX-Phe at 30 min. By 2 h, MTX levels in this tissue were 203 nmol/g, and prodrug was not detectable. Overall the data show that MTX-Phe was rapidly converted to MTX in the mice, and significant amounts of the generated MTX were then observed in circulation. This rapid systemic generation of large amounts of MTX appeared to us to make MTX-Phe unsuitable for ADEPT.

Table II. In vivo stability and biodistribution of MTX-Phe (GW1311) upon injection of 50 mg/kg to CD-1 nu/nu mice Time Tissue Plasma Liver Kidney Lg. int. Sm. int./panc.a Spleen 30 min % recovered as MTX-Pheb 31 95 100 65 17 81 MTX-phe level, nmol/gb 17.8 335 26.9 19.3 17.3 19.9 MTX level, nmol/gb 39.6 18 0 10.4 84.5 4.7 120 min % recovered as MTX-Pheb 3 8 0 22 0 0 MTX-Phe level, nmol/gb 1.1 0.6 0 1.1 0 0 MTX level, nmol/gb 35.7 7.1 0 3.9 203 1.4 a Small intestine and pancreas were analyzed as one tissue due to the difficulty of surgically resolving them. b Each tissue was analyzed for MTX-phe and MTX. Percentage (%) recovered as MTX-phe = 100(MTX-Phe)/(MTX-Phe + MTX) for the particular tissue analyzed. Limit of detection = 0.5-1 nmol/g = 3 × background.

To overcome the in vivo metabolism problem, the source of MTX-Phe metabolism was investigated. In vitro tissue metabolism experiments showed that the compound was stable in plasma and only slowly metabolized in Ref. 42. We reasoned that other potential sources of this metabolism are the pancreatic CPAs that are secreted into the small intestine. This reasoning is consistent with the large MTX accumulation we observed in the small intestine and pancreas (Table II). These pancreatic enzymes enter the small intestine through the duodenum in the solution known as pancreatic juice. The material can be obtained from patients with pancreatic fistulae and used as a source of the human enzymes; indeed, human CPA was originally purified from this source (33). We used this material intact as a source of all pancreatic enzymes secreted into the small intestine to predict the stability of MTX-Phe in the small intestine. MTX-Phe was rapidly hydrolyzed to MTX by this material. A 1:100 dilution of pancreatic juice, hydrolyzed 50% of 50 µM MTX-Phe in 17 min at 25 °C. MTX-Phe is not a good substrate for trypsin, chymotrypsin, or carboxypeptidase B, and since it is a good substrate for both hCPA1 and hCPA2 (31), the most likely sources of metabolism of MTX-Phe in pancreatic juice are the CPAs. Since this experiment suggested that human pancreatic juice is at least one source of in vivo metabolism of MTX-Phe, the stabilities of the novel prodrugs were determined in this material and compared with the stability of MTX-Phe. The comparison was then used as the primary test of new prodrugs designed and synthesized in the current program to be more stable in vivo (Table III). The results from the stability in pancreatic juice were then confirmed with subsequent in vivo experiments (42).

Table III. Comparison of rate of prodrug pancreatic juice hydrolysis with CPA enzyme kinetics Compound Pancreatic juice (% of MTX-Phe hydrolysis rate) hCPA1a hCPA2a Vmax Km kcat/Km Vmax Km kcat/Km µmol/min/mg mM 1/M(s) µmol/min/mg mM 1/M(s) MTX-Phe 100 3.24 0.0043 440,000 8.06 0.056 90,000 Negatively charged prodrugs   MTX-Glu 0.12 0.094 0.1 170 0  NDb 0   MTX-Asp 0.06 0.063 0.5 80 0 ND 0   MTX-2-carboxy-Phe 0 0.003 0.7 3 <0.0005@50 µM ND ND   MTX-3-carboxy-Tyr 0.008 0.009 0.5 10 0.01 0.2 40   MTX-3-carboxy-Phe 0.27 0.08 30 1550 0.63 120 3100 Bulky aromatic prodrugs (phenylalanine based)   MTX-2-iodo-Phe ND 3.42 0.017 120,000 36 0.05 420,000   MTX-naphthyl-Ala 67 0.16 0.065 1400 36.9 0.016 1,400,000   MTX-2-cyclopentyl-Phe 0.17 0.021 0.06 200 0.11 0.081 770   MTX-2-cyclohexyl-Phe 0.0067 0 ND 0 0 ND 0   MTX-3-cyclobutyl-Phe 0.053 0 ND 0 0.086 0.2 260   MTX-3-t-butyl-Phe 0.02 0 ND 0 0.04 0.3 80   MTX-3-cyclopentyl-Phe 0.004 0 ND 0 0.012 0.3 25   MTX-3-n-pentyl-Phe 0 0 ND 0 0 ND 0 Bulky aromatic prodrugs (tyrosine-based)   MTX-3,5-diiodo-Tyr 0.093 0 ND 0 0.31 0.3 610   MTX-2-cyclopentyl-Tyr 0.14 0.43 0.4 575 1 0.6 910   MTX-3-cyclobutyl-Tyr 0.0067 0 ND 0 0 ND 0   MTX-3-t-butyl-Tyr 0.0013 0 ND 0 0 ND 0   MTX-3-cyclopentyl-Tyr 0.002 0 ND 0 0 ND 0 a Limit of detection for Vo was 0.0006 µmol/min/mg for hCPA1 and 0.002 µmol/min/mg for hCPA2. Assuming a Km of ~50 µM, the limit of kcat/Km detection = 5 and 20/(M)(s), respectively. b ND, not determined.

