Bloconjugate Chem. 1003,4, 334-340
334
Antitumor Activities of a Cephalosporin Prodrug in Combination with Monoclonal Antibody-@-LactamaseConjugates Vivekananda M. Vrudhula, Hhkan P. Svensson, Karen A. Kennedy, Peter D. Senter,’ and Philip M. Wallace’ Bristol-Myers Squibb Pharmaceutical Research Institute, 3005 First Avenue, Seattle, Washington 98121. Received March 11, 1993
7-(Pheny1acetamido)cephalosporinmustard (CM) and 7-(4-carboxybutanamido)cepahalosporinmustard (CCM) were developed as anticancer prodrugs that could be activated site selectively by monoclonal antibody-8-lactamase conjugates targeted to antigens present on tumor cell surfaces. Both CM and CCM were hydrolyzed by purified 8-lactamases from Escherichia coli (ECPL),Bacillus cereus (BCPL), and Enterobacter cloacae (EClPL). This resulted in the release of phenylenediamine mustard (PDM), a potent cytotoxic drug. The K,,,and kat values of the reactions were determined, and it was found that EClj3L effected the hydrolysis of CM and CCM more rapidly than the other enzymes. Conjugates of EClpL were prepared by reacting maleimide-substituted F(ab’)n fragments of the monoclonalantibodies L6 and P1.17 to EClgL that had been modified with sulfhydryl groups. In vitro experiments indicated that CCM (IC60 = 25-45 pM) was less toxic than PDM (IC60 = 1.5 pM) to H2981 lung adenocarcinoma cells (L6 antigen positive, P1.17 antigen negative) and that immunologically specific prodrug activation took place when the cells were treated with LG-EClPL. In vivo experiments in nude mice demonstrated that CCM was less toxic than CM, and that both prodrugs were much less toxic than PDM. Neither CCM nor PDM exerted antitumor activity on subcutaneous H2981 tumors in vivo. However, a significant antitumor effect was obtained in mice that received L6-EClPL 96 h prior to the administration of CCM. The effect was immunologically specific (P< 0.051, since a smaller degree of antitumor activity was obtained in mice that received the nonbinding control conjugate P1.17-EClPL prior to CCM. These studies demonstrate the potential therapeutic utility of monoclonal antibody-0-lactamase conjugates for the activation of cephalosporin-containing prodrugs.
INTRODUCTION Several recent reports have described the use of monoclonal antibodies (mAbs)’ as delivery agents for enzymes that are capable of generating anticancer drugs from relatively noncytotoxic precursors (reviewed in ref 1).By introducing a high level of an enzyme activity within a tumor mass, it should be possible to convert a suitable prodrug substrate into an active anticancer agent and obtain a level of selectivity that cannot be achieved by systemic administration of the corresponding released drug. A considerable amount of evidence has accumulated indicating that substantial levels of in vitro and in vivo antitumor activities can be obtained using mAb-enzyme conjugates for prodrug activation (1). There are a number of factors that must be taken into account in selecting appropriate enzyme/prodrug combinations for this targeting strategy. Ideally, the prodrug should be stable, nontoxic, and converted to active drug only by the targeted enzyme. This suggests the use of nonendogenous enzymes that catalyze reactions not ordinarily occurring within the body. In addition, it would be advantageous for a single mAb-enzyme conjugate to activate a panel of prodrugs into anticancer drugs that can act synergistically in combination chemotherapy. Therefore, the targeted enzyme should have broad sub-
* Authors to whom correspondence should be addressed.
1 Abbreviations used: BC@L,Bacillus cereus @-lactamase(11); CCM, 7-(4-~arboxybutanamido)cephalosporinmustard; CM, 7-(phenylacetamido)cephalosporinmustard; EC@L, Escherichia coli TEM-2 @-lactamase;EClBL, Enterobacter cloacae P99 B-lactamase;mAb, monoclonal antibody; ICw,concentration that gives 50% cell kill; MTD, maximum tolerated dose; PDM, phenylenediamine mustard.
