Bbconjwte Chem. IS91, 2, 447-451
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Generation of 5-Fluorouracil from 5-Fluorocytosine by Monoclonal Antibody-Cytosine Deaminase Conjugates Peter D. Senter,' Peter C. D. Su, Tohoru Katsuragi,+ Takuo Sakai,' Wesley L. Cosand, Ingegerd Hellstrom, and Karl Erik Hellstrom Oncogen Division of the Bristol-Myers Squibb Pharmaceutical Research Institute, 3005 First Avenue, Seattle, Washington 98121, and University of Osaka Prefecture, College of Agriculture, Mozuumemachi, Sakai, Osaka, 591 Japan. Received June 24, 1991
Cytosine deaminase (CDase) catalyzes the conversion of cytosine to uracil and is also able to convert the clinically used antifungal agent 5-fluorocytosine (5FC) into the anticancer drug 5-fluorouracil (5FU). The enzyme was purified from bakers' yeast in a six-step procedure. Studies indicated that bakers' yeast CDase had a molecular weight of approximately 32 kDa and was composed of two subunits of equal molecular weights. Monoclonal antibodies were covalently attached to CDase, forming conjugates that could bind to antigens on tumor cell surfaces. The combination of L6-CDase and 5FC was equivalent in cytotoxic activity to 5FU when tested against the H2981 human lung adenocarcinoma cell line (L6 positive, 1F5 negative). 5FC alone was noncytotoxic. The activation of 5FC was immunologically specific since 1F5-CDase did not enhance 5FC activity.
INTRODUCTION Many of the drugs that are utilized in cancer chemotherapy have dose-limiting side effects that can lead to the destruction of nontarget tissues (I). As a result, much effort has been directed toward the discovery of pharmacologically inactive anticancer drug precursors (prodrugs) that can be activated by enzymes or physiological conditions preferentially found in cancer cells and tumor masses (2-4). For example, prodrugs have been developed to take advantage of the hypoxic nature of solid tumors (5-7), or the presence in some tumors of high levels of enzymes such as plasminogen activator (8). Unfortunately, progress in the development of such anticancer prodrugs for the clinic has been hindered by the lack of a suitable target for drug activation that is abundantly present within tumors compared to normal tissues. We (9-12)and others (13,14)have recently described approaches to prodrug activation in which monoclonal antibodies (mAbs)' that bind to tumor-associated antigens are used to localize enzymes a t tumor masses. The enzymes are selected for their abilities to convert prodrugs into active anticancer drugs. mAbs have been covalently attached to enzymes such as alkaline phosphatase (10, Il), carboxypeptidase G2 (13),penicillin amidase (I2), and 8-lactamase (14)for the activation of a wide range of prodrugs. Significant antitumor activities have been reported. One of the uncertainties surrounding the prodrug substrates for mAb-enzyme conjugates is that nothing is known about their pharmacological properties and toxicities in humans. Because of this, there may be advantages in utilizing anticancer prodrugs that have well-understood clinical properties. This paper describes the conversion of the clinically used antifungal agent 5-fluorocytosine
* Author to whom correspondence should be addressed at On-
cogen.
University of Osaka Prefecture. Abbreviations used: 5FC, 5-fluorocytosine; 5FU, 5-fluorouracil; CDase, cytosine deaminase; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; mAb, monoclonal antibody; PBS, phosphate-buffered saline, pH 7.2; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; Tris, tris(hydroxymethy1)aminomethane. +
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(5FC) into the known antitumor drug 5-fluorouracil (5FU) by mAb-cytosine deaminase (mAb-CDase) conjugates. The properties of both CDase and mAb-CDase conjugates, and the in vitro activities of mAb-CDase in combination with 5FC, are presented. EXPERIMENTAL PROCEDURES Materials. The antibodies used were L6 (IgGPa),which binds to an antigen on human carcinomas (I5),and 1F5 (IgG2a), which binds to the CD20 antigen on normal and neoplastic B-cells (16). The cellline H2981 was established a t Oncogen from a human lung adenocarcinoma. Fluorescence activated cell sorter analysis indicates that L6 binds strongly to H2981 cells, whereas 1F5 shows very weak binding. Purification of CDase. CDase was purified from Fleishmann's compressed bakers' yeast using procedures related to those of Katsuragi and co-workers (17). All steps in the purification were carried out a t 4 OC. The enzyme activity was determined with cytosine or 5FC a t 3 mM in phosphate-buffered saline (PBS) a t 37 "C as described earlier (18). Solutions of the enzyme were added, and the course of reaction was monitored spectrophotometrically on aliquots that were quenched with 0.1 N HC1. The millimolar concentrations of the drugs were calculated as follows: [5FC] = 0.119 X A290 - 0.025 X A255; [5FU] = 0.185 X A255 - 0.049 X A2m; [cytosine] = 0.110 X A2m 0.024 X A2s; [uracil] = 0.158 X A250 - 0.049 X A2w. One unit of enzyme activity is defined as 1pmol of 5FU formed/ min a t 37 "C. Protein concentrations were measured using the BCA assay available from Pierce (Rockford, IL). The step-by-step results of the purification are shown in Table
I. Step 1. Preparation of Yeast Autolysate. Bakers' yeast (2.25 kg) was mixed with ethyl acetate (225 mL) and stirred for 30 min. To this was added 2.25 L of 50 mM potassium phosphate buffer a t pH 7.2 containing 15% (NH412S04, 5 mM EDTA, and 0.1 mM dithiothreitol (DTT). The mixture was stirred for 3 days and the pH was adjusted daily to pH 7.2 with solid tris(hydroxymethy1)aminomethane (Tris). The cell debris was removed by centrifugation a t 17700g for 15 min. Step 2. Ammonium Sulfate Fractionation. Protein was precipitated from the supernatant of step 1 by 0 199 1 American Chemical Society
