(Received for publication, March 4, 1997, and in revised form, April 7, 1997)
From GlaxoWellcome Inc., Research Triangle Park, North Carolina 27709
Antibody-directed enzyme prodrug therapy (ADEPT)
has the potential of greatly enhancing antitumor selectivity of cancer
therapy by synthesizing chemotherapeutic agents selectively at tumor
sites. This therapy is based upon targeting a prodrug-activating enzyme to a tumor by attaching the enzyme to a tumor-selective antibody and
dosing the enzyme-antibody conjugate systemically. After the enzyme-antibody conjugate is localized to the tumor, the prodrug is
then also dosed systemically, and the previously targeted enzyme converts it to the active drug selectively at the tumor. Unfortunately, most enzymes capable of this specific, tumor site generation of drugs
are foreign to the human body and as such are expected to raise an
immune response when injected, which will limit their repeated
administration. We reasoned that with the power of crystallography, molecular modeling and site-directed mutagenesis, this problem could be
addressed through the development of a human enzyme that is capable of
catalyzing a reaction that is otherwise not carried out in the human
body. This would then allow use of prodrugs that are otherwise stable
in vivo but that are substrates for a tumor-targeted mutant
human enzyme. We report here the first test of this concept using the
human enzyme carboxypeptidase A1 (hCPA1) and prodrugs of methotrexate
(MTX). Based upon a computer model of the human enzyme built from the
well known crystal structure of bovine carboxypeptidase A, we have
designed and synthesized novel bulky phenylalanine- and tyrosine-based
prodrugs of MTX that are metabolically stable in vivo and
are not substrates for wild type human carboxypeptidases A. Two of
these analogs are MTX--3-cyclobutylphenylalanine and MTX-
-3-cyclopentyltyrosine. Also based upon the computer model, we
have designed and produced a mutant of human carboxypeptidase A1,
changed at position 268 from the wild type threonine to a glycine
(hCPA1-T268G). This novel enzyme is capable of using the in
vivo stable prodrugs, which are not substrates for the wild type
hCPA1, as efficiently as the wild type hCPA1 uses its best substrates
(i.e. MTX-
-phenylalanine). Thus, the
kcat/Km value for the wild
type hCPA1 with MTX-
-phenylalanine is 0.44 µM
1 s
1, and
kcat/Km values for
hCPA1-T268G with MTX-
-3-cyclobutylphenylalanine and
MTX-
-3-cyclopentyltyrosine are 1.8 and 0.16 µM
1 s
1, respectively. The
cytotoxic efficiency of hCPA1-268G was tested in an in
vitro ADEPT model. For this experiment, hCPA1-T268G was chemically conjugated to ING-1, an antibody that binds to the tumor
antigen Ep-Cam, or to Campath-1H, an antibody that binds to the T and B
cell antigen CDw52. These conjugates were then incubated with HT-29
human colon adenocarcinoma cells (which express Ep-Cam but not the
Campath 1H antigen) followed by incubation of the cells with the
in vivo stable prodrugs. The results showed that the
targeted ING-1:hCPA1-T268G conjugate produced excellent activation of
the MTX prodrugs to kill HT-29 cells as efficiently as MTX itself. By
contrast, the enzyme-Campath 1H conjugate was without effect. These
data strongly support the feasibility of ADEPT using a mutated human
enzyme with a single amino acid change.
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 × 10
6 to
6.3 × 10
8 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.
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
Synthesis of MTX ProdrugsThe synthesis of the MTX prodrugs shown in Table I is described elsewhere (40).
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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).
Purification of Expressed hCPA1 and hCPA2 EnzymesThe 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).
Spectrophotometric Enzymatic AssaysEnzymatic 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 M
1.
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 mM
1 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.
Stability of Prodrugs in Pancreatic JuiceThis 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.
In Vivo Stability of ProdrugsThis 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 of the Activated EnzymeMaleimide 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.
Coupling of Antibody with EnzymeThe 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.
Cell Culture ADEPT ExperimentsIC50 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.
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.
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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).