1043-1a0219312904-0334$04.0010
strate specificity. 8-Lactamases are enzymes that might meet these criteria. These enzymes cleave the B-lactams of a variety of penicillins and cephalosporins-many of which have been shown to possess considerable stability in the human body (2). Thus, for the approach described here, prodrugs that are activated as a result of &lactam cleavage would be expected to be stable when administered in vivo until they contact the targeted mAb-j3-lactamase conjugate. Promising in vitro data have been obtained with several P-lactamaselprodrug combinations (3-5). We have previously demonstrated that 7-(pheny1acetamido)cephalosporin mustard (CM) could be formed by linking the drug phenylenediamine mustard (PDM) to the 3’-position of a cephalosporin (5). CM was at least 50 times less toxic than PDM on H2981 human lung adenocarcinoma cells, but could be fully activated by a mAb-B-lactamase conjugate that bound to cell surface antigens. The 6-lactamases used for the activation of CM were obtained from Escherichia coli and Bacillus cereus. Here, we extend these findings to include 7-(4-carboxybutanamido)cephalosporin mustard (CCM), which is less toxic and easier to administer in vivo than CM. Activation of CCM is mediated both in vitro and in vivo in an immunologically specific manner by a P-lactamase from Enterobacter cloacae (EClPL) conjugated to a mAb that binds to tumorassociated antigens. EXPERIMENTAL PROCEDURES
Materials. CrudeB. cereus and E. cloacae 8-lactamases were obtained from Sigma Chemical Co. B. cereus p-lactamase (11) (BCBL) was purified according to a 0 1993 American Chemical Soclety
Activation of Cephalosporin Prodrugs
previously described procedure, which involved heat precipitation followedby cation-exchangechromatography (5). E. coli TEM-2 j3-lactamase (ECj3L) was purified on an affinity column as previously described (5). CM (5), 1 (6),and PDM (5,7)were prepared according to literature methods (structures shown in Figures 1and 2). The mAb L6 binds to antigens on many human carcinomas including the H2981 human lung adenocarcinoma (8). P1.17 shows no detectable binding to H2981 cells. Both mAbs are of the IgGzaisotype. The F(ab’)2 fragments of the mAbs were prepared by digestion with pepsinogen as previously described (5). Preparation of 7-(4-Carboxybutanamido)cephalosporin Mustard (CCM). The known cephalosporin alcohol, 2, was prepared through modifications of a literature method (6). A mixture of the monopotassium salt of 1 (4.73 g, 11.2 mmol) (6) and immobilized esterase (4.73 g, Bristol-Myers Squibb Industrial Division, Syracuse, NY) in 50 mL of 0.1 M bicarbonate (pH 9.0) was stirred overnight at room temperature. Analysis by TLC (SiOz, ethyl acetate-methanol-acetic acid, 8:l:l) revealed that the reaction was complete, and that a more polar product was formed. The reaction mixture was filtered, partially concentrated, and applied to a C-18 column (100 g, 3 x 24 cm), which was washed with H2O. Fractions that eluted between 150 and 220 mL contained the desired product. The H20 was removed by lyophilization, and the resulting pale yellow powder was triturated with methanol and filtered. The filtrate was evaporated to give 2 as the sodium salt. lH NMR (DMSO-de) indicated loss of the acetate methyl resonance and a new signal at 6 4.3 for the 3’-CH2. Formation of the bis(triethy1ammonium) salt of 2 was achieved by dissolving the compound in 0.1 M triethylammonium acetate buffer and applying the solution to a C-18 column (3 X 24 cm) equilbrated in the same buffer. Fractions containing 2 as the triethylammonium salt were combined and evaporated, and the residue was dried over P205 to give 4.27 g (70% yield) of a pale yellow gum that was used without further purification. To an ice-cold, stirred suspension of the HC1 salt of PDM (969 mg, 3.6 mmol) (7) in dry THF (40 mL) under an N2 atmosphere was added diisopropylethyl amine (0.63 mL, 3.6 mmol). After 10 min, a solution of phosgene in toluene (1.95 mL, 1.9 M) was added dropwise. TLC analysis (SiO2, ethyl acetate-hexane, 1:4) indicated completion of the reaction after 1 h with the formation of a less polar product. The isocyanate solution was introduced via a canula and Nz pressure into an ice-cold solution of