Bioconjugate Chem. lQ92, 3, 176-181
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Monoclonal Antibody-@-LactamaseConjugates for the Activation of a Cephalosporin Mustard Prodrug H&an P. Svensson,+John F. Kadow,* Vivekananda M. Vrudhula,’ Philip M. Wallace,+ and Peter D. Senter**+ Bristol-Myers Squibb Pharmaceutical Research Institute, 3005 First Avenue, Seattle, Washington 98121, and 5 Research Parkway, Wallingford, Connecticut 06492. Received December 20, 1991
Cephalosporin mustard (CM) was designed as an anticancer prodrug that could be activated in a sitespecific manner by monoclonal antibody-P-lactamase conjugates targeted to antigens present on tumor cell surfaces. Purified 8-lactamases from Bacillus cereus (BCPL) and Escherichia coli (ECBL)catalyzed the release of phenylenediamine mustard (PDM) from CM through a fragmentation reaction which occurs after the P-lactam ring of CM is hydrolyzed. The K, and V,, values were 5.7 pM and 201 pmol/min per mg for BCPL and 43 pM and 29 pmol/min per mg for ECPL, respectively. Conjugates of BCPL were prepared by combining the F(ab’)z fragments of the maleimide-substituted monoclonal antibodies L6 and 1F5 with thiolated BCPL. The conjugates showed little loss in enzymatic activity and bound nearly as well as the unmodified F(ab’)z monoclonal antibodies to antigens expressed on the H2981 human lung adenocarcinoma cell line (L6 positive, 1F5 negative). PDM was approximately 50-fold more cytotoxic than CM to H2981 cells. Treatment of the cells with L6-BCPL followed by CM resulted in a level of cytotoxic activity that was comparable to that of PDM. This was most likely due to activation of CM by conjugate that bound to cell-surface antigens, since pretreatment of H2981 cells with BCOL or lF5-BCPL enhanced the activity of CM to a lesser extent. Thus, we have shown that CM is a prodrug, and that it can be activated with immunological specificity by a monoclonal antibody@-lactamaseconjugate.
INTRODUCTION The treatment of cancer with conventional chemotherapy is often hampered by low drug specificity,dose-limiting side effects, and poor antitumor activities (I). To address these issues,we have explored a two-step approach in which relatively nontoxic drug precursors (prodrugs) are activated by targeted enzymes (for a review, see ref 2). In the first step, monoclonal antibodies (mAbs)l are used to deliver enzymes to antigens present on tumor cell surfaces. After the unbound conjugate has undergone a sufficient degree of clearance from nontarget tissues, a prodrug is administered that can be converted by the enzyme into an active anticancer drug. We initially tested the feasibility of this approach using mAb-alkaline phosphatase conjugates for the hydrolysis of phosphorylated anticancer drug derivatives (2-5). While significant in vivo antitumor activites were observed, in only one case was the prodrug substantially less toxic than the corresponding drug (5). A larger differential in toxicity will most likely require the use of enzymes that carry out reactions not readily occurring in the body, or are confined to areas inaccessible to the prodrug. Many such enzymes for prodrug activation have been described. They include carboxypeptidase G2 (6, 3,carboxypeptidase A (8), penicillin amidase (91, P-lactamase (10-12), cytosine deaminase (13),and nitroreductase (14). + Seattle, WA.