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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)
kcat/Km 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 kcat/Km values
for MTX-Phe with hCPA1 and hCPA2, respectively, were 440,000 M1 s
1 and 90,000 M
1 s
1. The
kcat/Km for MTX-naphthyl-Ala
with hCPA1 was 1,400 M
1 s
1
(0.3% of MTX-Phe) but was 1,400,000 M
1
s
1 with hCPA2. This excellent substrate activity with
hCPA2 resulted in MTX-naphthyl-Ala being nearly as unstable in
pancreatic juice as MTX-Phe. The
kcat/Km for
MTX-2-cyclopentyl-Phe with hCPA1 was 200 M
1
s
1, and for hCPA2 it was 770 M
1
s
1, which resulted in a pancreatic juice hydrolysis rate
of 0.17% of MTX-Phe. The
kcat/Km of
MTX-3-cyclopentyl-Phe with hCPA1 was not measurable, and with hCPA2 it
was 25 M
1 s
1, resulting in a
pancreatic juice hydrolysis rate of 0.004% of MTX-Phe. Finally, The
kcat/Km 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.
Enzyme StabilitySeveral 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.
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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.
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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 kcat/Km for the hydrolysis of MTX-3-carboxy-Phe, a 50-fold enhancement in the kcat/Km for hydrolysis of MTX-3-carboxy-Tyr and a 250-fold enhancement in the kcat/Km 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 kcat/Km values for these reactions were all quite small compared with that for hCPA1 with MTX-Phe (approximately 1000-fold less).
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Table VII shows the activity of the
hCPA mutants with the bulky hydrophobic prodrugs from Table I. The 268G
mutants were designed to remove unfavorable interaction between the
enzyme and 2- or 3-substituted MTX-Phe or MTX-Tyr, allowing their
efficient binding and hydrolysis. As shown in Table VII, this goal was
realized; the mutant hCPA1-T268G proved to efficiently hydrolyze a
variety of substrates with bulky 2- or 3-position substituents
(kcat/Km 100,000 M
1 s
1). In contrast, the T268A
mutant is a much poorer enzyme with all of these substrates. Thus, the
minimal replacement of methyl with hydrogen at residue 268 produced
dramatic rate enhancements. The hCPA2-A268G mutant also provided
dramatic rate enhancements for some prodrugs; however,
kcat/Km for the mutant with none of the stable prodrugs (pancreatic juice hydrolysis rate
0.01%
of MTX-Phe) exhibited kcat/Km
approaching 100,000 M
1 s
1.
Therefore, this enzyme was not as efficient as hCPA1-T268G with these
compounds.
Since the active site of the WT hCPA2 has Ala at position 268, the activity of the hCPA1-T268A mutant was expected to parallel that of WT hCPA2. Data in Tables VI and VII demonstrate that this was observed, since the activity of hCPA1-T268A followed that of WT hCPA2 for most prodrugs assayed.
The I255A and I255A/T268A mutants of hCPA1 did not prove useful for the hydrolysis of any of the bulky prodrugs (data not shown). Interestingly, the activity of the I255A mutants appeared in general to be dictated by the identity of the amino acid at position 268. Thus, substrate specificity of I255A was similar to that of WT hCPA1, and the substrate specificity of I255A/T268A was similar to that of T268A. This suggests that position 255 has little impact on substrate specificity in this class of aromatic substrates.
Overall the most useful mutant was hCPA1-T268G, since it was able to
efficiently metabolize several of the bulky, stable MTX prodrugs. The
most useful substrates for the enzyme, based upon large
kcat/Km for the mutant and
good pancreatic juice stability, were MTX-3-t-butyl-Phe,
MTX-3-cyclobutyl-Phe, MTX-3-cyclopentyl-Phe, MTX-3-cyclopentyl-Tyr, and
MTX-3-cyclobutyl-Tyr. All were approximately 4000-fold or more stable
in pancreatic juice than MTX-Phe, and all were hydrolyzed (at 25 °C)
with second order rate constants of or greater than 105
M1 s
1.
To test the utility of the enzyme prodrug combination for ADEPT, hCPA1-T268G was conjugated to two antibodies. ING-1 binds to Epcam, a molecule expressed on a variety of epithelial cell tumors (46). Campath-1H binds to CDw52, which is expressed on a variety of T and B cells but not on most epithelial cells or tumors (30, 47). To couple hCPA1-T268G to these antibodies, a maleimide was placed on the enzyme with Sulfo-SMCC; this process had no effect on enzyme activity. A free thiol was then placed on the antibody with 2-iminothiolane. The hCPA1-T268G·antibody conjugate was then generated upon combination of the activated proteins under nitrogen. Conjugate was purified from the small amount of protein aggregate formed in the reaction and from free hCPA1-T268G by gel filtration on Pharmacia Superose 12 HR 10/30.