the bis(triethy1ammonium) salt of 2 (1.64 g) in anhydrous DMF (10 mL) containing diisopropylethyl amine (1.6 mL, 9.2 mmol) under N2. The resulting orange solution was stirred on an ice bath for 3 h. Acetonitrile (30 mL) was added, followed by the addition of 10 g of (2-18 silica gel. Volatiles were removed, and the residue was applied to a C-18 column (2 X 12 cm), which was equilibrated with 30 % acetonitrile in 1% aqueous acetic acid. The column was washed with 200 mL of the above solvent, and then with 500 mL each of 40% and 50% acetonitrile in 1% aqueous acetic acid. The first 75 mL were discarded, and then 15-mL fractions were collected. Fractions 25-30 (containing CCM) were combined and evaporated to give 500 mg of a pale yellow gum. Addition of ethyl acetate (6 mL) resulted in the formation of a yellow solution from which CCM crystallized as a white fluffy solid (350 mg, 20% yield). HRMS: M+ 602.1022 (calcd, 602.1005). ‘H NMR (DMSO-& 300 MHz): 6 9.42 ( ~ H, , lNH), 8.83 (d, 1H, NH, J = 8.1 Hz), 7.26 (d, 2 H, ArH, J = 9.3 Hz), 6.68
Bloconlugate Chem., Vol. 4, No. 5, 1993 335
(d, 2 H, ArH, J = 9.3 Hz), 5.66 (dd, 2 H, 7-H, J = 4.1 and 8.1Hz), 5.10 (d, 1H, 6-H, J = 4.8 Hz), 5.02 (d, 1H, 3-CHz0, J = 12.6 Hz), 4.72 (d, 1H, 3-CH20, J = 12.6 Hz), 3.67 (8, 8 H, (NCHZCH2C1)2), 3.64 (d, 1H, 2-H, J = 18.0 Hz), 3.52 (d, l H , 2-H, J = 18.0 Hz), 2.21 (m, 4 H, 2’- and 4’-CH2), 1.71 (m, 2 H, 3’-CH2). Anal.: Found C, 47.51; H, 4.66; N, 9.37; C1,11.36; S, 5.30. Theory: C, 47.77; H, 4.68; N, 9.28; C1, 11.75; S, 5.31. Enzymatic Activity Assays. The activities of the j3-lactamases were measured using spectrophotometric assays with nitrocefin, CM, and CCM as substrates. Ae values were established by measuring the maximum change in absorbance of nitrocefin (M490m), CM ( M 2 6 6 n m ) , and CCM (AA2snm)of standard solutions that were treated with BCj3L (10 pg/mL) as described below. (A) Nitrocefin Assay. The enzymatic activities of the 8-lactamases were determined using nitrocefin as a substrate (5,9).Solutions containingEClj3L(5.0ngof enzyme/ mL for the EClj3L-containingconjugates, and 4.75 ng/mL for unconjugated EClj3L) were incubated at room temperature with nitrocefin (50 pM) in phosphate-buffered saline,pH 7.4 (PBS), containing 12.5pg/mL bovine serum albumin in a quartz cuvette. The absorbance at 490 nm was determined every 30 s for 5 min starting 30 s after the reaction was initiated. Under these conditions, the increase of absorbance at 490 nm ( A e 4 ~ n m= 19 500 M-’ cm-’) was linear. (B)Assays with CMand CCM. The kinetic parameters for the hydrolyses of CM and CCM (in PBS containing 12.5 pg/mL bovine serum albumin) with the 8-lactamases were obtained spectrophotometrically by determination of the initial velocities of the reactions as a function of substrate concentration. Lineweaver-Burk plots of the data were used to estimate the Km and Vm, values. Cleavage of the @-lactam ring resulted in a loss of absorbance at 266 nm for both CM (AC266nm = 1.8 X lo4 M-l cm-l) and CCM (AE266nm = 2.1 X 104M-lcm-l). The enzyme and substrate concentrations were as follows: ECj3L (1pg/mL) with CM (5.7-86 pM) or CCM (10.6-79.5 pM); BCj3L (50ng/mL) with CM (3.5-25.9 FM); BCj3L (25 ng/mL) with CCM (4.7-78.7 pM); EClj3L (100ng/mL) with CM (3.3-100.2 pM) or CCM (3.5-69.7 pM). The reactions were monitored at room temperature in a cuvette by reading A266nm every 0.1 min for 1.05 min starting at 0.15 min after the addition of enzyme. All assays were run in duplicate, and the experiments were repeated up to three times. Between experiments, the kinetic parameters differed by less than 105%. Purification of EClaL. All manipulations during the enzyme purification were carried out a t 4 OC. Crude EClgL (Sigma Chemical Co.) was taken up in ice-cold HzO and centrifuged for 30 min at 9OOg. The supernatant was applied to a boronic acid affinity column (hydrophilic-L type, IO)which was equilibrated in 20 mM triethanolamine hydrochloride containing 0.5 M NaCl (pH 7.0). The column was washed with this buffer until A2~nmwas zero, and the enzyme was eluted with 0.5 M sodium borate containing0.5 M NaCl (pH 7.0). Fractions that contained 8-lactamase activity (using nitrocefin as a substrate)were pooled and dialyzed against PBS. The purified enzyme (>95% pure by SDS-PAGE) was concentrated by ultrafiltration, sterile filtered (0.2-pm filter), and stored at 4 “C. HPLC Analysis of the Hydrolysis of CCM by EClBL. To 99.5 pL of CCM (1mM) in PBS was added EClj3L (13 pug in 5 pL PBS). Under these conditions, complete conversion of CCM to PDM occurred within 1min. The course of reaction was monitored by HPLC on an IBM