Wallingford, CT. Abbreviations used: BCPL, Bacillus cereus (3-lactamase(11); CM, cephalosporin mustard; DMF, dimethylformamide; EC(3L Escherichia coli p-lactamase; EDTA, ethylenediaminetetraacetic acid; FITC, fluorescein isothiocyanate; LFE, linear fluorescence equivalence; mAb, monoclonal antibody; PBS, phosphatebuffered saline; PDM, phenylenediamine mustard; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; SMCC,N-succinimidyl4- (maleimidomethyl)cyclohexane-l-carboxylate; 2-IT, 2-iminothiolane hydrochloride. f
Several factors must be taken into account in selecting an enzyme for prodrug activation. These include the molecular weight and physical properties of the enzyme, its activity and stability under physiological conditions, the presence of the enzyme or related enzymeswithin the body, and the nature of the drug that the enzyme generates. Ideally, the enzyme should be able to activate a panel of anticancer drugs that differ mechanistically and have synergistic activities. In these respects, many 8-lactamases hold a great deal of potential because of their favorable kinetics (15)and broad substrate specificities (161,as well as their abilities to effect the elimination of substituents appended to the 3’-position of cephalosporin substrates (10-12,17,18). This report describes the activation of CM, a cephalosporin derivative of phenylenediamine mustard (PDM), by p-lactamases from Escherichia coli, Bacillus cereus, and mAb-@-lactamase conjugates. EXPERIMENTAL PROCEDURES Materials. Crude B. cereus penicillinase (catalog number P0389) and pepsinogen were obtained from Sigma Chemical Co. The E . coli strain expressing the TEM-2 plasmid-mediated @-lactamasewas a gift from Dr. Karen Bush (Bristol-MyersSquibb, Princeton, NJ). L6 (19)and IF5 (20)are IgGze mAbs that bind to antigens on human carcinomas and to the CD20 antigen on normal and neoplastic B-cells, respectively. The human lung adenocarcinoma cell line H2981 has been described previously (19, 21). L6 binds strongly to these cells, while IF5 shows very weak binding. The F(ab’)z fragments of L6 and IF5 were obtained by digestion of the mAbs with 2.2% pepsinogen (w/w) in 0.2 M sodium acetate at pH 4.2 for 7-9 h a t 37 “C. The reaction mixture was brought to pH 7-8 with 1.0 M Tris base, and the F(ab’)Zfragments were purified on a Sephacryl S-200 (Pharmacia) column equilibrated in PBS. The purified product was concentrated by ultrafiltration to 5-10 mgl mL.
1043-1802/92/2903-0176$03.00/0 0 1992 American Chemical Society
&Lactamases for Prodrug Actlvatlon
Preparation of CM. To a solution of 4 g (9.7 mmol) of 3-acetoxy-7-(phenylacetamido)cephalosporanicacid sodium salt (22)in 50 mL of 0.1 M bicarbonate buffer at pH 9 was added 11.7g of immobilized esterase (Bristol-Myers Squibb Industrial Division, Syracuse, NY). After 48 h at room temperature, TLC analysis of the supernatant (Si02 plate, ethyl acetate/methanol/acetic acid, 8:l:l) indicated that the reaction was complete. The product was filtered, partially concentrated, and purified by chromatography on (2-18 silica gel (3 X 24 cm column) equilibrated with H20. The column was first washed with H2O (280 mL) and then with 20% methanol in HzO. Fractions 4-22 (10 mL each) contained the sodium salt of 3-(hydroxymethyl)7-(phenylacetamido)cephalosporanicacid. Lyophilization gave 3.55 g of a pale yellow powder which was used without further purification. The 'H NMR ( D M s 0 - d ~showed ) loss of the acetate methyl group and a new signal at 6 4.0 for the C-7 CH2 group. To a magnetically stirred green suspension of PDM hydrochloride (23) (650 mg, 2.4 mmol) in absolute THF (24 mL) under N2 at 0 "C was added diisopropylethylamine (0.42 mL, 2.4 mmol). After 10 min at 0 "C, a solution of phosgene in toluene (1.9 M, 1.3 mL) was added dropwise. Analysis by TLC (Si02 plate, ethyl acetate/ hexane, 1:4) indicated that the reaction was complete after 1 h and that a less polar product was formed. A suspension of the sodium salt of 3-(hydroxymethy1)7-(pheny1acetamido)cephalosporanic acid (750 mg) in anhydrous toluene (100 mL) was evaporated to dryness under reduced pressure. The residue was dissolved in DMF (40 mL) and cooled to 0 "C under N2, and 1.1 mL of diisopropylethylamine was added. After 5 min, the icecold solution of PDM isocyanate was introduced via a cannula into the DMF solution. The orange solution was allowed to warm to room temperature. After 2 h, the reaction mixture was treated with 1N HCl(l.5 mL), and volatiles were removed under reduced pressure. To the residue was added ethyl acetate (100 