The product was assayed for its carboxypeptidase activity and ability to bind to antigen. Table VIII shows the specific activity of conjugates toward hippuryl-Phe. Based upon the enzyme activity measured in this way, 1-1.5 mol of hCPA1-T268G appeared to be routinely associated with each mol of antibody. Table VIII also shows the percentage of the enzyme activity that bound to the relevant antigen along with the binding affinity as determined according to Lindmo (49). Both antibody conjugates expressed enzyme activity and bound to antigen. The percentage of conjugate that bound to antigen was 75-85%, and the Kd values are in agreement with literature indicating that the antigen combining region of the antibody was not adversely effected by the conjugation process (30, 46, 47).
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The conjugates were used in a series of in vitro ADEPT experiments. For these experiments, HT-29 cells were grown in 5% human serum. Preliminary experiments had shown that some component in fetal bovine serum was a competitive inhibitor of hCPA1 and hCPA1-T268G. This inhibitor greatly decreased the efficacy of both enzymes, resulting in the requirement for high concentrations of conjugate to produce an in vitro ADEPT response. Human serum also inhibited the enzymes, but the inhibition was less severe and most closely fit an uncompetitive kinetic model (data not shown). Since ADEPT was to be targeted to humans, the human serum conditions were considered the more relevant.
HT-29 cells, which were found to express Epcam but not significant
amounts of CDw52, were seeded and allowed to adhere for 24 h. The
cells on the plate were then incubated at 37 °C for 1 h with 0, 2, 10, or 50 µg/ml conjugate consisting of hCPA1-T268G coupled to
either ING-1 or Campath-1H. Cells were then washed free of unbound
antibody and allowed to grow for 72 h in the presence of varying
concentrations of MTX-Phe, MTX-3-cyclobutyl-Phe, or MTX-3-cyclopentyl-Tyr. The results of these experiments are shown in
Figs. 3 and 4 for the ING-1 and
Campath-1H conjugates, respectively. The IC50 for MTX in
these cells under these conditions was 10 nM. In the
absence of conjugate, all three prodrugs were considerably less toxic
than MTX. The stable prodrugs, MTX-3-cyclobutyl-Phe and
MTX-3-cyclopentyl-Tyr, were 800 times less toxic than MTX (IC50 values for the two prodrugs were 8.5 and 7.8 µM, respectively). MTX-Phe was 200 times less toxic than
MTX under these conditions.
When the cells were preincubated with the Epcam-specific conjugate ING-1·hCPA1-T268G, as expected all three prodrugs became more potent. Even the lowest dose of conjugate, 2 µg/ml, increased the toxicity of all three compounds. At this dose, the IC50 values became 0.01, 0.066, and 0.16 for MTX-Phe, MTX-3-cyclobutyl-Phe, and MTX-3-cyclopentyl-Tyr, respectively, in good agreement with the relative activities of the T268G mutant enzyme with these prodrugs shown in Table VII. At 10 and 50 µg/ml conjugate, the IC50 values for all three prodrugs approached that of MTX itself, indicating excellent conversion of all three prodrugs to the drug at these conjugate doses.
In contrast, when the cells were preincubated with the control conjugate Campath-1H·hCPA1-T268G, the IC50 values did not change with increasing conjugate. Thus, in the presence of 50 µg/ml Campath-1H·hCPA1-T268G conjugate, IC50 values for the three prodrugs were 2.5, 13, and 15 µM, respectively, not different from the prodrugs in the absence of conjugate. Therefore, activity of the ING-1·hCPA1-T268G conjugate was immunospecific. In summary, the data demonstrate that an immune specific conjugate of the hCPA mutant hCPA1-T268G, can convert in vivo stable MTX prodrugs to MTX in a cell culture ADEPT experiment.