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Bioconjugate Chem., Vol. 4, No. 5, 1993
5-pm ODS C-18 column (4.6 mm X 250 mm) with a linear gradient (20 min) of 40-100 % CH3CN in 0.1 M NH40Ac. The eluent was monitored a t 260 nm, and the flow rate was 0.5 mL/min. PDM and CCM eluted at 13.2 and 4.6 min, respectively. Conjugate Preparation. The conjugates were prepared as described earlier for the preparation of mAbBCPL conjugates (5) with modifications in the method used for conjugate purification. Briefly, the F(ab')z mAbs (5-10 mg/mL) were modified with maleimide groups by the reaction with N-succinimidyl 4-(maleimidomethy1)cyclohexane-1-carboxylate (SMCC, Pierce Chemical Co., final concentration 0.25 mM) and then combined with EClPL (1:1 molar ratio) that had been modified with 2-iminothiolane (1.7 mM). After 50 min at room temperature, the reaction was terminated by the addition of 2-aminoethanethiol(O.1 mM final concentration) followed by N-ethylmaleimide (0.2 mM final concentration) in rapid succession. The mixture was concentrated by ultrafiltration and applied to a Sephacryl S-300 column equilibrated with 20 mM triethanolamine hydrochloride containing 0.5 M NaCl (pH 7.0) in order to remove aggregates and unbound enzyme. Fractions containing monomeric mAb-EClPL adducts (determined by SDS-PAGE and enzymatic activity) were pooled and applied to a boronic acid affinity column (hydrophilic-L type, IO). The column was washed with the above buffer until A2wnmwas zero to remove unconjugated mAb. mAb-EClflL conjugates were eluted with 0.5 M borate containing 0.5 M NaCl (pH 7.0). Fractions were analyzed by SDS-PAGE and relative enzyme activity. Conjugate containing fractions were pooled, dialyzed into PBS, concentrated by ultrafiltration, sterile filtered, and stored a t -70 "C. Concentrations of mAb and EClBL were determined spectrophotometrically at 280 nm using El" values of 14 and 15.3, respectively (obtained with the BCA protein ass8.y from Pierce Chemical Co.using bovine serum albumin to obtain a standard curve). The purity of the conjugates (>95 % ) was assessed by SDS-PAGE, and enzyme activities were measured using nitrocefin as a substrate. The ability of the LG-EClPL conjugate to bind to antigens on the H2981 human lung adenocarcinoma cell line was determined using a previously described method (5). The yield of the purified conjugates ranged from 16 to 18% in three separate experiments. In Vitro Cytotoxicity. H2981 cells (5 X lo5) were suspended in 0.4 mL of Roswell Park Memorial Institute 1640 media containing 10% (v/v) fetal bovine serum with or without the mAb-EClBL conjugates at 0.316 pg/mL. Blocking experiments were performed by preincubating the cells with unconjugated L6 (1mg/mL) for 10 min prior to the addition of LG-EClBL in the above medium. The cells were incubated for 30 min at 4 "C, rinsed three times with media, resuspended, and plated into 96-well tissue culture plates at IO4 cells/well (0.1 mL volume). CCM and PDM (freshly diluted in media) were added to the wells (0.1 mL volume). After 1h at 37 "C, the cells were washed three times with Iscove's modified Dulbecco's medium with 10% (v/v) fetal bovine serum, 200 units/mL penicillin, 0.1 mg/mL streptomycin, and 2 mM glutamine (IMDM) and then incubated overnight in IMDM (0.1mL/ well). The media was removed, and the cells were pulsed with t3H]thymidine (1pCi in 0.1 mL media) for 24 h. The cells were then washed with PBS, trypsinized, and harvested onto glass-fiber filters. The filters were counted on an LKB Beta plate cell harvester. In Vivo Experiments. Female athymic nude mice (8weeks-old from Harlan-Sprague Dawley, Indianapolis, IN) were implanted subcutaneously in the flank with H2981
COOH Compound
R
1
HOOC(CHz),CO
2
HOOC(CH&C 0
CCM
HOOC(CH,),CO
R1
OCOCH,
OH
CI
Figure 1. Structures of the cephalosporin derivatives used in
this study.