mL) and H20 (50 mL), and the pH was adjusted to 3.0 with 1 N HC1 with vigorous stirring. The layers were separated, and the aqueous portion was extracted with ethyl acetate (50 mL). The combined organic layers solution was concentrated to about 25 mL, 10 g of C-18 silica gel was added, and volatiles were removed under reduced pressure. The residual brown powder was applied to a C-18 column (2 X 12 cm) and eluted with 50 % and 70% acetonitrile in 1% aqueous acetic acid (250 mL each). A total of 448 mg (38% yield) of CM was obtained. A portion (300 mg) was triturated with dichloromethane (5 mL) and filtered to give CM (280 mg) as a white fluffy solid. High-resolution mass spectrometry: M+ 606.1116 (obsd),606.1107 (calcd). 'H NMR (DMSO-d6),300 MHz): 6 9.42 (s, 1 H), 9.10 (d, 1H,NH, J = 6Hz),7.24 (m, 7H,Ar-H),6.68 (d, 2 H,Ar-H, J = 9 Hz), 5.66 (dd, 2 H, H-7, J = 6 Hz and J = 9 Hz), 5.09 (d, 1 H, H-6, J = 6 Hz), 4.88 (dd, 2 H, C3-CH20, J = 12 Hz), 3.67 ( ~ , H, 8 ( N C H Z C H ~ C ~3.53 ) ~ )(dd, , 2 H, H-2, J = 15 Hz). Enzymatic Activity. Enzymatic activities of BCPL and mAb conjugates of BCPL were determined using nitrocefin as a substrate (24). Solutions containing BCPL (50 ng/mL) were incubated with nitrocefin (40 pM) in PBS containing 75 pg/mL bovine serum albumin in 96well polystyrene microtiter plates. The absorbance at 490 nm was determined every 2.0 min for 10 min starting 2.0 min after the reaction was initiated. Under these conditions, the increase of absorbance at 490 nm was linear and was directly proportional to the specific activities of the BCPL-containing solutions. Enzyme Purification. (A) From B. cereus. BCPL was purified using a modification of the method of Davies et
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al. (25). Crude 0-lactamase from Sigma was taken up in H20, dialyzed into 10 mM sodium phosphate at pH 7.5 containing 50 mM NaC1, and incubated for 30 min at 60 "C. Precipitated material was removed by centrifugation (2 X 9OOg for 30 min) and the supernatant was applied to a Mono S column (Pharmacia) which was equilibrated with the above phosphate buffer. Purified enzyme was eluted off the column with 10 mM sodium phosphate at pH 7.5 containing 0.3 M NaC1. The protein was concentrated to 2-4 mg/mL by ultrafiltration, filtered through a 0.2-pm filter, and stored at 4 "C. The purity of the BCPL thus obtained was assessed by SDS-PAGE and by specific activity measurements. P-Lactamase concentration was determined spectrophotometrically at 280 nm using El% of 10.5. ( B )From E. coli. Tryptic soybean broth was inoculated with pure colonies of @-lactamaseexpressing E . coli. The cultures were incubated for 18 h at 37 "C and centrifuged (6400g for 15 min at 4 "C). The pellet was washed with PBS, resuspended in PBS, frozen, and then quickly thawed. The debris was centrifuged off (21500g for 20 min at 4 "C) and resuspended in 0.2 M sodium acetate at pH 5.0. Supernatants from four such freeze-thaw cycles were pooled and dialyzed extensively against 20 mM triethanolamine hydrochloride containing 0.5 M NaCl at pH 7.0. After centrifugation (9OOg for 30 min at 4 "C), the supernatant was applied to a boronic acid affinity column (26) which was equilibrated in 20 mM triethanolamine hydrochloride containing 0.5 M NaCl at pH 7.0. The column was washed with this buffer until A280 nm was zero, and the enzyme was then eluted with 0.5 M sodium borate containing 0.5 M NaCl at pH 7.0. ECBL was concentrated by ultrafiltration and the protein concentration was determined using the BCA assay (Pierce Chemical Co.). HPLC Analysis of Drug Release. The release of PDM from CM (50 pM in PBS at 37 "C) by crude BCPL (0.3 pg/mL total protein) was investigated by HPLC using a (2-18 column (4.6 X 250 mm). The gradient conditions were 45-90% acetonitrile in 0.08% diethylamine and 0.08% phosphoric acid (pH 2.3) over 15 min at 1mL/min. The eluant was monitored at 254 nm. Under these conditions, the retention times of PDM and CM were 6.4 and 8.8 min, respectively. Enzyme Kinetics with CM. Kinetic parameters for the P-lactamases (25 ng/mL for BCPL and 1 pg/mL for ECOL) using CM (in PBS containing 12.5 pg/mL bovine serum albumin) as a substrate (4-65 pM with BCPL, 5-50 pM with ECBL) were obtained by determination of the initial velocity of the reaction as a function of substrate concentration. This was estimated from the loss of absorbance at 266 nm (AC266nm = 1.8 X lo4 M-' cm-') that occurs upon P-lactam hydrolysis. The reaction was monitored in a cuvette at room temperature by reading A266nm every 