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Table I. Purification of CDase from 2.25 kg of Bakers' Yeast total total specific protein0 activityb activity fold step (mg) (units) (units/mg) purification 1. cell-free extract 12oooO 1750 0.014 1 1270 0.035 2.5 2. (NHJ)PSOJ 36000 3.Q-Sepharose 4200 685 0.16 11.4 4. G-75 Sephadex 184 648 3.5 250 5.octyl-Sepharose 20 495 25 1800 6.G-75-Sephadex 6 394 67 4800 Protein concentration determined using a BCA assay (Pierce). * One unit of enzyme activity is defined as 1 pmol5FU formed/min at 37 "C. adjusting the EDTA concentration to 5 mM and adding (NH4)2S04 to a final concentration of 70% saturation. After 16 h, the precipitate was collected by centrifugation. The pellet was dissolved in 1.5 L of 50 mM potassium phosphate a t pH 7.2 containing 5 mM EDTA and 0.1 mM DTT, and dialysis against this buffer was allowed to proceed overnight. Additional (NH4)2S04 was added to reach 50% saturation. After 1 h, the precipitate was centrifuged and additional (NH4)2S04 was added to the supernatant so that the final concentration of (NH4)2S04 was 73 % saturation. The precipitate was collected, dissolved in 1L of 20 mM Tris buffer a t pH 8.0 containing 0.1 mM DTT, and dialyzed extensively with this buffer. Step 3. Anion-Exchange Chromatography. The dialysate from step 2 (1.5 L) was applied to a 4.8 X 25 cm Q-Sepharose(Pharmacia) column equilibrated with 20 mM Tris containing 0.1 mM DTT a t pH 8.0. The column was washed with 900 mL of 20 mM Tris containing 0.1 mM DTT, and the enzyme was eluted with a linear gradient (1 L each) of 0-0.3 M KC1 in the above buffer. The fractions containing CDase activity were pooled and concentrated to approximately 20 mL by ultrafiltration (Amicon, PM-30 filter). Step 4. Gel-Permeation Chromatography. The CDase from step 3 was applied to a G-75 Sephadex column (2.5 X 100 cm) and eluted with PBS containing 0.1 mM DTT. The fractions containing CDase activity were pooled and then dialyzed against 4 L of 100mM potassium phosphate buffer containing 1.8 M (NH4)2S04 a t pH 7.0. Step 5. Hydrophobic Interact ion Chromatography. The material from step 4 was applied to a 2.5 X 15 cm octyl-Sepharose(Pharmacia) column equilibrated with 100 mM potassium phosphate containing 1.8 M (NH4)2S04a t pH 7.0. The enzyme was eluted with a linear gradient (300mL each) of 1.8-0 M (NH4)2S04in 100mM potassium phosphate a t pH 7.0. The fractions containing CDase activity were combined and concentrated by ultrafiltration (Amicon, YM-5 filter). Step 6. Gel-Permeation Chromatography. A final gel filtration of the material from step 5 on G-75 Sephadex using PBS as eluant was performed as described in step 4. The purified enzyme was concentrated by ultrdiltration and stored a t -70 "C. Preparation of mAb-CDase Conjugates. To a solution of CDase (2 mL a t 1.5 mg/mL) in 100 mM sodium phosphate a t pH 7 was added 100 pL of succinimidyl4-(Nmaleimidomethy1)cyclohexane-1-carboxylate(Pierce, 20 mM in DMF). After 30 min a t 30 "C, the protein was separated from low molecular weight materials on a G-25 Sephadex column that was eluted with 40 mM sodium phosphate containing 500 mM NaCl a t pH 7.2. Thiol groups were introduced into the mAbs, L6 and 1F5 (1-2 thiols/protein molecule),as previously described (IO). The maleimide- and thiol-modified proteins were
123456789 94, 67, 43
r?
2 x
30
L
= 20.1. 14.4.
Figure 1. Composition of proteins (as determined on a 14% SDS-PAGE gel under nonreducing conditions) during CDase purification. Lanes 1 and 9, molecular weight standards; lane 2, autolysis supernatant (step 1, Table I); lane 3,70% (NHJ)ILSOJ precipitation (step 2, Table I); lane 4, 50-73% (NHd2SO4 precipitation (step 2, Table I); lane 5, Q-Sepharose chromatography (step 3, Table I); lane 6, G-75 Sephadex chromatography (step 4, Table I); lane 7, octyl-Sepharose chromatography (step 5, Table I); lane8, G-75 Sephadex chromatography (step6, Table
I). combined immediately following preparation and characterization in a 1:l molar ratio and allowed to stand at 4 "C overnight. The conjugates were purified by gel filtration on S-300 Sepharose (Pharmacia) a t 4 "C using PBS as eluant. Fractions were monitored at 280 nm and for CDase activity as previouslydescribed. Those fractions consisting mainly of 1:l mAb-CDase adducts by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) were combined, concentrated by ultrafiltration (Amicon, PM-30 filter), and sterile filtered. A t 4 "C the conjugates thus prepared were stable and showed no loss of enzyme activity after several weeks. In Vitro CytotoxicityAssays. H2981 cells in medium (Iscoves' modified Dulbeco's medium with 10%v/v fetal calf serum, 200 units/mL penicillin, 100 pg/mL streptomycin, and 2 mM glutamine) were plated into 96-well microtiter plates a t 10 000 cells/well and allowed to adhere overnight at 37 "C. The cells were washed, and conjugate was added (100 pL a t 10 pg/mL containing 10 unit/mg CDase activity) in medium. After 30 min a t 4 "C, the cells were washed four times, and 0.15 mL of leucine-free RPMI media containing dilutions of 5FC was added to each of the wells. 5FU was added to cells untreated with conjugate. After 18h a t 37 "C, the cells were pulsed with [3H]leucine (1pCi in 50 pL of leucine-free RPMI), and incubation a t 37 "C was continued for 6 h. The cells were frozen at -70 "C, thawed, and harvested onto glass fiber disks. The filters were counted on a Beckman 3701 scintillation counter. RESULTS
Purificationand Characterization of CDase. The six-step procedure used for the purification of CDase is shown in Table I. Starting from 2.25 kg of bakers' yeast, 6 mg of a highly purified preparation of CDase was obtained. This represents a 4800-fold purification, and a 22% overall recovery of enzyme activity. SDS-PAGE analysis (nonreducing) of the protein sample obtained during enzyme purification is shown in Figure 1. Two distinct protein bands a t 17 and 19 kDa were observed for the purified protein sample (lane 8) in a ratio of approximately 41. Complete separation of these two proteins has been achieved by reversed-phase HPLC (H. Marquardt, unpublished results). Analyses of the purified proteins indicated that the major band a t 17 kDa possesses the CDase activity. The molecular weight of CDase was determined, under nondenaturing conditions, by application of the purified
Activation of 5FC by mAb-CDase Conjugate
Bioconjumte Chem., Vol. 2, No. 6, 1991 449 Protein lrubslrale)
0
CDose (cytosine) lF5-CDose Icylosine) L6-CDose lcylosme)
CDose(5FC) IFS.CDoseI5FCI A L6-CDose l 5 F C ) 0 6
Time (min 1
Figure 4. Activity of CDase and mAb-CDase conjugates using cytosine and 5FC (3 mM in PBS at 23 "C) as substrates. The
free CDase solutions contained 5 pg/mL total protein from step 4, Table I. The protein concentrations in the mAb-CDase
conjugates were correctedusing the factor 35 kDa/ 195kDa, which represents the proportion of CDase in the conjugate. L6-CDase IF5-CDase
0
60 I20 180 Volume Eluted (ml)
Figure 2. Elution of CDase off of G-50 Sephadex (1.5 X 100cm). (A) 27 units of CDase (from step 6, Table I) was mixed with 2 mg each of ribonuclease A and ovalbumin and 1 mg of chymotrypsinogen A and eluted with PBS. Fractions were monitored at 280 nm, to determine total protein content, and at 290 nm using the CDase assay with 5FC as a substrate. (B) Elution of CDase off of the same G-50 Sephadex column without the calibration standards.