tumors (ca. 32 mm sections from in vivo passaging). Treatment was initiated on day 20, when the tumors were established and growing. Conjugates (1 mg mAb component/kg in PBS containing 0.1 % bovine serum albumin) were injected iv. After 96 h, CCM (46.7 mg/kg/injection in PBS containing 10% DMSO) was administered in 3 equal doses spaced 4 h apart. Other groups were treated with CCM (62.2 mg/kg/injection), PDM (1.4 mg/kg/ injection in saline), or PBS containing 10% DMSO according to the same schedule, but without having received prior conjugate treatment. Tumor volumes were eBtimated by the formula: longest length X (perpendicular widthI2/2. Each treatment group consisted of eight mice. No premature deaths resulting from the tumor implants or therapeutic regimen occurred during the course of the experiment. Toxicity experiments were performed as indicated in Table I1 using three to six mice/dose. Maximum tolerated doses (MTDs) resulted in no deaths, and were within 30% or less of doses where drug-related deaths occurred. Statistical analyseswere performed using the Student t-test, and P values that were less than 0.05 were deemed as being statistically significant.
RESULTS P r e p a r a t i o n of CM a n d CCM. Hydrolysis by 8-Lactamases. The preparation of CM (Figure 1)was carried out as previously described (5). CCM was prepared from the monopotassium salt of 1(6)by enzymatic removal of the acetoxy group, followed by condensation of the resulting 3-(hydroxymethy1)-containingproduct (2) with the isocyanate derivative of PDM (5,7). The structure of CCM wm confirmedby 3 0 0 - m lI-I ~ NMR, high-resolution mass spectrometry, and elemental analysis. Enzymatic hydrolyses of CM and CCM were investigated using 8-lactamases isolated from B. cereus, E. coli,and E. cloacae. The enzymes from B. cereus (BCBL) and E. coli (ECj3L) were purified from crude extracts as previously described (5).E. cloacae 8-lactamase (ECl/3Wwas purified from a commercially available crude preparation using a boronic acid affinity column (10). The purified enzymes appeared as single bands on SDS-PAGE gels, and had the following apparent molecular weights: BCPL, 25 kDa; ECBL, 30 kDa; EClpL, 43 kDa (data not shown). This is consistent with the literature values (11-13).
Actbation of Cephalosporin Prodrugs
Bloconlugate Chem., Vol. 4, No. 5, 1993 917
p-lactamase
loo
CM or CCM 80
COOH
r
R-NH
1 -
CI
1
CI
Is)
c
60-
E c .
E
$? 4 0 -
--o-
Figure 2. Mechanism of drug release catalyzed by 8-lactamase.