0.1 min for 1.05min starting at 0.15 min after the addition of enzyme. Lineweaver-Burk plots of the data were used to estimate the Km and V,, values. The rate of hydrolysis of CM by BCPL in the presence of EDTA was investigated by incubating the enzyme (1.0 pg/mL in PBS) with 0 (control), 1.0, and 2.5 mM EDTA for 1 h at room temperature. Upon addition of CM (40 p M in PBS), the reaction rate was determined as previously described. Conjugate Preparation. SMCC (Pierce Chemical Co.) was dissolved in DMF at 5.0 mM and added to solutions of the F(ab')~mAbs at 5-10 mg/mL in PBS to give a final SMCC concentration of 0.25 mM. The solution was incubated for 30 min at 30 "C. Unreacted SMCC was removed by gel filtration through Sephadex G-25 (PD-10, Pharmacia) equilibrated in 40 mM sodium phosphate at pH 7.5 containing 0.6 M NaCl. The number of maleim-
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ides (1.1-1.4) incorporated into the mAbs was estimated by adding an excess amount of mercaptoethanol to an aEquot of the solution, followed by titration of free thiols with 5,5'-dithiobis(2-nitrobenzoic acid). 2-IT (Pierce Chemical Co.) was dissolved in 0.5 M sodium borate at pH 8.5 (final concentration 18.7 mM) and added to BCPL at 2-4 mg/mL in 10 mM sodium phosphate containing 0.3 M NaCl at pH 7.5 to give a final 2-IT concentration of 1.7 mM. After incubation for 90 min at 4 "C, unreacted 2-ITwas removed by gel filtration through Sephadex G-25 (PD-10) equilibrated in 40 mM sodium phosphate at pH 7.5 containing 0.6 M NaC1. The number of thiol groups (1.1-1.4) was determined using 5,5'-dithiobis(2-nitrobenzoic acid). Maleimide-substituted F(ab')z and thiolated BCPL were mixed at a 1:l molar ratio and incubated for 1h at room temperature. The solution was cooled to 4 OC, and subsequent manipulations were carried out at this temperature. The reaction was terminated by the addition of 2-aminoethanethiol hydrochloride (100 pM final concentration) and N-ethylmaleimide (200 pM final concentration) in rapid succession. The mixture was concentrated by ultrafiltration and purified on a Sephacryl S-300column equilibrated with 10 mM Tris HC1 at pH 7.5 containing 150 mM NaC1. Fractions containing monomeric mAbBCPL adducts (determined by SDS-PAGE and relative activity) were pooled, dialyzed into 10 mM Tris HCl at pH 7.5 containing 50 mM NaC1, and applied to a CMSephadex column (Pharmacia) equilibrated in the above Tris HC1 buffer. The conjugate was eluted with a linear gradient of 50-300 mM NaCl(10 X column volume) in 10 mM Tris HClat pH 7.5. Alternatively, a stepwise gradient (10 mM Tris HC1 at pH 7.5 containing first 0.1 M NaCl and then 0.3 M NaC1) was used to elute the bound material from the column. The conjugate eluted in the high-salt fractions. Fractions were analyzed by SDS-PAGE and relative enzyme activity. Those fractions containing primarily 1:l conjugated proteins were pooled, dialyzed into saline, concentrated by ultrafiltration to 1.5-3.0 mg/ mL, filtered through a 0.2-pm filter, and stored at -70 "C. Concentrations of conjugated mAb and BCPL were determined spectrophotometrically at 280 nm using an El%of 16.6and 66, respectively. The purity of conjugates was assessed by SDS-PAGE. Cell Binding. LG-F(ab')z-BCOL was tested for its ability to bind to H2981 cells relative to L6-F(ab')z. Cells (7.5 X 105) in 0.2 mL of incomplete modified Dulbecco's medium with 10% fetal bovine serum (v/v)were incubated at 4 "C for 30 min wit1 different proportions of FITClabeled whole L6 and either unlabeled L6-F(ab')2 or L6F(ab')z-BCPL. The total concentration of L6 for the solution was kept constant at 400 nM. After the incubation, the cells were washed twice with medium and analyzed on a fluorescence activated cell sorter for fluorescence intensity. The mean channel number of fluorescence was converted into linear fluorescence equivalence (LFE) and percent of binding was calculated using the following formula: % binding = 100 - 100[(LFEIWw LG-FITC - LFE,,,,,)/ (LFEIW%
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In Vitro Cytotoxicity. H2981 cells in incomplete modified Dulbecco's medium with 10% fetal bovine serum (v/v) were plated into 96-well microtiter plates at 8000 cells/well and allowed to adhere overnight at 37 "C. The cells were exposed to analytically pure CM or PDM for 1 h a t 37 "C, washed three times, and incubated an additional 18 h at 37 "C. This was followed by a 6-h pulse with PHIthymidine (1 pCi/well). The cells were detached by