ADEPT has the potential to greatly enhance the tumor selectivity
of cancer chemotherapy by generating a means for tumor-specific synthesis of chemotherapeutic or other toxic agents. This is
accomplished through staged, systemic delivery of components that
localize to tumor sites and selectively generate active drug(s) at the tumor (2-7). System requirements for a successful ADEPT strategy include (a) a prodrug that is not activated to drug in
vivo by host endogenous enzymes, (b) an enzyme capable
of producing the drug from the prodrug at a rate sufficient to produce
toxic levels of the drug at the specific site, and (c) an
antibody capable of targeting and localizing the enzyme to the tumor. A
number of enzymes have been reported for use in ADEPT. These include bovine carboxypeptidase A, Pseudomonas carboxypeptidase G,
several bacterial -lactamases, E. coli penicillin G
amidase, yeast cytosine deaminase, human and E. coli
-glucuronidase, bovine alkaline phosphatase, and others (2-29).
To permit repeat dosing with the ADEPT, an additional parameter is
needed. A mechanism to decrease immune response to the ADEPT system is
desirable. This goal is being approached by use of immune suppressing
agents (5, 28) and, in one example, the use of a human enzyme (24). The
latter system makes use of -glucuronidase and takes advantage of the
intralysosomal location of
-glucuronidase in mammalian cells by
utilizing prodrugs with poor lysosomal access (24). Unfortunately, the
enzyme is most active at acidic pH values not normal in the
extracellular environment, decreasing the effectiveness of the
extracellular targeted enzyme.
In an attempt to increase the repertoire of human enzymes available to ADEPT, we have investigated the potential utility of human CPA1 and CPA2. Heunnekens and colleagues (19-21) have suggested the use of bovine CPA along with amino acid prodrugs of MTX. Our initial experiments investigated the utility of this system directly. For this, we synthesized MTX-Phe and tested its substrate activity with the human CPA1 and CPA2. As reported for the bovine CPA, MTX-Phe is a good hCPA1 substrate and a fair hCPA2 substrate (21, 31). We also confirmed that the prodrug was stable in fetal bovine serum- and human serum-based growth media and was approximately 200 times less toxic than MTX in cell culture. However, MTX-Phe proved to be unstable in vivo, rapidly generating circulating and intestinal MTX. Indeed, even MTX-naphthyl-Ala, which is a poor hCPA1 substrate but is readily cleaved by hCPA2 (31), is also unstable in vivo or in human pancreatic juice. Thus, it appeared to us that substrates for the WT hCPAs would not be optimal for ADEPT. To overcome the in vivo stability problem, we investigated the potential of (a) designing MTX prodrugs that would not be hCPA substrates and therefore would be stable in vivo and (b) in parallel designing an hCPA mutant that could efficiently utilize these prodrugs.
Design of Prodrugs That Are Not Hydrolyzed by WT hCPA and Are Hydrolyzed by Mutant hCPAs
Charged ProdrugsWell known mammalian CPAs exist with
substrate specificities for hydrophobic aromatic/aliphatic amino acids
(those of the CPA class) and for basic amino acids (those of the
carboxypeptidase B class). However, to our knowledge, mammalian
carboxypeptidases that efficiently hydrolyze acidic C-terminal amino
acids have not been reported. Thus, one of our first attempts to
produce stable prodrugs was to produce natural and novel acidic amino acid -peptides of MTX. Indeed, the prodrugs were considerably more
stable in human pancreatic juice than MTX-Phe; MTX-2-carboxy-Phe and
MTX-3-carboxy-Tyr, made for this purpose, were among the most stable
prodrugs produced in our program.
To accommodate these negatively charged prodrugs in the hCPA hydrophobic binding pocket, Ile255 or Thr268 were mutated to Lys or His. However, we were unable to isolate these proteins from our yeast expression system. Presumably, the charged moiety in the hydrophobic environment prevented folding or active site alignment. Our inability to observe any protein from these mutants suggests that the three-dimensional structure was severely distorted.
One hCPA mutant did show some activity with the negatively charged
aromatic prodrugs. The
kcat/Km values for
MTX-3-carboxy-Tyr and MTX-2-carboxy-Phe with hCPA1-T268G are 50- and
200-fold greater, respectively, than with WT hCPA. These two prodrugs
are unique among the MTX prodrugs reported here in that they carry a
negative charge and are bulkier than their respective Phe or
Tyr parent. Thus, the rate enhancement observed with the T268G mutant
using these prodrugs may be a result of either the mutant's ability to
accommodate a negative charge or increased bulk. Enhancement via
accommodation of increased bulk is most likely. This is supported by
the observation that T268G was no better than WT hCPA1 with either
MTX-Asp or MTX-Glu, which are charged but are not bulky. Nonetheless,
the absolute kcat/Km values
for MTX-3-carboxy-Tyr and MTX-2-carboxy-Phe were each approximately 500 M1 s
1. Since this is 1000-fold
poorer than WT hCPA1 with MTX-Phe, this enzyme/prodrug combination was
not considered adequate for ADEPT, and improvements were sought.