The active site model of hCPA1 was generated as described elsewhere (40) from the crystal structure of bovine CPA (32) using the Composer modeling program. This model is shown in Figs. 1 and 2A. The hCPA2 model is similar. The exceptions to this are a Gly in place of Ser at 253, Ser in place of Thr at 254, and an Ala in place of Thr at residue 268 (31). These substitutions participate to make a larger binding pocket in hCPA2 compared with hCPA1 and are presumably responsible for the hydrolysis of bulkier substrates by hCPA2 versus hCPA1 as has been reported for both the murine and human enzymes (31, 37).

The models of hCPA1 and hCPA2 were used to design MTX prodrugs that would not be substrates for either enzyme. Suitability of the designed and synthesized prodrugs for ADEPT was assessed by assaying them with WT hCPA1 and hCPA2 and testing their stability in pancreatic juice (and in mice; Ref. 42). Three general types of structural modification were explored for enhancement of prodrug stability. These included (a) the introduction of a negative charge to the prodrug amino acid moiety, (b) the addition of bulk to Phe- or Tyr-based prodrugs, and (c) the addition of both a charge and bulk.

Fig. 2 shows that the active sites of hCPA1 and hCPA2 are highly hydrophobic. Therefore, MTX-Glu, MTX-Asp, MTX-2-carboxy-Phe, MTX-3-carboxy-Phe, and MTX-3-carboxy-Tyr were produced. All of these compounds would introduce a negatively charged carboxyl group into the hydrophobic pocket and as such were not expected to be good hCPA substrates. The activities of these compounds with hCPAs and their relative stabilities in human pancreatic juice (compared with MTX-Phe with a defined rate of 100%) are shown in Table III. All of these charged compounds are poor substrates for both of the CPAs and are considerably more stable in pancreatic juice than is MTX-Phe. The greatest pancreatic juice stability and poorest enzyme activity was found with the 2-carboxy-Phe and 3-carboxy-Tyr prodrugs, which introduce both bulk and a negative charge into the prodrug. Surprising, MTX-3-carboxy-Phe was a much better substrate than either MTX-2-carboxy-Phe or MTX-3-carboxy-Tyr and consequently far less stable in pancreatic juice. The reasons for this are not clear but may represent an ability of the 3-carboxy-Phe substituent to access the para-hydroxyl binding site of tyrosine-based substrates.

Fig. 2A shows that Thr²⁶⁸ of hCPA1 closely abuts the 2- or 3-hydrogen of the glycyl-Tyr slow substrate bound in this model. Ala²⁶⁸ of hCPA2 provides more space. This size restriction was used to design bulky prodrugs that were not expected to be either hCPA1 or hCPA2 substrates. This was accomplished by adding hydrocarbon substituents to the 2- or 3-positions of Phe or Tyr. The prodrugs produced, along with their enzyme activities and pancreatic juice stabilities are shown in Table III. Bulky substituents were very effective in decreasing the activity of the prodrugs toward hCPA1, hCPA2, and pancreatic juice hydrolysis. As predicted from our model and from previous work (31, 37), bulky prodrugs were in general poorer substrates for hCPA1 than hCPA2. Both Phe- and Tyr-based prodrugs were made with 2-cyclopentyl, 3-cyclobutyl, 3-t-butyl, and 3-cyclopentyl substituents. The data indicate that (when the activities were measurable, non-zero) k_cat/K_m for both hCPA1 and hCPA2 and relative pancreatic juice hydrolysis followed the order 2-cyclopentyl > 3-cyclobutyl > 3-t-butyl 3-cyclopentyl for both the Phe and Tyr prodrugs.