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Figure 1. Structures of CM and PDM and the mechanism of drug release catalyzed by 6-lactamase. freezing at -20 "C and then harvested onto filter mats using an LKB WALLAC 1295-001 cell harvester. The filter mats were counted on an LKB WALLAC 1205liquid scintillation counter. Cells that were pretreated with mAb-8-lactamase conjugates were exposed to the conjugates at 10 pg/mL (mAb concentration) for 30-40 min at 4 "C, washed five times with cold medium, and then treated with CM. The cells were then washed, incubated, and pulsed as described above. A control experiment included cells that were pretreated with BCPL (2.5pg/mL), washed five times, and then exposed to CM. RESULTS
Preparation and Reactivity of CM. CM (Figure 1) was prepared in two steps from 3-acetoxy-7-(phenylacetamido)cephalosporanic acid (22) by enzymatic removal of the acetoxy group followed by condensation of the resulting 3-(hydroxymethy)-containing product with the isocyanate derivative of PDM (23). The structure of CM was confirmed using high-field NMR and high-resolution mass spectrometry. Preliminary studies were undertaken in order to identify enzymes that were capable of effecting the transformation depicted in Figure 1. Commercially available crude 8lactamase preparations from E. coli, B. cereus, and Enterobacter cloacae were incubated with CM, and the formation of PDM was monitored by analytical methods such as HPLC and UV spectroscopy. It was found that all three enzyme preparations catalyzed the hydrolysis of CM and that PDM was released. With crude B. cereus P-lactamase (0.3 pg/mL), the half-life for the conversion of CM (50 pM in PBS) to PDM was 10 min. Identity of the product formed was determined by HPLC comparison with an authentic sample of PDM. Purification and Characterization of P-Lactamases. Crude p-lactamase from B. cereus was obtained commercially as a mixture of proteins having penicilli-
Bioconjugate Chem., Vol. 3, No. 2, 1992
&Lactamases for Prodrug Activation
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nase and cephalosporinase activities. Analysis of the mixture by SDS-PAGE (Figure 2A, lane 1)revealed the presence of a major component at 30 kDa and a minor component at 25 kDa. These proteins could be separated using a modification of the method of Davies et al. (25). The 30-kDa protein underwent precipitation when the protein mixture was heated at 60 “C. After centrifugation (Figure 2A, lane 2), purified B. cereus P-lactamase (11) (BCPL) was obtained by cation-exchange chromatography (Figure 2A, lane 3). E. coli P-lactamase(ECPL),from a strain that produced a TEM-2 plasmid-mediated enzyme (16), was isolated according to a previously described procedure (26). Briefly, several freeze-thaw cycles of the cells, followed by centrifugation, provided a crude enzyme preparation (Figure 2B, lane 1). Purification was achieved by passing the material through a boronic acid affinity column. SDSPAGE indicated that the enzyme purified in this manner had an apparent molecular weight of 30 kDa (Figure 2B, lane 2). It was possible to spectrophotometrically follow the 6lactam hydrolysis by measuring the decrease in absorbance at 266 nm. With CM as the substrate, the K m and Vm, values for both enzymes were obtained from Lineweaver-Burk plots (Figure 3). BCPL had a Kmof 5.7 pM and a Vm, of 201 pmol/min per mg (Figure 3A). The K m and Vm, values obtained for ECpL were 43 pM and 29 pmol/min per mg, respectively (Figure 3B). The two enzymes were compared with each other using an in vitro cytotoxicity assay. H2981 human lung adenocarcinoma cells were incubated with CM and varying concentrations of either BCPL or ECPL. The concentration required to achieve 50 % cell death was significantly lower for BCPL (4 ng/mL) compared to ECPL (30 ng/mL) (Figure 4). These results are in agreement with the kinetic analyses which show that CM is more rapidly hydrolyzed by BCPL than by ECPL. At this stage, it became necessary to more fully characterizeBCPL and to compare its properties with those of the previously reported B. cereus 0-lactamase (25). Incubation of the purified enzyme with 2.5 mM EDTA prior to addition of CM resulted in complete inhibition of enzyme activity, indicating the presence of an essential metal cofactor. This is consistent with a previous report indicating that BCPL is a zinc metalloenzyme (25). Further confirmation of the identity of the enzyme was obtained by N-terminal amino acid sequence analysis (10 residues, data not shown), which correlated to the published amino acid sequence for the B. cereus enzyme (27).