4-
t-
Table I. Kinetic Constants for B-Lactamases
CM enzyme ECDL BCDL ECIj3L
Km (pM)
kat (8-l)
25.6 6.2 7.0
63
8.3
119
CCM k a t (E-’) (pM) 61.2 1.6 31.1 116 43.2 337
Km
A spectrophotometric method was used to quantify the rate of 8-lactam hydrolysis of CM and CCM catalyzed by each of the three enzymes. This involved determination of the loss of absorbance at 266 nm, which is most likely associated with the migration of the conjugated double bond when the &lactam ring is hydrolyzed (5,141. The kinetic constants, derived from Lineweaver-Burk plots, indicated that CM had higher affinity for the enzymes than CCM (Table I). EClj3L displayed the highest kcaJ K , ratios and absolute turnover numbers with both substrates. The proposed reaction mechanism (Figure 2) is based on HPLC identification of PDM as the released product (see Experimental Procedures and ref 51, and on detailed mechanistic studies reported elsewhere with related molecules (14, 15). mAb-8-Lactamase Conjugates. On the basis of the kinetic properties shown in Table I, mAb conjugates of EClBL were prepared for further evaluation. The F(ab’)2 fragments of the IgGza mAbs L6 (anticarcinoma, 8) and P1.17 (nonbinding control) were modified with maleimide groups through the reaction with SMCC. These were combined with EClgL that contained free sulfhydryl groups as a result of the reaction with 24minothiolane. The thioether-linked conjugates thus formed were subjected to a two-step purification procedure. Gel filtration served to remove unconjugated enzyme and high molecular weight aggregates. A boronic acid affinity column (10) was then used to remove the unconjugated mAbs from the mAb-EClj3L conjugates. The principle products consisted of monomeric adducts of the mAbs to ECl@L having molecular weights of approximately 140 kDa (data not shown). Binding studies were performed using a competitive binding assay as previously reported for L6-BCBL conjugates (5). Briefly, the L6 conjugates were allowed to compete with a L6fluorescein isothiocyanate adduct (L6FITC) for binding to antigens on the H2981 human lung adenocarcinoma cell line (L6 antigen positive). Fluorescence-activated cell sorter (FACS) analysis was used to determine the extent to which L6-FITC bound to the H2981 cells, and from this, conjugate binding data was obtained. LG-EClBL was able to compete with L6-FITC for binding to H2981 cells, but was less effective in doing so compared to L6-BCPL (Figure 3). FACS analysis indicated that P1.17 and P1.17-EClBL showed no measurable specific binding to H2981 cells (data not shown). The 8-lactamase activities of the conjugates were determined using nitrocefin as a substrate ( 5 , 9 ) . Figure
0
20
40
60
80
100 1
% Test Sample Figure 3. Competitivebinding assay. H2981 cellswere incubated with various concentrations of the test samples (L6 (IgGd, L6BCj3L, and L6-ECVL) mixed with L6(I&+fluorescein ieothiccyanate. Fluorescence activated cell sorter analysis was used to determine H2981 fluorescence intensity. ECiPL L6-ECIPL
Time (minutes)
Figure 4. Enzymatic activities of ECML (4.75 ng/mL) and mAbEClSL conjugates (5 ng/mL EClj3L component) using nitrocefii (50 NM) as a substrate.
4 shows that the enzyme activities of the mAb-ECl@L conjugates were approximately the same as unmodified EClSL. Thus, the procedures used for the preparation and purification of the conjugates had little effect on enzyme activity. This is consistent with what waa previously observed with mAb-BCBL conjugates (5). In Vitro Cytotoxicity. The cytotoxic effects of CCM, PDM, and combinations of the mAb-ECl@L conjugates with CCM were determined on the H2981 cell line (L6 antigen positive, P1.17 antigen negative). Cells were exposed to CCM or PDM for 1h, washed, and incubated for a total of 48 h. Viability was determined by measuring the incorporation of 13H]thymidine into DNA. The cytotoxic activity of CCM (IC50 = 25-45 pM)was much lower than that of PDM (IC50 = 1.5 pM, Figure 5). A significant increase in the cytotoxic activity of CCM was obtained on cells that were pretreated with LGEClBL. The activity of this combination (IC50 = 3.2 pM) was comparable to that for PDM. Prodrug activation occurred in an immunologically specificmanner as evidenced by the fact that P1.17-ECvL did not increase the cytotoxic activity of CCM. A second method to show immunological specificity involved sat-
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Vrudhula et el.
Bloconjugate Chem., Vol. 4, No, 5, 1993 T
Table 11. Maximum Tolerated Doses of CM, CCM, and PDM in Nude Mice. compd
k\
I
CM CCM CCM PDM PDM
\
schedule 3 doses, 12 h apart 3 doses, 12 h apart 3 doses, 4 h apart 3 doses, 12 h apart 3 doses, 4 h apart
dose (mgikglinjection)
23 >62* 62 2.8 1.4
The agents listed were administered iv (tail vein) in female athymic nude mice (3 to 6 animalsigroup). Highest dose tested.