The width of the phenyl ring binding pocket in hCPA1 is minimally defined on one side of the ring by Thr268 (Fig. 2). The side chain for this amino acid is within 3.5 Å of the ortho- and meta-positions of the model inhibitor N-acetyl-Gly-Tyr. In hCPA2, this amino acid is Ala (31, 35), which according to our model of hCPA2 adds 0.5 Å to the width of the hCPA2 binding pocket compared with hCPA1 (although the change from Thr to Ala also increases peripheral space, which is not accounted for in this simple analysis). Consistent with a wider pocket, hCPA2 accepts bulkier aromatic substrates than hCPA1, as has been reported by others for Trp-based model substrates (37), as we have reported for MTX-naphthyl-Ala (31) and show in Table III for several compounds. To determine if this enlarged hCPA2 binding pocket compared with hCPA1 could be replicated in hCPA1, hCPA1-T268A was generated. We observed that the mutation increased the kcat/Km for hCPA1 approximately 500-fold for MTX-naphthyl-Ala to within a factor of 2 of WT hCPA2 with this compound, and a similar, although less dramatic, effect was also observed for MTX-3,5-diiodo-Tyr. Indeed, activity with hCPA1-T268A paralleled that of WT hCPA2 for most prodrugs made. The notable exception for this was MTX-Phe itself, which was used 250-fold more efficiently by hCPA1-T268A than WT hCPA2, suggesting that additional factors (such as amino acids 253 and 254) were also involved in substrate alignment.
To make use of the information that position 268 was important in defining the substrate pocket and to increase the pocket dimensions further, the size of the amino acid side chain at 268 in hCPA1 was decreased to "H" by mutating Thr to Gly. Based upon our model, this should increase the pocket width by 1.5 Å. This change only produced a modest, 2-fold, enhancement in the activity of the enzyme with MTX-naphthyl-Ala compared with hCPA1-T268A, suggesting that Ala at position 268 was sufficient for proper substrate binding and alignment of MTX-naphthyl-Ala. To explore this enlarged binding pocket further, the bulky amino acid prodrugs in Table I were designed to fit into the active site of our structural model for hCPA1-T268G. The compounds were synthesized and tested.
Determination of Optimal Binding Pocket Limits for Bulky Prodrugs
Tables III and VII show that the 2-iodo substituent on
2-iodo-MTX-Phe was accommodated by both WT hCPA1 and WT hCPA2. Indeed, kcat/Km of WT hCPA2 with
MTX-2-iodo-Phe was 4-fold larger than with MTX-Phe. In the case of WT
hCPA1, however, the kcat/Km for the iodo-substituted compound was approximately 4-fold smaller than
that for MTX-Phe. This suggested that for hCPA1 the added size of the
iodo substituent, which has a van der Waals radius of 4.23 Å along the
C-I bond, was at the space limit of the binding pocket. The 1000-fold
decrease in kcat/Km for the
2-cyclopentyl-substituted MTX-Phe, which has a van der Waals radius of
4.97 Å, measured along the bond connecting the substituent to the ring
in the minimum energy cyclopentyl conformation, supports this notion.
Incremental increases in the binding site for the 2-position
substituents were observed upon mutation of the Thr268 to
Ala and Gly. Thus, mutation of WT hCPA1 to hCPA1-T268A increased the
kcat/Km for
MTX-2-cyclopentyl-Phe 8-fold, and mutation to hCPA1-T268G increased
kcat/Km another 100-fold to yield a kcat/Km within 3-fold
of that of WT hCPA1 with MTX-Phe. The size of the 2-cyclopentyl
substituent appears to be at the limit of the hCPA1-T268G binding
pocket size, since kcat/Km
for T268G with MTX-2-cyclohexyl-Phe (2-cyclohexyl with a minimum energy
conformation van der Waals radius of 6.17 Å) was 200-fold lower than
that for MTX-2-cyclopentyl-Phe. These data suggest that the T268G
mutation in hCPA1 functionally increased the space at the
ortho-position by at least 0.74 Å (4.97 4.23) to less than
1.94 Å (6.17
4.23). For comparison, calculations from our
model give an added length in this dimension of 1.5 Å, in good
agreement with the structure-activity relationship.