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Conjugate Preparation, Purification, and Characterization. The F(ab’)2 fragments of the mAbs L6 (anticarcinoma, 19) and 1F5 (anti B-lymphoma, 20) were covalently linked to BCPL through stable thioether bonds. This was achieved by combining maleimidesubstituted mAbs with thiolated BCPL. The formation of high molecular weight aggregates could be minimized by carefully controlling the extent to which each of the proteins was modified. Optimal results were obtained when 1.1-1.4 modifyinggroups per protein molecule were introduced. The composition of the crude conjugation mixture is shown in Figure 5 (lane 2). Purification of the conjugate was achieved using a two-step procedure. Gel filtration was used to separate high molecular weight aggregates and unreacted enzyme from the conjugate-containing fractions (Figure 5, lane 3). Unconjugated mAbs could be removed by cation-exchange chromatography. The resulting conjugateconsisted almost exclusively of a 1:lmAb BCPL adduct which had a molecular weight of approximately 125 kDa (Figure 5, lane 4). A competitive binding assay was used to establish how effectively the conjugates could bind to cell-surface antigens. In this assay, H2981 cells (L6 positive) were exposed to different proportions of L6-BCPL and FITCmodified L6. It was found that L6-BCPL and unmodified L6 were nearly equal in their abilities to compete
180 Bloconjugate Chem., Vol. 3, No. 2, 1992 1
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with FITC-modified L6 for cell-surface binding (Figure 6), indicating that the conjugation chemistry used did not significantlyalter the capability of the L6 moiety to bind to cell surface antigens. 1F5 binds very weakly to H2981 cells (3-5). Enzymatic activity measurements with the purified conjugates were done using nitrocefin as a substrate (24). Typically,the conjugates retained 70-85 % of the enzyme’s specific activity compared to unmodified BCPL (Figure 7). This small loss was probably not directly due to the addition of thiol groups to BCPL with 2-IT, since the thiolated enzyme retained 90-100% of its original activity (data not shown). Conjugate-MediatedCytotoxicity. In vitro cytotoxicity experiments were performed using the H2981human lung adenocarcinomacell line (L6 positive, 1F5 negative).
Figure 8. In vitro cytotoxicity of CM and PDM, on previously untreated H2981 cells (L6 positive, 1F5 negative), and of CM on cells that were pretreated with either L6-BCPL, lF5-BCBL, or BCPL (2.5 pg of BCPL component/mL).
Cells were treated with CM or PDM for 1h, washed, and incubated for a total of 24 h. The incorporation of [3H]thymidine into DNA was used as a measure of cell viability. It was found that CM (IC50 > 30 pM) was significantlyless cytotoxic than PDM (IC50 = 1.5 pM, Figure 8). The cytotoxic activity of CM on cells that were pretreated with L6-BCPL was equal to that of PDM. This activation showed signs of immunological specificity based on the finding that the control conjugate, 1F5-BCPL, caused a smaller degree of enhancement. The cytotoxic effect of CM on 1F5-BCPL-treated cells might be due to insufficient washing or to interactions between the enzyme and the cell surface, since a similar degree of activation was observed when cells were treated with BCPL before exposure to CM. DISCUSSION