*
40
-
20
-
\
-
d CCM PDM
* CCM + L6-ECIPL CCM + L6-ECIPL + L6 CCM + P1.17-ECIPL
O I 1
I
1
10
100
Concentration (pM) Figure 5. In vitro cytotoxicity. H2981 cells (L6 antigen positive, P1.17 antigen negative) were treated with L6-EClj3L or P1.17EClSL (0.316 pg/mL), washed, and then treated with CCM. Antigen blocking was achieved by treating the cells with saturating amounts of L6 (1 mg/mL) prior to treatment with L6-ECl@L.
urating H2981 cells with unmodified L6 prior to exposure to LG-EClpL. Again, there was no enhancement in the cytotoxic activity of CCM (IC50 = 30 pM). These experiments establish that CCM is a prodrug and that prodrug activation is mediated in an immunologically specific manner by conjugate that binds to cell-surface antigens. In Vivo Studies. Initial experiments in H2981 tumor bearing mice that were treated with mAb-8-lactamase conjugates indicated that the antitumor activities of CM and CCM were significantly higher when the prodrugs were administered intravenously (iv) compared to intraperitoneally (datanotshown). Therefore iv administration was used to establish maximum tolerated doses (MTDs) of CM, CCM, and PDM. The toxicities of CM, CCM, and PDM in nude mice were determined by injecting the agents at a variety of doses using two different dosing schedules. The MTD is defined as the dose that resulted in no deaths, and where drug-related deaths occurred with dose increases of 30% or less. Table I1 shows the MTDs for the various agents. It was found that both CM and CCM had significantly higher MTDs than PDM. When administered in three doses spaced 12 h apart, the MTDs of CM and CCM were 3.6 and >9.9 times greater than PDM on a molar basis. However, CM caused extensive necrosis at the site of
injection, which limited subsequent prodrug administration. In contrast, no apparent necrosis was associated with the administration of CCM. Consequently, therapy experiments with CCM in combination with the mAb-EClpL conjugates were the subject of subsequent investigation. In vivo therapy experiments were performed on mice that had established subcutaneous H2981 tumors. The mice were randomized just before the initiation of therapy so that there were no significant differences in tumor sizes between any of the groups. Treatment was initiated 20 days after implantation, a t which time the tumors were established (approximately 180 mm3 in volume) and growing. The mAb-EClpL conjugates were administered iv at 1 mg mAb component/kg, which corresponds to approximately 25 pg/mouse. After 96 h, the prodrug CCM (46.7 mg/kg/injection) was then administered in three separate doses spaced 4 h apart. This dosing schedule was selected since at 96 h after conjugate administration the blood levels of enzymatic activity were low. In addition, it was possible to administer more CCM cumulatively in three separate doses compared to a single bolus injection. Other groups were treated with CCM (62 mg/kg/injection) or PDM (1.4 mg/kg/injection) without prior conjugate treatment. In these experiments, the drug and prodrug doses used were at or very near to the MTDs. The MTD of CCM in conjugate-treated mice (46.7 mg/kg/injection) was lower than when CCM was given alone (62 mg/kg/ injection), owing most likely to the formation of PDM by the mAb-enzyme conjugate. There were no treatment or tumor related toxicities during the entire course of the experiment. The prodrug, CCM, and drug, PDM, had little to no effect on tumor growth (Figure 6). In contrast, asignificant level of antitumor activity was obtained in mice treated with L6-EClj3L prior to the administration of CCM. The antitumor effect appeared to be immunologicallyspecific, since a lower level of antitumor activity was obtained when CCM was administered to animals that had previously been treated with the nonbinding control conjugate P1.17ECWL. Statistical analysis of the data from day 34 onward indicated that the tumors in LG-ECl@L/CCMtreated mice were smaller than in any of the other treatment groups (P < 0.05). DISCUSSION
Previously, it was demonstrated that CM was a prodrug that released PDM upon treatment with 8-lactamases from E. coli and B. cereus (5). Invitro cytotoxicityexperiments indicated that CM was approximately 50 times less cytotoxic to H2981 cells than PDM, and that L6-BCpL could activate CM in an immunologically specific manner. These results prompted us to investigate the in vivo activities of CM in combination with mAb-p-lactamase conjugates. In vivo experiments indicated that CM was significantly less toxic to mice than PDM (Table 11). However, treatment with CM was hampered by severe tail necrosis followingiv injection. We reasoned that the necrosis might
1-
Bioconlugete Chem., Vol. 4, No. 5, 1993 338
Activation of Cephalosporln Prodrugs 16007
1400
h
1200
-
E E
1000
-
800
-
m
--O-
Control L6-ECIPL + CCM
f
P1.17-ECIPL+ CCM
v
600 400
-
200
-
0 4 12
I
19
26
33
40
47
Days Post Implant Figure 6. Effects of PDM, CCM, and mAb-EClgL conjugates in combination with CCM on subcutaneous H2981 tumors in nude mice (eight mice/group). Conjugates (1 mg/kg) were administered on day 20. This was followed 96 h later by CCM (46.7 mg/kg/injection). The therapeutic effects were compared to those of CCM (62.2 mg/kg/injection) and PDM (1.4 mg/kg/ injection) in mice that did not receive prior conjugate treatment. All drugs were administered in three doses spaced 4 h apart.