CDase
a I
404
. " 0
0
10
20
30
Time (hours)
Figure 5. Stability of CDase and mAb-CDase conjugates at 37 "C. The conjugates and CDase (from step 4, Table I) in PBS were incubated at 37 O C . At various intervals,the CDase activity
was determined on aliquots as described in the experimental section.
0
60
120
180
240
Time (hours)
Figure 3. Stability of CDase at 37 "C. Purified CDase (from step 6, Table I) in PBS (0.04 mg/mL) containing protease-free BSA (1mg/mL) was incubated at 37 O C in a polypropylenetube. At various intervals, the CDase activity was determined using 10 MLof the enzyme solution.
enzyme (lane 8, Figure 1)to a G-50 Sephadex column along with other calibration proteins (Figure 2). Fractions were monitored at 280 nm to measure total protein as well as for CDase activity using 5FC as a substrate. CDase activity was centered at 32 kDa, or approximately twice the molecular weight observed by SDS-PAGE (Figure l). This is consistent with the purified enzyme existing in a dimeric noncovalently associated form, but does not rule out the possibility that a disulfide bond between the two chains was broken by reducing agents during purification. Under nondenaturing conditions, purified CDase contained no detectable free thiol groups. Prolonged storage (8months at 4 "C in PBS) did not alter the enzyme's activity or composition by SDS-PAGE. The stability of the CDase activity in the preparation (after step 6, Table I) was determined in PBS at pH 7.2. Using 5FC as a substrate, a slow decrease in enzyme activity was observed upon prolonged incubation a t 37 "C (Figure 3). Under these conditions, the enzyme lost half of its activity after 5.2 days. There was no apparent loss of CDase activity in the first 4 h of incubation (Figure 3, inset). CDase Immunoconjugates. Immunoconjugates of CDase (from step 4 in Table I) were prepared with the mAbs L6 (15) and 1F5 (16) using chemistry described
earlier for the preparation of alkaline phosphatase conjugates (10). Stable thioether bonds between the two proteins were formed by combining mAbs and CDase that were modified with thiols and maleimides, respectively. The crude conjugate preparations were purified by gel filtration and characterized by SDS-PAGE, CDase activity, and binding to cell surface antigens on human H2981 lung adenocarcinoma cells. Yields using this conjugation procedure are consistently around 20%. The remaining protein consists of high molecular weight aggregates, unconjugated material, and mixed fractions. Fluorescence activated cell sorter analysis indicated that L6 bound strongly to the H2981 cells and that antigen saturation occurred at ca. 10pg/mL. At this concentration, the LG-CDase/LG linear fluorescence equivalence ratio was 120/158, indicating that the conjugate bound 82% as efficiently as the unmodified mAb. At a subsaturating concentration (1 pg/mL), the ratio was 67/75, again indicating good binding activity for the L6-CDase conjugate. 1F5-CDase showed no detectable binding up to an antibody concentration of 10 pg/mL. Both the free enzyme and the mAb-CDase conjugates were capable of converting 5FC into 5FU and cytosine into uracil (Figure 4). The rates of product formation catalyzed by the conjugates were approximately equal to that with free CDase. Thus, there was no apparent loss in enzyme activity as a result of conjugation. The finding that the activity was somewhat lower with 5FC than with cytosine is consistent with what has been observed with CDase from Escherichia coli (18). The stability of CDase activity in the mAb-CDase conjugates was compared to that of free CDase at 37 "C in PBS. After 29 h, the conjugates lost very little activity and under these conditions they were a t least as active as the free enzyme (Figure 5). The thermalstability of CDase is not lost as a result of conjugation.
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Bioconjugate Chem., Vol. 2, No. 6, 1991 -loo[
5F,U
/4
Y'. L6-CO t 5FC
-. b
.IFS-CD 4*5FC 100
1000
Drug Concentrotion (pM)
Figure 6. In vitro cytotoxicity of 5FC, 5FU, and mAb-CDase plus 5FC against H2981 cells.
In Vitro Cytotoxicity. The cytotoxic effects of 5FC, 5FU, and combinations of the mAb-CDase conjugates with 5FC were determined by measuring the incorporation of [3H]leucineinto the protein of H2981 cells (Figure 6). 5FU (IC50 = 20 pM)was much more cytotoxic than 5FC (noncytotoxic up to 200 mM). The combination of L6-CDase and 5FC proved to be as cytotoxic as 5FU. The nonbinding conjugate 1F5-CDase did not enhance the activity of 5FC. DISCUSSION 5FU and its deoxyribonucleoside derivative are used extensively in the treatment of metastatic cancer, either as single agents or in combination chemotherapy (19).The drugs are toxic and frequently cause leukopenia and gastrointestinal disorders (20). In contrast, 5FC is lacking in antineoplastic activity and is used in humans for the treatment of fungal infections (21). This activity is most likely due to the conversion of the noncytotoxic prodrug 5FC into 5FU by the CDase present in the fungal cells, but not in mammalian cells (22, 23). Localized generation of 5FU from 5FC has been achieved by surgically implanting encapsulated CDase into subcutaneous tumors growing in rats followed by intraperitoneal treatment with 5FC (22). This approach was designed to minimize the toxic effects of systemically administered 5FU and to improve the activity of the drug by generating it directly a t the tumor. Considerable antitumor activity for the immobilized enzyme-5FC combination was observed. For the treatment of cancers that are widely disseminated, or surgically inaccessible, we felt that a more suitable strategy for localized 5FU generation would be to deliver CDase with mAbs that bind to tumorassociated antigens. The physical properties of CDase from various organisms have been shown to differ significantly in molecular weights, stabilities, activities, and subunit compositions. The thermostable CDase enzymes from Salmonella typhimurium (24)and E . coli (18)have molecular weights above 200 kDa and are composed of several subunits. On the other hand, bakers' yeast CDase has been reported as having a molecular weight of 32-41 kDa based on gel filtration (17, 25), SDS-PAGE (26), and amino acid analysis (26). Immunoconjugates of bakers' yeast CDase were therefore expected to have sufficiently low molecular weights to allow for good penetration into tumors. The CDase isolated, according to the procedures indicated in Table I, displays properties that distinguish it from previously isolated CDase enzymes from bakers' yeast. Gel filtration (Figure 2) indicated that the enzyme had a molecular weight of approximately 32 kDa, but SDSPAGE displayed a single band a t 17 kDa corresponding to CDase activity. Based on these observations, it appears that the purified CDase isolated here is dimeric and that each of the subunits has molecular weights of 17 kDa. In addition, although previous reports indicate bakers' yeast CDase to be thermolabile ( 1 7,23,27),the enzyme purified
according to the methods described in this work has considerable stability a t 37 "C (Figure 3). Enzyme thermostability is a necessary feature for in vivo applications of this targeting strategy, because there may be several days between the administrations of conjugate and prodrug (10, 1 1 , 13). From the in vitro data presented in Figure 5, it is evident that 5FU is formed from 5FC in an immunologically specific manner by L6-CDase that is bound to cell-surface antigens. These studies provide the basisof current studies involving the in vivo activities of mAb-CDase conjugates. ACKNOWLEDGMENT We wish to thank David Hirschberg for performing the cytotoxicity assays, Peter Linsley for valuable discussions, and Ana Wieman for preparing the manuscript. LITERATURE CITED (1) Zubrod, C. G. (1982) Principles of Chemotherapy, Cancer
Medicine (J. F. Holland, and E. Frei, Eds.) pp 627-632, Lea,
and Febiger, Philadelphia. (2) Wilman, D. E. V. (1986) Prodrugs in cancer chemotherapy.