The reaction of P-lactamases with appropriately substituted cephalosporins can result in the fragmentation of the 3’-substituentthrough the mechanism shown in Figure 1. This has led to the development of “dual-action cephalosporins” for the treatment of bacterial strains that are antibiotic resistant due to their levels of P-lactamases. Antibacterial activity was obtained when such strains were treated with 3’-substituted cephalosporins that released antibiotics when the P-lactam ring was hydrolyzed (17, 18). Application of this strategy for the release of anticancer drugs such as PDM (12),other nitrogen mustards (12), and vinca alkaloids (IO,11)from corresponding 3’substituted cephalosporins have recently been reported. The 0-lactamasesused to catalyze the release of these drugs were obtained from E. cloacae. We have demonstrated that the mechanistically different (25, 26) P-lactamases from B. cereus and E. coli catalyze the conversion of the prodrug CM into PDM. These enzymes have pronounced structural differences. They share no significant amino acid sequence homology (27,28),and would not be expected to have immunological cross-reactivity. In therapeutic applications,it may therefore be possible to switch enzymes if an immune response against the P-lactamase used initially precludes further treatment. One of the greatest advantages of using P-lactamases for prodrug activation is that a variety of drugs can be released upon hydrolysis of the @-lactamring (10-12,17, 18). Many anticancer agents with amino groups that are essential for activity would be rendered inactive when attached to cephalosporins through carbamate linkages as shown in this paper. Our finding that CM was at least 50 times less cytotoxic than PDM is consistent with what others have reported concerning the effect that electron-
j3-Lactamases for Prodrug Activation
withdrawing groups have on the activities of NJV-bis(2chloroethy1)anilines (29, 30). The activation of CM by L6-BCj3L is a further example of a targeted enzyme capable of activating a relatively noncytotoxic prodrug (2-14). We are currently investigating the in vivo activities of CM combined with monoclonal antibody-BCj3L conjugates. In addition, we are developinga variety of other prodrugs that can be activated by j3-lactamases. ACKNOWLEDGMENT
We wish to thank Karl Erik and Ingegerd Hellstrom for their support throughout the course of this work, Hans Marquardt and Steven Klohr for analyses, and Karen Bush and David Lowe for discussions and useful reagents. 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) Senter, P. D. (1990) Activation of prodrugs by antibodyenzyme conjugates: A new approach t o cancer therapy. FASEB J . 4, 199-193. (3) Senter, P. D., Saulnier, M. G., Schreiber, G. J., Hirschberg, D. L., Brown, J. P., Hellstrom, I., and Hellstrom, K. E. (1988) Antitumor effects of antibody-alkaline phosphatase conjugates in combination with etoposide phosphate. Proc. Natl. Acad. Sci. U.S.A. 85, 4842-4846. (4) Senter,P. D., Schreiber,G. J., Hirschberg,D. L.,Ashe, S.A., Hellstrom, K. E., and Hellstrom, I. (1988) 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. 49,57895792. (5) Wallace, P. M., and Senter P. D. (1991) In vitro and in vivo activities of monoclonal antibody-alkaline phosphatase conjugates in combination with phenol mustard phosphate. Bioconjugate Chem. 2, 349-352. (6) Bagshawe, K. D., Springer, C. J., Searle, F., Antoniw, P., Sharma, S. K., Melton, R. F. (1988) A cytotoxic agent can be generated selectively a t cancer sites. Br. J . Cancer 58, 700703. (7) Springer, C. J., Bagshawe, K. D., Sharma, S. K., Searle, F., Boden, J. A., Antoniw, P., Burke, P. J., Rogers, G. T., Sherwood, R. F., and Melton, R. G. (1991) Ablation of human choriocarcinoma xenografts in nude mice by antibody-directed enzyme prodrug therapy (ADEPT) with three novel compounds. Eur. J . Cancer 27,1361-1366. (8) Esswein, A., Hanseler, E., Montejano, Y., and Huennekens, F. M. (1991) Construction and chemotherapeutic potential of carboxypeptidase-A/monoclonalantibody conjugate. Adv. Enzyme Regul. 31, 3-12. (9) Kerr, D. E., Senter, P. D., Burnett, W. V., Hirschberg, D. L., Hellstrom, I., and Hellstrom, K. E. (1990) Antibody-penicillinV amidase conjugates kill antigen-positive tumor cells when combined with doxorubicin phenoxyacetamide. Cancer Zmmunol. Zmmunother. 31, 202-206. (10) Shepherd,T. A., Jungheim, L. N., Meyer, D. L., and Starling, J. J. (1991) A novel targeted delivery system utilizing a cephalosporin-oncolytic prodrug activated by an antibody P-lactamase conjugate for the treatment of cancer. Bioorg. Med. Chem. Lett. I, 21-26. (11) Meyer,D. L., Jungheim,L. N.,Mikolajczyk, S. D., Shepherd, T. A., Starling, J. J., and Ahlem, C. N. (1992) Preparation and characterization of P-lactamase-Fab' conjugates for the site-