The activities of both hCPA1 and hCPA2 contributed to the pancreatic juice hydrolysis rate; therefore, pancreatic juice stability required that a prodrug be stable toward both hCPAs. This can be seen for example with MTX-Phe, MTX-naphthyl-Ala, MTX-2-cyclopentyl-Phe, MTX-3-cyclopentyl-Phe, and MTX-3-cyclopentyl-Tyr, which had decreasing total hCPA1 plus hCPA2 activity and increasing pancreatic juice stability. The k_cat/K_m values for MTX-Phe with hCPA1 and hCPA2, respectively, were 440,000 M¹ s¹ and 90,000 M¹ s¹. The k_cat/K_m for MTX-naphthyl-Ala with hCPA1 was 1,400 M¹ s¹ (0.3% of MTX-Phe) but was 1,400,000 M¹ s¹ with hCPA2. This excellent substrate activity with hCPA2 resulted in MTX-naphthyl-Ala being nearly as unstable in pancreatic juice as MTX-Phe. The k_cat/K_m for MTX-2-cyclopentyl-Phe with hCPA1 was 200 M¹ s¹, and for hCPA2 it was 770 M¹ s¹, which resulted in a pancreatic juice hydrolysis rate of 0.17% of MTX-Phe. The k_cat/K_m of MTX-3-cyclopentyl-Phe with hCPA1 was not measurable, and with hCPA2 it was 25 M¹ s¹, resulting in a pancreatic juice hydrolysis rate of 0.004% of MTX-Phe. Finally, The k_cat/K_m values of MTX-3-cyclopentyl-Tyr with both hCPA1 and hCPA2 were not measurable, and this compound was hydrolyzed by pancreatic juice at 0.002% the rate of MTX-Phe. As shown elsewhere (42), compounds with pancreatic juice hydrolysis rates in the range of 0.01% of MTX-Phe or less had excellent in vivo stability.

Our next goal was to utilize our structural models of hCPA1 and hCPA2 to model mutant hCPAs into which the "stable prodrugs" could be docked. Fig. 2, B and C, shows models of two such mutants of hCPA1, I255H and T268G. The former was designed to introduce a positive charge into the active site to bind the carboxyl-containing prodrugs. The latter was designed to produce an active site that would bind Phe- or Tyr-based prodrugs substituted at the 2- or 3-position. I255K and I255H/T268G were also designed for the charged prodrugs, and T268A, I255A, and I255A/T268A were also designed to accept bulk. The A268G mutant of hCPA2 was also predicted and made for the bulky prodrugs. Expression and isolation of the hCPA1 mutants T268A, T268G, I255A, and T268A/I255A and the hCPA2 mutant A268G were accomplished using the yeast expression system described under "Experimental Procedures." All of these mutants were synthesized and secreted by the yeast as catalytically active enzymes. Unfortunately, no hCPA protein or enzymatic activity could be found in yeast expressing I255K, I255H, or I255H/T268G. Presumably, these three proteins misfolded and were degraded prior to secretion.

Several of the WT and mutant enzymes were characterized for their stability in phosphate-buffered saline at 4, 25, 37, and 50 °C. The results from these experiments are shown in Table IV. All enzymes are quite stable at 4 °C. The I255A mutants were the least stable, since they exhibited half-lives of 8 h at 25 °C while all other mutants were stable for several days at this temperature. At 37 °C, WT hCPAs as well as hCPA1-T268A and hCPA2-A268G lost little activity after 2 days, and while hCPA1-T268G was less stable it also had a half-life of 24 h. At 50 °C, differential stabilities were clearer. At this temperature, the order of stability was WT hCPA2 > hCPA2-A268G > hCPA1-T268A > WT hCPA1 > hCPA1-T268G. In summary, these data show that the mutants of hCPA are active and stable.

Table IV. Thermal stability of WT and mutant hCPAs Sample t1/2 4 °C 25 °C 37 °C 50°C h WT hCPA1 Longa >170b 140 0.77 hCPA1-T268A Long >170b >96b 0.93 hCPA1-T268G Long 80 24 0.17 hCPA1-I255A Long 8 1 hCPA1-I255A/T268A Long 8 1 WT hCPA2 Long >170b >170b 8.75 hCPA2-A268G Long >170b 140 5.1 a At 4 °C, all the mutants were stable for at least 2 months. In addition, both WT enzymes, the 268G mutants, and the T268A mutant have been stored at this temperature for 2 years with little or no loss. b Less than 10% decrease in activity of the enzyme over the stated time period.

Specific activities of these enzymes with the model substrates hippuryl-Phe and hippuryl-Phe lactate are shown in Table V. As can be seen, all of these enzymes hydrolyzed these substrates efficiently. Interestingly, WT CPA2 and its 268G mutant had very large esterase hydrolytic rates.