None of the meta-substituted prodrugs were WT hCPA1 substrates
(kcat/Km 10 M
1 s
1) including the smaller
compounds, 3-t-butyl- (van der Waals radius of 4.11 Å) and
3-cyclobutyl- (van der Waals radius of 4.5 Å). As in the case of the
2-substituted prodrugs, however, the hCPA1-T268G mutation exhibited
activity with several of the bulky prodrugs with
kcat/Km values similar to or
better than WT hCPA1 with MTX-Phe. These included
3-t-butyl-, 3-cyclobutyl-, and 3-cyclopentyl-Phe and
3-cyclopentyl-Tyr. The 3-n-pentyl- (length of 7.11 Å)
substituted prodrug appears to be beyond the limit of optimal
substituent size for the meta-position. Since none of the 3-substituted
compounds were active with the WT hCPA1, a functional measure of the
hCPA1-T268 pocket increase at the meta-position binding site is not
feasible, but calculations from our model yield a 1.5-Å increased
length at this position as well.
The activities of WT hCPA2 with the 3-substituted compounds were all low, but detectable activity with the 3-cyclobutyl-, 3-t-butyl-, and 3-cyclopentyl-Phe, which were inactive with WT hCPA1, suggests the presence of more space in the meta-position binding site in hCPA2 than in the corresponding site in hCPA1, as expected. As with the 2-substituted prodrugs, the activity of hCPA1-T268A mimicked that of WT hCPA2, supporting the apparent similarities of the hCPA1-T268A and WT hCPA2 binding sites, although the correlation was not as tight as for the 2-substituted compounds.
Mutation of hCPA2 to A268G did not prove as effective as the T268G
mutation for hCPA1. Thus, while the
kcat/Km values for
hCPA1-T268G with several of the 3-substituted prodrugs were over
100,000 M1 s
1, the
kcat/Km for hCPA2-A268G with
these compounds were all
10,000 M
1
s
1. The prodrug that exhibited the largest
kcat/Km enhancement with this
enzyme was MTX-3,5-diiodo-Tyr
(kcat/Km for the mutant = 520,000 M
1 s
1 compared with
kcat/Km for the WT hCPA2 = 610). However, this compound was hydrolyzed too rapidly by pancreatic
juice and as such was not useful. The compound may suggest, however,
that other 3,5-disubstituted prodrugs should be explored for
hCPA2-A268G.
Summary Analysis of Optimal Enzyme/Prodrug Combination
The 3-cyclopentyl substituent on either Tyr- or Phe-based prodrugs produced very poor enzyme activity with both hCPA1 and hCPA2. This poor enzyme activity resulted in excellent stability of the 3-cyclopentyl-substituted prodrugs in pancreatic juice. The hCPA1-T268G mutation returned the enzyme activity of both the Tyr- and Phe-based 3-cyclopentyl prodrugs to produce kcat/Km values approaching that of WT hCPA1 with MTX-Phe. The similar mutation on hCPA2 was not effective. Therefore, the optimal enzyme/prodrug combination from the current analysis is hCPA1-T268G with either Tyr- or Phe-based 3-cyclopentyl prodrugs.
Anomalies in the Model
The effects of the 268G mutation on hCPA1 versus hCPA2 enzyme demonstrate differences between hCPA1 and hCPA2 that cannot be accounted for in our model. Thus, the kcat/Km of hCPA1-T268G with the 3-cyclobutyl prodrug was at least 100,000 times greater than that of WT hCPA1 with this prodrug and actually 4 times better than WT hCPA1 with MTX-Phe. In contrast, mutation of hCPA2 to hCPA2-A268G enhanced the activity with this compound less than 10 times, and the absolute kcat/Km for the mutant was about 40 times poorer than WT hCPA2 with MTX-Phe. Similarly, the 3-cyclopentyl-Phe, 3-n-pentyl-Phe, 3-t-butyl-Phe, 3-cyclopentyl-Tyr, 3-cyclobutyl-Tyr, and 3-t-butyl-Tyr prodrugs were all 10-1000-fold better substrates for hCPA1-T268G than for hCPA2-A268G. These differences define some as yet undetermined deficiency in our model and stress the importance of result-directed model refinement, as used here, for this type of enzyme and prodrug design process.