Bioconjugate Chem., Voi. 3, No. 2, 1992 181
specific activation of oncolytic agents. Bioconjugate Chem. 3, 42-48. (12) Alexander, R. P., Beeley, N. R. A.; O'Driscoll, M., ONeill, F. P., Millican, T. A., Pratt, A. J., and Willenbrock, F. W. (1991) Cephalosporin nitrogen mustard carbamate prodrugs for "ADEPT". Tetrahedron Lett. 32, 3269-3272. (13) Senter, P. D., Su, P. C. D., Katsuragi, T., Sakai, T., Cosand, W. L., Hellstrom, I., and Hellstrom, K. E. (1991) Generation of 5-fluorouracil from 5-fluorocytosineby monoclonal antibodycytosine deaminase conjugates. Bioconjugate Chem. 2,447451. (14) Sunters, A., Baer, J., and Bagshawe, K. D. (1991) Cytotoxicity and activation of CB1954 in a human tumour cell line. Biochem. Pharmacol. 9, 1293-1298. (15) Waley, S. G. (1988) P-Lactamases: A major cause of antibiotic resistance. Sci. Prog. (Oxford) 72, 579-597. (16) Bush, K. (1989) Characterization of P-lactamases. Antimicrob. Agents Chemother. 33, 259-263. (17) Mobashery, S., Lerner, S. A., and Johnston, M. (1986) Conscripting P-lactamase for use in drug delivery. Synthesis and biological activity of a cephalosporin C10-ester of an 108, 1685-1686. antibiotic dipeptide. J. Am. Chem. SOC. (18) Albrecht, H. A., Beskid, G., Christenson, J. G., Durkin, J. W., Fallet, V., Georgopapadakau, N. H., Keith, D. D., Konzelmann, F. M., Lipschitz, E. R., McGarry, D. H., Siebelist, J., Wei, C. C., Weigele, M., and Yang, R. (1991) Dual-action cephalosporins: Cephalosporin 3'-quaternary ammonium quinolones. J. Med. Chem. 34, 669-675. (19) 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. (20) 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. (21) Hellstrom, I., Beaumier, P. L., and Hellstrom, K. E. (1986) Antitumor effects of L6,an IgGza antibody that reacts with most human carcinomas. Proc. Natl. Acad. Sci. U.S.A. 83, 7059-7063. (22) Cocker, J. D., Cowley, B. R., Cox, J. S. G., Eardley, S., Gregory, G. I., Lazenby, J. K., Long, A. G., Sly, J. C. P., and Somerfield, G. A. (1965) Cephalosporanic acids. Part 11. Displacement of the acetoxy-group by nucleophiles. J . Chem. SOC. 1965, 5015-5031. (23) Everett, J. L., and Ross, W. C. J. (1949)Aryl-2-halogenoalkylamines. Part 11. J. Chem. SOC. 1949, 1972-1983. (24) Madgwick, P. J., and Waley, S. G. (1987) P-Lactamase I from Bacillus cereus. Biochem. J. 248, 657-662. (25) Davies, R. B., and Abraham, E. P. (1974) Separation, purification and properties of P-lactamase I and j3-lactamase I1 from Bacillus cereus 569/H/9. Biochem. J . 143, 115-127. (26) Cartwright, S. J., and Waley, S. G. (1984) Purification of p-lactamases by affinity chromatography on phenylboronic acid-agarose. Biochem. J. 221, 505-512. (27) Ambler, R. P., Daniel, M., Fleming, J., Hermoso, J. M., Pang, C., and Waley, S. G. (1985) The amino acid sequence of the zinc-requiring P-lactamase from the bacterium Bacillus cereus 569. FEBS Lett. 189, 207-211. (28) Ambler, R. P., and Scott, G. K. (1978) Partial amino acid sequence of penicillinase coded by Escherichia coli plasmid R6K. Proc. Natl. Acad. Sci. U.S.A. 75, 3732-3736. (29) Palmer, B. D., Wilson, W. R., Pullen, S. M., and Denny, W. (1990) Hypoxia-selective antitumor agents. 3. Relationships between structure and cytotoxicity against cultured tumor cells for substituted N,N-bis(2-chloroethyl)anilines.J.Med. Chem. 33,112-121. (30) Chakravarty, P. K., Carl, P. L., Weber, M. J., and Katzenellenbogen, J. A. (1983) Plasmin-activated prodrugs for cancer therapy. 1. Synthesis and biological activity of peptidylacivicin and peptidylphenylenediamine mustard. J. Med. Chem. 26,633-638.