Biochem. SOC. Trans. 14, 375-382. (3) Denny, W. A. (1988) New directions in the design, and evaluation of anti-cancer drugs. Drug Des. Delivery 3, 99124. (4) Conners, T. A. (1986)Prodrugs in cancer chemotherapy. X e nobiotica 16, 975-988. (5) Ross, W. C. J., and Warwick, G. P. (1955) Reduction of cytotoxic compounds by hydrazine and by the xanthine oxidase system. Nature 176, 298-299. (6) Senter, P. D., Pearce, W. E., and Greenfield, R. S. (1990) Development of a drug-releese strategy based on the reductive fragmentation of benzyl carbamate disulfides. J. Org. Chem. 55, 2975-2978. (7) Kennedy, K. A. (1987)Hypoxic cells as specific drug targets for chemotherapy. Anti-Cancer Drug Des. 2, 181-194. (8) Chakravarty, P. K., Carl, P. L., Weber, M. J., and Katzenellenbogen, J. A. (1983) Plasmin-activated prodrugs for cancer chemotherapy. Synthesis and biological activity of peptidylacivicin and peptidylphenylenediamine mustard. J. Med. Chem. 26,633-638. (9) Senter, P. D. (1990) Activation of prodrugs by antibodyenzyme conjugates: A new approach to cancer therapy. FASEB J. 4, 188-193. (10) Senter, P. D., Saulnier, M. G., Schreiber, G. J., Hirschberg, D. L., Brown, J. P., Hellstrom, I., and Hellstrom, K. E. (1988) Anti-tumor effects of antibody-alkaline phosphatase conjugates in combination with etoposide phosphate. Proc. Natl. Acad. Sci. U.S.A. 85,4842-4846. (11) Senter, P. D., Schreiber, G. J., Hirschberg, D. L., Ashe, S. A., Hellstrom, K. E., and Hellstrom, I. (1989) Enhancement of the in vitro and in vivo antitumor activities of phosphorylated mitomycin c and etoposide derivatives by monoclonal antibody-alkaline phosphatase conjugates. Cancer Res. 49, 5789-5792. (12) Kerr, D. E., Senter, P. D., Burnett, W. V., Hirschberg, D. L., Hellstrom, I., and Hellstrom, K. E. (1990) Antibodypenicillin-V-amidase conjugates kill antigen-positive tumor cells when combined with doxorubicin phenoxyacetamide. Cancer Immunol. Immunother. 31, 202-206. 3 ) Bagshawe, K. D., Springer, C. J., Searle, F., Antoniw, P., Sharma, S. K., Melton, R. G., and Sherwood, R. F. (1988) A cytotoxic agent can be generated selectively at cancer sites. Br. J. Cancer 58, 700-703. 4) Shepherd,T. A., Jungheim, L. N.,Meyer,D. L.,andStarling, J. J. (1991)A novel targeted delivery system utilizing a cephalosporin-oncolytic prodrug activated by an antibody 8-lactamase conjugate for the treatment of cancer. Bioorg. Med. Chem. Lett. 1, 21-26.
Actlvatlon of 5FC by mAb-CDase Conjugate (15) Hellstrom, I., Horn, D., Linsley, P. S., Brown, J. P., Brankovan, V., and Hellstrom, K. E. (1986) Monoclonal antibodies raised against human lung carcinoma. Cancer Res. 46,39173923. (16) Clark, E. A., Shu, G., and Ledbetter, J. A. (1985) Role of the Bp35 cell surface polypeptide in human B cell activation. Proc. Natl. Acad. Sci. U.S.A. 82, 1766-1770. (17) Katsuragi, T., Sonoda, T., Matsumoto, K., Sakai, T., and Tonomura, K. (1989) Purification and some properties of cytosine deaminase from bakers’ yeast. Agric. Biol. Chem. 53, 1313-1319. (18) Katsuragi, T., Sakai, T., Matsumoto, K., and Tonomura, K. (1986) Cytosinedeaminase fromEscherichia coli-production, purification, and some characteristics. Agric. Biol. Chem. 50, 1721-1730. (19) Heidelberger, C. (1975) Fluorinated Pyrimidines and Their Nucleosides. Handb. Exp. Pharmacol. 38, 193-231. (20) Heidelberger, C. (1982) Pyrimidine and pyrimidine nucle-
oside antimetabolites. Cancer Medicine (J. F. Holland, and E. Frei, Eds.) pp 801-824, Lea and Febiger, Philadelphia. (21) Steer, P. L., Marks, M. I., Klite, P. D., and Eickhoff, T. C. (1972) 5-Fluorocytosine: An oral antifungal compound. A report on clinical and laboratory experience. Ann. Intern. Med. 76, 15-22. (22) Nishiyama, T., Kawamura, Y., Kawamoto, K., Matsumura, H., Yamamoto, N., Ito, T., Ohyama, A.; Katsuragi, T., and
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Sakai, T. (1985) Antineoplastic effects in rats of B-fluorocytosine in combination with cytosine deaminase capsules. Cancer Res. 45, 1753-1761. (23) Kream, J., and Chargaff, E. (1952) On the cytosine deaminase of yeast. J. Am. Chem. Soc. 74, 5157-5160. (24) West, T. P., Shanley, M. S., and O’Donovan, G. A. (1982) Purification and some properties of cytosine deaminase from Salmonella typhimurium. Biochim.Biophys. Acta 149,11711174. (25) Ipata, P. L., Marmocchi, F., Magni, G., Felicioli, R., and Polidoro, G. (1971) Bakers’ yeast cytosine deaminase. Some
enzymic properties and allosteric inhibition by nucleosides and nucleotides. Biochemistry 10, 4270-4276. (26) Yergatian, S., Lee, J. B., Geisow, M. J., and Ireson, J. C. (1977) Cytosine deaminase: Structural modification studies. Experientia 33, 1570-1571. (27) Katsuragi, T., Sakai, T., and Tonomura, K. (1987) Implantable enzyme capsules for cancer chemotherapy from bakers’yeast cytosine deaminase immobilizedon epoxy-acrylic resin and urethane prepolymer. Appl. Biochem. Biotechnol. 16,61-69.
Registry No. 5FC, 2022-85-7; 5FU, 51-21-8; CDase, 902505-2.