Cell Culture ADEPT
For the purposes of ADEPT, the kinetic data indicate that hCPA1-T268G is the best suited enzyme. This conclusion is based on the excellent stability of 3-substituted Phe and Tyr prodrugs in pancreatic juice and their equally excellent hydrolytic rates with the hCPA1-T268G. This conclusion led us to test the combinations further in a series of in vitro ADEPT experiments. In these experiments we showed that only antigen-specific binding of conjugate to tumor cells (i.e. the ING-1·hCPA1-T268G conjugate with HT-29 cells) was able to generate an ADEPT response. Importantly, the data indicate that the tumor cell-bound enzyme is capable of generating MTX from otherwise in vivo stable prodrugs at relevant conjugate and prodrug concentrations. Indeed, in the presence of sufficient conjugate, the IC50 of the prodrugs tested approached that of MTX itself. Since MTX efficacy is dependent upon both the concentration and time of exposure, the IC50 data indicate that MTX was rapidly and quantitatively generated in these cases.
The relative efficiency at which ING-1·hCPA1-T268G used the prodrugs
tested in the cell culture experiments can be assessed by comparing the
amount of conjugate required to drop the prodrug IC50 to
that of MTX itself. For MTX-3-cyclopentyl-Tyr, the amount of conjugate
needed was 10 µg/ml. In comparison, for MTX-Phe the amount needed was
less than 2 µg/ml, since at all conjugate doses tested, MTX-Phe was
as active as MTX itself. This is consistent with the relative
kcat/Km values for these
compounds with hCPA1-T268G (0.16 versus 7.35 × 106 M1 s
1 for
MTX-3-cyclopentyl-Tyr and MTX-Phe, respectively).
The conjugate doses of 2-10 µg/ml required here can be compared with
those reported for other enzyme prodrug systems. Thus, others have
reported the requirement of 0.25-10 µg/ml for antibody-enzyme conjugates using -lactamases (12, 13), 1-10 µg/ml for E. coli
-glucuronidase (22, 23), 10-50 µg/ml for bovine
alkaline phosphatase, and 10-100 µg/ml for penicillin-G amidase (8, 9). This comparison suggests the efficiency of the current system is
comparable with that of most other systems.
The -lactamase system with a doxorubicin prodrug appears to be more
efficient than that reported here. However, the selectivity of the
current system appears to be greater than that of the
-lactamase/doxorubicin system, since in the absence of enzyme, the
doxorubicin prodrug was only 7-fold less toxic than doxorubicin itself,
while the MTX prodrugs were 800-fold less toxic than MTX (13).
Nonetheless, the greater efficiency of the
-lactamase/doxorubicin
system suggests that further efficiency refinements may be warranted in
the current enzyme/prodrug system. Additional refinement also may be
suggested by the inhibition of both WT and mutant hCPAs by both bovine
and human serum. While inhibition by bovine serum was the more severe, inhibition of these enzymes by human serum was significant. Further site-directed or random mutagenesis experiments may permit these refinements. The important advantage of the current system in using a
human enzyme warrants this additional refinement.
In summary, we have generated a novel human enzyme/prodrug combination for MTX (and potentially other antifolates) prodrugs. This system was realized through structure-driven site-directed mutagenesis of hCPA1 in parallel with structure-driven design of in vivo stable prodrugs of MTX. The work provides the first steps for the concept of generating in vivo stable prodrugs of MTX and a human enzyme capable of hydrolyzing them. The utility of this concept may extend well beyond the current enzyme prodrug system and provide a general methodology to make use of the wealth of structural data now available on a variety of human enzymes to design other in vivo stable prodrugs and mutate these enzymes to accept new prodrugs. The advantage that the current system may have for clinical use is the likely low immunogenicity of the mutant hCPA1 along with the known toxicity profile of MTX.
Parts of this work have been reported in the patent literature (40) and in abstract form (41, 42).