be due to in vivo precipitation of the prodrug, and that precipitation would not occur with more water-soluble analogues. This provided the rationale for the synthesis of CCM, which has a carboxylic acid containing side chain appended to the 7-position of the cephalosporin. In CM, this position is substituted with a hydrophobic phenylacetyl group. CCM was shown to be less cytotoxic than PDM on H2981 cells (Figure 5). This was anticipated, since previous studies have demonstrated that electronwithdrawing or highly polar substituentsappended to the aromatic rings on nitrogen mustards lead to reductions in cytotoxic activity (5, 16-19). ECPL, BCPL, and EClPL were all able to catalyze P-lactam hydrolysis of CCM. On the basis of the kinetic data shown in Table I, mAbEClBL conjugates were prepared and tested for their abilities to activate CCM. In vitro studies indicated that LG-EClPL effected the activation of CCM on L6 antigen positive H2981 cells (Figure 5). Prodrug activation occurred in an immunologically specific manner, since no such enhancement took place on P1.17-EClBL treated cells. Thus, while the binding of L6-EClPL is impaired compared to L6 (Figure 31, enough binding activity is retained by the conjugate to selectively convert CCM to PDM. The reduced binding of L6-EClPL compared to LG-BCPL was reproducibly found in three separate experiments We believe that this is due to steric interaction between EClBL with one of the L6 antigen binding sites. Efforts are currently underway to explore the effects that other conjugation strategies have on the antigen binding and prodrug activation characteristics of L6-EClBL. The approach of using targeted enzymes for prodrug activation will be therapeutically useful only if it clearly has advantages over conventional cytotoxic chemotherapy. One way to establish such an advantage is to compare the activities on subcutaneous tumor xenografts, even though such tumor models bear little resemblance to actual tumors that grow and metastasize in humans. With this limitation in mind, it is encouraging that LG-EClPL in combination with CCM was superior in activity to PDM in vivo (Figure 6). This is noteworthy, since H2981 tumor xenografts were well-established and unresponsive to therapy with MTDs of PDM or CCM without prior conjugate administration.
These results are consistent with other mAb-enzyme/ prodrug combinations that have also demonstrated superior in vivo activities compared to conventional drug therapy (1, 16, 17,20-22). The fact that some degree of antitumor activity was mediated by P1.17-EClbL in combination with CCM (Figure 6) may be due to residual levels of systemic and tumor-associated enzyme activity when the prodrug was administered. Previous in vivo studies with conjugates of alkaline phosphatase in combination with mitomycin phosphate have given similar results (20). Even though the in vivo therapeutic effects of the L6EClBLICCM combination are superior to systemic drug administration, it is apparent from the data shown in Figure 6 that continued efforts to enhance the activities yet further are required. It should be noted that the effects of conjugate dose and timing of prodrug administration have not been extensively explored. Parameters such as these will likely have strong influences on antitumor activity (22)and immunological specificity. Also, in vivo experiments with other cephalosporin-containing prodrugs, such as cephalosporin doxorubicin (151,are currently the subject of investigation, since the nature of the released drug will undoubtedly play an important role in therapeutic efficacy. ACKNOWLEDGMENT
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