447
Generation of 5-Fluorouracil from 5-Fluorocytosine by Monoclonal Antibody-Cytosine Deaminase Conjugates Peter D. Senter,' Peter C. D. Su, Tohoru Katsuragi,+ Takuo Sakai,' Wesley L. Cosand, Ingegerd Hellstrom, and Karl Erik Hellstrom Oncogen Division of the Bristol-Myers Squibb Pharmaceutical Research Institute, 3005 First Avenue, Seattle, Washington 98121, and University of Osaka Prefecture, College of Agriculture, Mozuumemachi, Sakai, Osaka, 591 Japan. Received June 24, 1991
Cytosine deaminase (CDase) catalyzes the conversion of cytosine to uracil and is also able to convert the clinically used antifungal agent 5-fluorocytosine (5FC) into the anticancer drug 5-fluorouracil (5FU). The enzyme was purified from bakers' yeast in a six-step procedure. Studies indicated that bakers' yeast CDase had a molecular weight of approximately 32 kDa and was composed of two subunits of equal molecular weights. Monoclonal antibodies were covalently attached to CDase, forming conjugates that could bind to antigens on tumor cell surfaces. The combination of L6-CDase and 5FC was equivalent in cytotoxic activity to 5FU when tested against the H2981 human lung adenocarcinoma cell line (L6 positive, 1F5 negative). 5FC alone was noncytotoxic. The activation of 5FC was immunologically specific since 1F5-CDase did not enhance 5FC activity.
INTRODUCTION Many of the drugs that are utilized in cancer chemotherapy have dose-limiting side effects that can lead to the destruction of nontarget tissues (I). As a result, much effort has been directed toward the discovery of pharmacologically inactive anticancer drug precursors (prodrugs) that can be activated by enzymes or physiological conditions preferentially found in cancer cells and tumor masses (2-4). For example, prodrugs have been developed to take advantage of the hypoxic nature of solid tumors (5-7), or the presence in some tumors of high levels of enzymes such as plasminogen activator (8). Unfortunately, progress in the development of such anticancer prodrugs for the clinic has been hindered by the lack of a suitable target for drug activation that is abundantly present within tumors compared to normal tissues. We (9-12)and others (13,14)have recently described approaches to prodrug activation in which monoclonal antibodies (mAbs)' that bind to tumor-associated antigens are used to localize enzymes a t tumor masses. The enzymes are selected for their abilities to convert prodrugs into active anticancer drugs. mAbs have been covalently attached to enzymes such as alkaline phosphatase (10, Il), carboxypeptidase G2 (13),penicillin amidase (I2), and 8-lactamase (14)for the activation of a wide range of prodrugs. Significant antitumor activities have been reported. One of the uncertainties surrounding the prodrug substrates for mAb-enzyme conjugates is that nothing is known about their pharmacological properties and toxicities in humans. Because of this, there may be advantages in utilizing anticancer prodrugs that have well-understood clinical properties. This paper describes the conversion of the clinically used antifungal agent 5-fluorocytosine
* Author to whom correspondence should be addressed at On-
cogen.
University of Osaka Prefecture. Abbreviations used: 5FC, 5-fluorocytosine; 5FU, 5-fluorouracil; CDase, cytosine deaminase; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; mAb, monoclonal antibody; PBS, phosphate-buffered saline, pH 7.2; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; Tris, tris(hydroxymethy1)aminomethane. +
1043-1002/91/2902-0447$02.50/0
(5FC) into the known antitumor drug 5-fluorouracil (5FU) by mAb-cytosine deaminase (mAb-CDase) conjugates. The properties of both CDase and mAb-CDase conjugates, and the in vitro activities of mAb-CDase in combination with 5FC, are presented. EXPERIMENTAL PROCEDURES Materials. The antibodies used were L6 (IgGPa),which binds to an antigen on human carcinomas (I5),and 1F5 (IgG2a), which binds to the CD20 antigen on normal and neoplastic B-cells (16). The cellline H2981 was established a t Oncogen from a human lung adenocarcinoma. Fluorescence activated cell sorter analysis indicates that L6 binds strongly to H2981 cells, whereas 1F5 shows very weak binding. Purification of CDase. CDase was purified from Fleishmann's compressed bakers' yeast using procedures related to those of Katsuragi and co-workers (17). All steps in the purification were carried out a t 4 OC. The enzyme activity was determined with cytosine or 5FC a t 3 mM in phosphate-buffered saline (PBS) a t 37 "C as described earlier (18). Solutions of the enzyme were added, and the course of reaction was monitored spectrophotometrically on aliquots that were quenched with 0.1 N HC1. The millimolar concentrations of the drugs were calculated as follows: [5FC] = 0.119 X A290 - 0.025 X A255; [5FU] = 0.185 X A255 - 0.049 X A2m; [cytosine] = 0.110 X A2m 0.024 X A2s; [uracil] = 0.158 X A250 - 0.049 X A2w. One unit of enzyme activity is defined as 1pmol of 5FU formed/ min a t 37 "C. Protein concentrations were measured using the BCA assay available from Pierce (Rockford, IL). The step-by-step results of the purification are shown in Table
I. Step 1. Preparation of Yeast Autolysate. Bakers' yeast (2.25 kg) was mixed with ethyl acetate (225 mL) and stirred for 30 min. To this was added 2.25 L of 50 mM potassium phosphate buffer a t pH 7.2 containing 15% (NH412S04, 5 mM EDTA, and 0.1 mM dithiothreitol (DTT). The mixture was stirred for 3 days and the pH was adjusted daily to pH 7.2 with solid tris(hydroxymethy1)aminomethane (Tris). The cell debris was removed by centrifugation a t 17700g for 15 min. Step 2. Ammonium Sulfate Fractionation. Protein was precipitated from the supernatant of step 1 by 0 199 1 American Chemical Society