Table V. Specific activity of WT and mutant hCPAs with hippuryl-Phe and hippuryl-Phe lactate Assay conditions were as follows: 0.5 mM L-hippuryl-Phe or 1 mM DL-hippuryl-Phe lactate, at 25 °C in 25 mM Tris-HCl, 100 mM NaCl, pH 7.4.  Enzyme Specific acitvity Hippuryl-Phe Hippuryl-Phe lactate µmol/min/mg WT hCPA1 7.2 790 hCPA1-T268A 7.4 160 hCPA1-T268G 20 60 hCPA1-I255A 8 350 hCPA1-I255A/T268A 12 50 WT hCPA2 8.7 2800 hCPA2-A268G 4.9 1900 Bovine CPA 24.1 310 Rat CPA1 11 560

Kinetics of hCPA1-T268G, hCPA1-T268A, and hCPA2-A268G with the MTX prodrugs are shown in Tables VI and VII. Table VI shows the activity of the mutant hCPAs with the charged prodrugs from Table I. As indicated above, the hCPA mutants designed to hydrolyze these prodrugs (I255H, I255K, and I255H/T268G) could not be isolated. The hCPA mutants designed for the bulky hydrophobic prodrugs were not expected to efficiently hydrolyze charged prodrugs. However, mutation of hCPA1 to T268G produced a 5-fold enhancement in k_cat/K_m for the hydrolysis of MTX-3-carboxy-Phe, a 50-fold enhancement in the k_cat/K_m for hydrolysis of MTX-3-carboxy-Tyr and a 250-fold enhancement in the k_cat/K_m for hydrolysis of MTX-2-carboxy-Phe. The A268G mutation of hCPA2 was without effect on any of the charged prodrugs measured. The mutation of hCPA1 to T268A produced a modest enhancement in the hydrolysis of two of these prodrugs intermediate between the WT and T268G rates. These rate enhancements are likely due to the additional tolerance in the T268G and T268A active sites for bulk at the 2- and 3-positions of the substrate aromatic ring (as described below). The enhancements by hCPA1-T268G are probably inadequate for ADEPT, however, since the absolute k_cat/K_m values for these reactions were all quite small compared with that for hCPA1 with MTX-Phe (approximately 1000-fold less).

Table VI. Kinetics of charged prodrugs with hCPA1-T268G, hCPA1-T268A, and hCPA2-A268G Compound hCPA1-T268G hCPA1-T268A hCPA2-A268G Vmax Km kcat/ Km) (kcat/Km)mut/ (kcat/Km)wt Vmax Km kcat/ Km (kcat/Km)mut/ (kcat/Km)wt Vmax Km kcat/ Km (kcat/Km)mut/ (kcat/Km)wt µmol/ min/mg mM 1/M(s) µmol/ min/mg mM 1/M(s) µmol/ min/mg mM 1/M(s) MTX-Glu 0.012 0.06 120 0.7 NDa ND ND ND 0 ND ND MTX-ASP 0.024 0.25 60 0.75 ND  ND ND ND 0.12 0.9 76 MTX-2-carboxy-Phe 0.1 0.09 670 250 0.007 0.06 60 20 <0.002@50 µM ND ND MTX-3-carboxy-Tyr 0.06 0.07 500 50 0.01 0.1 60 6 0.02 0.2 70 2 MTX-3-carboxy-Phe 0.87 0.07 7200 4.6 0.11 0.05 1300 0.8 1.1 0.25 2500 0.8 a ND, not determined. Detection limits are as in Table III.

Table VII. Kinetics of bulky aromatic prodrugs with hCPA1-T268G, hCPA1-T268A, and hCPA2-A268G Compound hCPA1-T268G hCPA1-T268A hCPA2-A268G Vmax Km kcat/ Km) (kcat/Km)mut/ (kcat/Km)wt Vmax Km kcat/ Km (kcat/Km)mut/ (kcat/Km)wt Vmax Km kcat/ Km (kcat/Km)mut/ (kcat/Km)wt µmol/ min/mg mM 1/M(s) µmol/ min/mg mM 1/M(s) µmol/ min/mg mM 1/M(s) Phenylalanine-based compounds   MTX-Phe 3.74 0.0003 7,350,000 17 3.46 0.0009 2,250,000 5.1 3.1 0.045 41,000 0.45   MTX-2-iodo-Phe 13.7 0.0004 2.7E+07 230 7.56 0.009 480,000 4.1 NDa ND ND ND   MTX-naphthyl-Ala 1.15 0.0005 1,400,000 1,000 10.8 0.01 640,000 460 21.5 0.002 5,500,000 4   MTX-2-cyclopentyl-Phe 4.25 0.016 160,000 790 0.15 0.06 1,600 8 2.6 0.008 190,000 250   MTX-2-cyclohexyl-Phe 0.094 0.08 700 >100b 0.009 0.06 100 >5b 0.3 0.19 900 >50b   MTX-3-cyclobutyl-Phe 5.2 0.002 1,800,000 >400,000b 0.21 0.16 750 >40b 0.163 0.047 2100 8   MTX-3-t-butyl-Phe