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Table I. Purification of CDase from 2.25 kg of Bakers' Yeast total total specific protein0 activityb activity fold step (mg) (units) (units/mg) purification 1. cell-free extract 12oooO 1750 0.014 1 1270 0.035 2.5 2. (NHJ)PSOJ 36000 3.Q-Sepharose 4200 685 0.16 11.4 4. G-75 Sephadex 184 648 3.5 250 5.octyl-Sepharose 20 495 25 1800 6.G-75-Sephadex 6 394 67 4800 Protein concentration determined using a BCA assay (Pierce). * One unit of enzyme activity is defined as 1 pmol5FU formed/min at 37 "C. adjusting the EDTA concentration to 5 mM and adding (NH4)2S04 to a final concentration of 70% saturation. After 16 h, the precipitate was collected by centrifugation. The pellet was dissolved in 1.5 L of 50 mM potassium phosphate a t pH 7.2 containing 5 mM EDTA and 0.1 mM DTT, and dialysis against this buffer was allowed to proceed overnight. Additional (NH4)2S04 was added to reach 50% saturation. After 1 h, the precipitate was centrifuged and additional (NH4)2S04 was added to the supernatant so that the final concentration of (NH4)2S04 was 73 % saturation. The precipitate was collected, dissolved in 1L of 20 mM Tris buffer a t pH 8.0 containing 0.1 mM DTT, and dialyzed extensively with this buffer. Step 3. Anion-Exchange Chromatography. The dialysate from step 2 (1.5 L) was applied to a 4.8 X 25 cm Q-Sepharose(Pharmacia) column equilibrated with 20 mM Tris containing 0.1 mM DTT a t pH 8.0. The column was washed with 900 mL of 20 mM Tris containing 0.1 mM DTT, and the enzyme was eluted with a linear gradient (1 L each) of 0-0.3 M KC1 in the above buffer. The fractions containing CDase activity were pooled and concentrated to approximately 20 mL by ultrafiltration (Amicon, PM-30 filter). Step 4. Gel-Permeation Chromatography. The CDase from step 3 was applied to a G-75 Sephadex column (2.5 X 100 cm) and eluted with PBS containing 0.1 mM DTT. The fractions containing CDase activity were pooled and then dialyzed against 4 L of 100mM potassium phosphate buffer containing 1.8 M (NH4)2S04 a t pH 7.0. Step 5. Hydrophobic Interact ion Chromatography. The material from step 4 was applied to a 2.5 X 15 cm octyl-Sepharose(Pharmacia) column equilibrated with 100 mM potassium phosphate containing 1.8 M (NH4)2S04a t pH 7.0. The enzyme was eluted with a linear gradient (300mL each) of 1.8-0 M (NH4)2S04in 100mM potassium phosphate a t pH 7.0. The fractions containing CDase activity were combined and concentrated by ultrafiltration (Amicon, YM-5 filter). Step 6. Gel-Permeation Chromatography. A final gel filtration of the material from step 5 on G-75 Sephadex using PBS as eluant was performed as described in step 4. The purified enzyme was concentrated by ultrdiltration and stored a t -70 "C. Preparation of mAb-CDase Conjugates. To a solution of CDase (2 mL a t 1.5 mg/mL) in 100 mM sodium phosphate a t pH 7 was added 100 pL of succinimidyl4-(Nmaleimidomethy1)cyclohexane-1-carboxylate(Pierce, 20 mM in DMF). After 30 min a t 30 "C, the protein was separated from low molecular weight materials on a G-25 Sephadex column that was eluted with 40 mM sodium phosphate containing 500 mM NaCl a t pH 7.2. Thiol groups were introduced into the mAbs, L6 and 1F5 (1-2 thiols/protein molecule),as previously described (IO). The maleimide- and thiol-modified proteins were
123456789 94, 67, 43
r?
2 x
30
L
= 20.1. 14.4.
Figure 1. Composition of proteins (as determined on a 14% SDS-PAGE gel under nonreducing conditions) during CDase purification. Lanes 1 and 9, molecular weight standards; lane 2, autolysis supernatant (step 1, Table I); lane 3,70% (NHJ)ILSOJ precipitation (step 2, Table I); lane 4, 50-73% (NHd2SO4 precipitation (step 2, Table I); lane 5, Q-Sepharose chromatography (step 3, Table I); lane 6, G-75 Sephadex chromatography (step 4, Table I); lane 7, octyl-Sepharose chromatography (step 5, Table I); lane8, G-75 Sephadex chromatography (step6, Table
I). combined immediately following preparation and characterization in a 1:l molar ratio and allowed to stand at 4 "C overnight. The conjugates were purified by gel filtration on S-300 Sepharose (Pharmacia) a t 4 "C using PBS as eluant. Fractions were monitored at 280 nm and for CDase activity as previouslydescribed. Those fractions consisting mainly of 1:l mAb-CDase adducts by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) were combined, concentrated by ultrafiltration (Amicon, PM-30 filter), and sterile filtered. A t 4 "C the conjugates thus prepared were stable and showed no loss of enzyme activity after several weeks. In Vitro CytotoxicityAssays. H2981 cells in medium (Iscoves' modified Dulbeco's medium with 10%v/v fetal calf serum, 200 units/mL penicillin, 100 pg/mL streptomycin, and 2 mM glutamine) were plated into 96-well microtiter plates a t 10 000 cells/well and allowed to adhere overnight at 37 "C. The cells were washed, and conjugate was added (100 pL a t 10 pg/mL containing 10 unit/mg CDase activity) in medium. After 30 min a t 4 "C, the cells were washed four times, and 0.15 mL of leucine-free RPMI media containing dilutions of 5FC was added to each of the wells. 5FU was added to cells untreated with conjugate. After 18h a t 37 "C, the cells were pulsed with [3H]leucine (1pCi in 50 pL of leucine-free RPMI), and incubation a t 37 "C was continued for 6 h. The cells were frozen at -70 "C, thawed, and harvested onto glass fiber disks. The filters were counted on a Beckman 3701 scintillation counter. RESULTS
Purificationand Characterization of CDase. The six-step procedure used for the purification of CDase is shown in Table I. Starting from 2.25 kg of bakers' yeast, 6 mg of a highly purified preparation of CDase was obtained. This represents a 4800-fold purification, and a 22% overall recovery of enzyme activity. SDS-PAGE analysis (nonreducing) of the protein sample obtained during enzyme purification is shown in Figure 1. Two distinct protein bands a t 17 and 19 kDa were observed for the purified protein sample (lane 8) in a ratio of approximately 41. Complete separation of these two proteins has been achieved by reversed-phase HPLC (H. Marquardt, unpublished results). Analyses of the purified proteins indicated that the major band a t 17 kDa possesses the CDase activity. The molecular weight of CDase was determined, under nondenaturing conditions, by application of the purified
Activation of 5FC by mAb-CDase Conjugate
Bioconjumte Chem., Vol. 2, No. 6, 1991 449 Protein lrubslrale)
0
CDose (cytosine) lF5-CDose Icylosine) L6-CDose lcylosme)
CDose(5FC) IFS.CDoseI5FCI A L6-CDose l 5 F C ) 0 6
Time (min 1
Figure 4. Activity of CDase and mAb-CDase conjugates using cytosine and 5FC (3 mM in PBS at 23 "C) as substrates. The
free CDase solutions contained 5 pg/mL total protein from step 4, Table I. The protein concentrations in the mAb-CDase
conjugates were correctedusing the factor 35 kDa/ 195kDa, which represents the proportion of CDase in the conjugate. L6-CDase IF5-CDase
0
60 I20 180 Volume Eluted (ml)
Figure 2. Elution of CDase off of G-50 Sephadex (1.5 X 100cm). (A) 27 units of CDase (from step 6, Table I) was mixed with 2 mg each of ribonuclease A and ovalbumin and 1 mg of chymotrypsinogen A and eluted with PBS. Fractions were monitored at 280 nm, to determine total protein content, and at 290 nm using the CDase assay with 5FC as a substrate. (B) Elution of CDase off of the same G-50 Sephadex column without the calibration standards.
CDase
a I
404
. " 0
0
10
20
30
Time (hours)
Figure 5. Stability of CDase and mAb-CDase conjugates at 37 "C. The conjugates and CDase (from step 4, Table I) in PBS were incubated at 37 O C . At various intervals,the CDase activity
was determined on aliquots as described in the experimental section.
0
60
120
180
240
Time (hours)
Figure 3. Stability of CDase at 37 "C. Purified CDase (from step 6, Table I) in PBS (0.04 mg/mL) containing protease-free BSA (1mg/mL) was incubated at 37 O C in a polypropylenetube. At various intervals, the CDase activity was determined using 10 MLof the enzyme solution.
enzyme (lane 8, Figure 1)to a G-50 Sephadex column along with other calibration proteins (Figure 2). Fractions were monitored at 280 nm to measure total protein as well as for CDase activity using 5FC as a substrate. CDase activity was centered at 32 kDa, or approximately twice the molecular weight observed by SDS-PAGE (Figure l). This is consistent with the purified enzyme existing in a dimeric noncovalently associated form, but does not rule out the possibility that a disulfide bond between the two chains was broken by reducing agents during purification. Under nondenaturing conditions, purified CDase contained no detectable free thiol groups. Prolonged storage (8months at 4 "C in PBS) did not alter the enzyme's activity or composition by SDS-PAGE. The stability of the CDase activity in the preparation (after step 6, Table I) was determined in PBS at pH 7.2. Using 5FC as a substrate, a slow decrease in enzyme activity was observed upon prolonged incubation a t 37 "C (Figure 3). Under these conditions, the enzyme lost half of its activity after 5.2 days. There was no apparent loss of CDase activity in the first 4 h of incubation (Figure 3, inset). CDase Immunoconjugates. Immunoconjugates of CDase (from step 4 in Table I) were prepared with the mAbs L6 (15) and 1F5 (16) using chemistry described
earlier for the preparation of alkaline phosphatase conjugates (10). Stable thioether bonds between the two proteins were formed by combining mAbs and CDase that were modified with thiols and maleimides, respectively. The crude conjugate preparations were purified by gel filtration and characterized by SDS-PAGE, CDase activity, and binding to cell surface antigens on human H2981 lung adenocarcinoma cells. Yields using this conjugation procedure are consistently around 20%. The remaining protein consists of high molecular weight aggregates, unconjugated material, and mixed fractions. Fluorescence activated cell sorter analysis indicated that L6 bound strongly to the H2981 cells and that antigen saturation occurred at ca. 10pg/mL. At this concentration, the LG-CDase/LG linear fluorescence equivalence ratio was 120/158, indicating that the conjugate bound 82% as efficiently as the unmodified mAb. At a subsaturating concentration (1 pg/mL), the ratio was 67/75, again indicating good binding activity for the L6-CDase conjugate. 1F5-CDase showed no detectable binding up to an antibody concentration of 10 pg/mL. Both the free enzyme and the mAb-CDase conjugates were capable of converting 5FC into 5FU and cytosine into uracil (Figure 4). The rates of product formation catalyzed by the conjugates were approximately equal to that with free CDase. Thus, there was no apparent loss in enzyme activity as a result of conjugation. The finding that the activity was somewhat lower with 5FC than with cytosine is consistent with what has been observed with CDase from Escherichia coli (18). The stability of CDase activity in the mAb-CDase conjugates was compared to that of free CDase at 37 "C in PBS. After 29 h, the conjugates lost very little activity and under these conditions they were a t least as active as the free enzyme (Figure 5). The thermalstability of CDase is not lost as a result of conjugation.
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Bioconjugate Chem., Vol. 2, No. 6, 1991 -loo[
5F,U
/4
Y'. L6-CO t 5FC
-. b
.IFS-CD 4*5FC 100
1000
Drug Concentrotion (pM)
Figure 6. In vitro cytotoxicity of 5FC, 5FU, and mAb-CDase plus 5FC against H2981 cells.
In Vitro Cytotoxicity. The cytotoxic effects of 5FC, 5FU, and combinations of the mAb-CDase conjugates with 5FC were determined by measuring the incorporation of [3H]leucineinto the protein of H2981 cells (Figure 6). 5FU (IC50 = 20 pM)was much more cytotoxic than 5FC (noncytotoxic up to 200 mM). The combination of L6-CDase and 5FC proved to be as cytotoxic as 5FU. The nonbinding conjugate 1F5-CDase did not enhance the activity of 5FC. DISCUSSION 5FU and its deoxyribonucleoside derivative are used extensively in the treatment of metastatic cancer, either as single agents or in combination chemotherapy (19).The drugs are toxic and frequently cause leukopenia and gastrointestinal disorders (20). In contrast, 5FC is lacking in antineoplastic activity and is used in humans for the treatment of fungal infections (21). This activity is most likely due to the conversion of the noncytotoxic prodrug 5FC into 5FU by the CDase present in the fungal cells, but not in mammalian cells (22, 23). Localized generation of 5FU from 5FC has been achieved by surgically implanting encapsulated CDase into subcutaneous tumors growing in rats followed by intraperitoneal treatment with 5FC (22). This approach was designed to minimize the toxic effects of systemically administered 5FU and to improve the activity of the drug by generating it directly a t the tumor. Considerable antitumor activity for the immobilized enzyme-5FC combination was observed. For the treatment of cancers that are widely disseminated, or surgically inaccessible, we felt that a more suitable strategy for localized 5FU generation would be to deliver CDase with mAbs that bind to tumorassociated antigens. The physical properties of CDase from various organisms have been shown to differ significantly in molecular weights, stabilities, activities, and subunit compositions. The thermostable CDase enzymes from Salmonella typhimurium (24)and E . coli (18)have molecular weights above 200 kDa and are composed of several subunits. On the other hand, bakers' yeast CDase has been reported as having a molecular weight of 32-41 kDa based on gel filtration (17, 25), SDS-PAGE (26), and amino acid analysis (26). Immunoconjugates of bakers' yeast CDase were therefore expected to have sufficiently low molecular weights to allow for good penetration into tumors. The CDase isolated, according to the procedures indicated in Table I, displays properties that distinguish it from previously isolated CDase enzymes from bakers' yeast. Gel filtration (Figure 2) indicated that the enzyme had a molecular weight of approximately 32 kDa, but SDSPAGE displayed a single band a t 17 kDa corresponding to CDase activity. Based on these observations, it appears that the purified CDase isolated here is dimeric and that each of the subunits has molecular weights of 17 kDa. In addition, although previous reports indicate bakers' yeast CDase to be thermolabile ( 1 7,23,27),the enzyme purified
according to the methods described in this work has considerable stability a t 37 "C (Figure 3). Enzyme thermostability is a necessary feature for in vivo applications of this targeting strategy, because there may be several days between the administrations of conjugate and prodrug (10, 1 1 , 13). From the in vitro data presented in Figure 5, it is evident that 5FU is formed from 5FC in an immunologically specific manner by L6-CDase that is bound to cell-surface antigens. These studies provide the basisof current studies involving the in vivo activities of mAb-CDase conjugates. ACKNOWLEDGMENT We wish to thank David Hirschberg for performing the cytotoxicity assays, Peter Linsley for valuable discussions, and Ana Wieman for preparing the manuscript. LITERATURE CITED (1) Zubrod, C. G. (1982) Principles of Chemotherapy, Cancer
Medicine (J. F. Holland, and E. Frei, Eds.) pp 627-632, Lea,
and Febiger, Philadelphia. (2) Wilman, D. E. V. (1986) Prodrugs in cancer chemotherapy.
Biochem. SOC. Trans. 14, 375-382. (3) Denny, W. A. (1988) New directions in the design, and evaluation of anti-cancer drugs. Drug Des. Delivery 3, 99124. (4) Conners, T. A. (1986)Prodrugs in cancer chemotherapy. X e nobiotica 16, 975-988. (5) Ross, W. C. J., and Warwick, G. P. (1955) Reduction of cytotoxic compounds by hydrazine and by the xanthine oxidase system. Nature 176, 298-299. (6) Senter, P. D., Pearce, W. E., and Greenfield, R. S. (1990) Development of a drug-releese strategy based on the reductive fragmentation of benzyl carbamate disulfides. J. Org. Chem. 55, 2975-2978. (7) Kennedy, K. A. (1987)Hypoxic cells as specific drug targets for chemotherapy. Anti-Cancer Drug Des. 2, 181-194. (8) Chakravarty, P. K., Carl, P. L., Weber, M. J., and Katzenellenbogen, J. A. (1983) Plasmin-activated prodrugs for cancer chemotherapy. Synthesis and biological activity of peptidylacivicin and peptidylphenylenediamine mustard. J. Med. Chem. 26,633-638. (9) Senter, P. D. (1990) Activation of prodrugs by antibodyenzyme conjugates: A new approach to cancer therapy. FASEB J. 4, 188-193. (10) Senter, P. D., Saulnier, M. G., Schreiber, G. J., Hirschberg, D. L., Brown, J. P., Hellstrom, I., and Hellstrom, K. E. (1988) Anti-tumor effects of antibody-alkaline phosphatase conjugates in combination with etoposide phosphate. Proc. Natl. Acad. Sci. U.S.A. 85,4842-4846. (11) Senter, P. D., Schreiber, G. J., Hirschberg, D. L., Ashe, S. A., Hellstrom, K. E., and Hellstrom, I. (1989) Enhancement of the in vitro and in vivo antitumor activities of phosphorylated mitomycin c and etoposide derivatives by monoclonal antibody-alkaline phosphatase conjugates. Cancer Res. 49, 5789-5792. (12) Kerr, D. E., Senter, P. D., Burnett, W. V., Hirschberg, D. L., Hellstrom, I., and Hellstrom, K. E. (1990) Antibodypenicillin-V-amidase conjugates kill antigen-positive tumor cells when combined with doxorubicin phenoxyacetamide. Cancer Immunol. Immunother. 31, 202-206. 3 ) Bagshawe, K. D., Springer, C. J., Searle, F., Antoniw, P., Sharma, S. K., Melton, R. G., and Sherwood, R. F. (1988) A cytotoxic agent can be generated selectively at cancer sites. Br. J. Cancer 58, 700-703. 4) Shepherd,T. A., Jungheim, L. N.,Meyer,D. L.,andStarling, J. J. (1991)A novel targeted delivery system utilizing a cephalosporin-oncolytic prodrug activated by an antibody 8-lactamase conjugate for the treatment of cancer. Bioorg. Med. Chem. Lett. 1, 21-26.
Actlvatlon of 5FC by mAb-CDase Conjugate (15) Hellstrom, I., Horn, D., Linsley, P. S., Brown, J. P., Brankovan, V., and Hellstrom, K. E. (1986) Monoclonal antibodies raised against human lung carcinoma. Cancer Res. 46,39173923. (16) Clark, E. A., Shu, G., and Ledbetter, J. A. (1985) Role of the Bp35 cell surface polypeptide in human B cell activation. Proc. Natl. Acad. Sci. U.S.A. 82, 1766-1770. (17) Katsuragi, T., Sonoda, T., Matsumoto, K., Sakai, T., and Tonomura, K. (1989) Purification and some properties of cytosine deaminase from bakers’ yeast. Agric. Biol. Chem. 53, 1313-1319. (18) Katsuragi, T., Sakai, T., Matsumoto, K., and Tonomura, K. (1986) Cytosinedeaminase fromEscherichia coli-production, purification, and some characteristics. Agric. Biol. Chem. 50, 1721-1730. (19) Heidelberger, C. (1975) Fluorinated Pyrimidines and Their Nucleosides. Handb. Exp. Pharmacol. 38, 193-231. (20) Heidelberger, C. (1982) Pyrimidine and pyrimidine nucle-
oside antimetabolites. Cancer Medicine (J. F. Holland, and E. Frei, Eds.) pp 801-824, Lea and Febiger, Philadelphia. (21) Steer, P. L., Marks, M. I., Klite, P. D., and Eickhoff, T. C. (1972) 5-Fluorocytosine: An oral antifungal compound. A report on clinical and laboratory experience. Ann. Intern. Med. 76, 15-22. (22) Nishiyama, T., Kawamura, Y., Kawamoto, K., Matsumura, H., Yamamoto, N., Ito, T., Ohyama, A.; Katsuragi, T., and
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Sakai, T. (1985) Antineoplastic effects in rats of B-fluorocytosine in combination with cytosine deaminase capsules. Cancer Res. 45, 1753-1761. (23) Kream, J., and Chargaff, E. (1952) On the cytosine deaminase of yeast. J. Am. Chem. Soc. 74, 5157-5160. (24) West, T. P., Shanley, M. S., and O’Donovan, G. A. (1982) Purification and some properties of cytosine deaminase from Salmonella typhimurium. Biochim.Biophys. Acta 149,11711174. (25) Ipata, P. L., Marmocchi, F., Magni, G., Felicioli, R., and Polidoro, G. (1971) Bakers’ yeast cytosine deaminase. Some
enzymic properties and allosteric inhibition by nucleosides and nucleotides. Biochemistry 10, 4270-4276. (26) Yergatian, S., Lee, J. B., Geisow, M. J., and Ireson, J. C. (1977) Cytosine deaminase: Structural modification studies. Experientia 33, 1570-1571. (27) Katsuragi, T., Sakai, T., and Tonomura, K. (1987) Implantable enzyme capsules for cancer chemotherapy from bakers’yeast cytosine deaminase immobilizedon epoxy-acrylic resin and urethane prepolymer. Appl. Biochem. Biotechnol. 16,61-69.
Registry No. 5FC, 2022-85-7; 5FU, 51-21-8; CDase, 902505-2.
